bioRxiv preprint doi: https://doi.org/10.1101/2023.05.01.538969; this version posted May 1, 2023. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Molecular dissection of PI3Kb synergistic activation by receptor tyrosine kinases, GbGg, and Rho-family GTPases Benjamin R. Duewell*, Naomi E. Wilson*, Gabriela M. Bailey, Sarah E. Peabody, & Scott D. Hansen# Department of Chemistry and Biochemistry, Institute of Molecular Biology, University of Oregon, Eugene, OR 97403 * these authors contributed equally to this work # corresponding author: shansen5@uoregon.edu The class 1A phosphoinositide 3-kinase (PI3K) beta (PI3Kβ) is functionally unique in the ability to integrate signals derived from receptor tyrosine kinases (RTKs), heterotrimeric guanine nucleotide- binding protein (G-protein)-coupled receptors (GPCRs), and Rho-family GTPases. The mechanism by which PI3Kβ prioritizes interactions with various membrane tethered signaling inputs, however, remains unclear. Previous experiments have not been able to elucidate whether interactions with membrane- tethered proteins primarily control PI3Kβ localization versus directly modulate lipid kinase activity. To address this gap in our understanding of PI3Kβ regulation, we established an assay to directly visualize and decipher how three binding interactions regulate PI3Kβ when presented to the kinase in a biologically relevant configuration on supported lipid bilayers. Using single molecule Total Internal Reflection Fluorescence (TIRF) Microscopy, we determined the mechanism controlling membrane localization of PI3Kβ, prioritization of signaling inputs, and lipid kinase activation. We find that auto-inhibited PI3Kβ must first cooperatively engage a single RTK-derived tyrosine phosphorylated (pY) peptide before it can engage either GβGγ or Rac1(GTP). Although pY peptides strongly localize PI3Kβ to membranes, they only modestly stimulate lipid kinase activity. In the presence of either pY/GβGγ or pY/Rac1(GTP), PI3Kβ activity is dramatically enhanced beyond what can be explained by the increase in membrane avidity for these complexes. Instead, PI3Kβ is synergistically activated by pY/GβGγ and pY/Rac1(GTP) through a mechanism of allosteric regulation. INTRODUCTION Graziano et al. 2017), sustained PI(3,4,5)P3 signaling Critical for cellular organization, phosphatidylinositol is known to drive cancer cell metastasis (Hanker et al. phosphate (PIP) lipids regulate the localization and 2013). Elevated PI(3,4,5)P3 levels also stimulates the activity of numerous proteins across intracellular AKT signaling pathway and Tec family kinases, which membranes in eukaryotic cells (Di Paolo and De can drive cellular proliferation and tumorigenesis Camilli 2006). The interconversion between various (Manning and Cantley 2007; Fruman et al. 2017). PIP lipid species through the phosphorylation and While much work has been dedicated in determining dephosphorylation of inositol head groups is regulated the factors that participate in the PI3K signaling by lipid kinases and phosphatases (Balla 2013; Burke pathway, how these molecules collaborate to rapidly 2018). Serving a critical role in cell signaling, the synthesize PI(3,4,5)P3 remains an important open class I family of phosphoinositide 3-kinases (PI3Ks) question. To decipher how amplification of PI(3,4,5)P3 catalyze the phosphorylation of phosphatidylinositol arises from the relay of signals between cell surface 4,5-bisphosphate [PI(4,5)P2] to generate PI(3,4,5) receptors, lipids, and peripheral membrane proteins, P3. Although a low-abundance lipid (< 0.05%) in the we must understand how membrane localization and plasma membrane (Wenk et al. 2003; Nasuhoglu et activity of PI3Ks is regulated by different signaling al. 2002; Stephens, Jackson, and Hawkins 1993), inputs. Determining how these biochemical reactions PI(3,4,5)P3 can increase 40-fold following receptor are orchestrated will provide new insight concerning activation (Stephens, Hughes, and Irvine 1991; Parent the molecular basis of asymmetric cell division, cell et al. 1998; Insall and Weiner 2001). Although signal migration, and tissue organization, which are critical adaptation mechanisms typically restore PI(3,4,5)P3 to for understanding development and tumorigenesis. the basal level following receptor activation (Funamoto In the absence of a stimulatory input, the class IA et al. 2002; Yip et al. 2008; Auger et al. 1989), family of PI3Ks (PI3Kα, PI3Kβ, PI3Kδ) are thought to misregulation of the PI3K signaling pathway can result reside in the cytoplasm as auto-inhibited heterodimeric in constitutively high levels of PI(3,4,5)P3 that are protein complexes composed of a catalytic (p110α, detrimental to cell health. Since PI(3,4,5)P3 lipids serve p110β, or p110δ) and regulatory subunit (p85α, an instructive role in driving actin based membrane p85β, p55γ, p50α, or p55α) (Burke 2018; Vadas et protrusions (Howard and Oresajo 1985; Weiner 2002; al. 2011). The catalytic subunits of class IA PI3Ks bioRxiv preprint doi: https://doi.org/10.1101/2023.05.01.538969; this version posted May 1, 2023. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. contain an N-terminal adaptor binding domain (ABD), GTPases, or GβGγ remains unclear. In the case of a Ras/Rho binding domain (RBD), a C2 domain synergistic PI3Kβ activation, it’s unclear which protein- (C2), and an adenosine triphosphate (ATP) binding protein interactions regulate membrane localization pocket (Vadas et al. 2011). The inter-SH2 (iSH2) versus stimulate lipid kinase activity. No studies domain of the regulatory subunit tightly associates have simultaneously measured PI3Kβ membrane with the ABD of the catalytic subunit (Yu et al. 1998), association and lipid kinase activity to decipher providing structural integrity, while limiting dynamic potential mechanisms of allosteric regulation. Previous conformational changes. The nSH2 and cSH2 domains studies concerning the synergistic activation of PI3Ks of the regulatory subunit form additional inhibitory are challenging to interpret because RTK derived contacts that limit the conformational dynamics of the peptides are always presented in solution alongside catalytic subunit (Zhang et al. 2011a; Mandelker et al. membrane anchored signaling inputs. However, all 2009; Burke et al. 2011; Carpenter et al. 1993; Yu et the common signaling inputs for PI3K activation (i.e. al. 1998). A clearer understanding of the how various RTKs, GβGγ, Rac1/Cdc42) are membrane associated proteins control PI3K localization and activity would proteins. Activation of class 1A PI3Ks has never help facilitate the development of drugs that perturb been reconstituted using solely membrane tethered specific protein-protein binding interfaces that are activators conjugated to membranes in a biologically critical for membrane targeting and lipid kinase activity. relevant configuration. As a result, we currently lack Among the class IA PI3Ks, PI3Kβ is uniquely a comprehensive description of PI3Kβ membrane capable of interacting with Rho-family GTPases recruitment and catalysis. (Fritsch et al. 2013a), Rab GTPases (Christoforidis et To decipher the mechanisms controlling PI3Kβ al. 1999; Heitz et al. 2019), heterotrimeric G-protein membrane binding and activation, we established complexes (GβGγ) (Kurosu et al. 1997; Maier, Babich, a biochemical reconstitution using supported lipid and Nürnberg 1999; Guillermet-Guibert et al. 2008), bilayers (SLBs). We used single molecule Total Internal and phosphorylated receptor tyrosine kinases (RTKs) Reflection Fluorescence (TIRF) microscopy to quantify (Zhang et al. 2011a; Carpenter et al. 1993). Like other the relationship between PI3Kβ localization, lipid class IA PI3Ks, interactions with receptor tyrosine kinase activity, and the density of various membrane- kinase (RTK) derived phosphotyrosine peptides tethered signaling inputs. This approach allowed us release nSH2 and cSH2-mediated inhibition of the to measure the dwell time, binding frequency, and catalytic subunit to stimulate PI3Kβ lipid kinase activity diffusion coefficients of single fluorescently labeled (H. A. Dbouk et al. 2012; Zhang et al. 2011b). GβGγ PI3Kβ in the presence of RTK derived peptides, and Rac1(GTP) in solution have also been shown Rac1(GTP), and GβGγ. Simultaneous measurements to stimulate PI3Kβ lipid kinase activity (Hashem of PI3Kβ membrane recruitment and lipid kinase A. Dbouk et al. 2012; Fritsch et al. 2013a; Maier, activity allowed us to define the relationship between Babich, and Nürnberg 1999). Similarly, activation PI3Kβ localization and PI(3,4,5)P3 production in the of Rho-family GTPases (Fritsch et al. 2013a) and presence of different regulators. Overall, we found that G-protein coupled receptors (Houslay et al. 2016) membrane docking of PI3Kβ first requires interactions stimulate PI3Kβ lipid kinase activity in cells. However, with RTK-derived tyrosine phosphorylated (pY) it’s unclear how individual interactions with GβGγ or peptides, while PI3Kβ localization is insensitive to Rac1(GTP) can bypass autoinhibition of full-length membranes that contain either Rac1(GTP) or GβGγ PI3Kβ (p110β-p85α/β). Studies in neutrophils and in alone. Following engagement with a pY peptide, vitro biochemistry suggest that PI3Kβ is synergistically PI3Kβ can associate with either GβGγ or Rac1(GTP). activated through coincidence detection of RTKs In the case of synergistic PI3Kβ localization mediated and GβGγ (Houslay et al. 2016; Hashem A. Dbouk by pY/GβGγ, it’s essential for the nSH2 domain to et al. 2012). Similarly, Rac1(GTP) and GβGγ have move away from the GβGγ binding site. Although been reported to synergistically activate PI3Kβ in both the PI3Kβ-pY-Rac1(GTP) and PI3Kβ-pY-GβGγ cells (Erami et al. 2017). An enhanced membrane complexes display a ~2-fold increase in membrane recruitment mechanism is the most prominent model localization, the corresponding increase in catalytic used to explain synergistic activation of PI3Ks. efficiency is much greater. Overall, our results indicate There is limited kinetic data examining how PI3Kβ that synergistic activation of PI3Kβ depends on is regulated by different membrane-tethered proteins. allosteric modulation of lipid kinase activity. Previous biochemical studies of PI3Kβ have utilized solution-based assays to measure P(3,4,5)P3 RESULTS production. As a result, the mechanisms that determine how PI3Kβ prioritizes interactions with RTKs, small PI3Kβ prioritizes interactions with pY peptides over Rac1(GTP) and GβGγ bioRxiv preprint doi: https://doi.org/10.1101/2023.05.01.538969; this version posted May 1, 2023. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. A PIK3CB (p110β) B PIK3R1(p85α) Dy647 nSH2 Cy5-nSH2 Cy3-p67/phox SH3 BH cSH2 iSH2 SNAP pY Gβ Gβ AF488 Rac1 Gγ Signal Gγ Inputs GEFGTP GDP GTP pY +GTP farnesyl DOPC MCC PIP2 Glass Glass TIRF microscopy C D nSH2-Cy5 Cy3-p67/Phox AF488-GβGγ E Dy647-PI3Kγ 8000 – pY +GDP [low] 7000 6000 5000 4000 3000 +pY +GTP [high] 2000 + P-Rex1 GDP GTP 1000 0 0 100 200 300 400 Time (seconds) 5 µm 5 µm F no inputs Rac1(GTP) GβGγ pY G 8000 +pY 7000 6000 5000 4000 3000 2000 +Rac1(GTP) 1000 +GβGγ 0 0 200 400 600 800 5 µm Time (seconds) Figure 1 PI3Kβ prioritizes membrane interactions with RTK-derived pY peptides over Rac1(GTP) and GβGγ (A) Cartoon schematic showing membrane tethered signaling inputs (i.e. pY, Rac1(GTP), and GβGγ) attached to a supported lipid bilayer and visualized by TIRF-M. Heterodimeric Dy647-PI3Kβ (p110β-p85α) in solution can dynamically associate with membrane bound proteins. (B) Cartoon schematic showing method for visualizing membrane tethered signaling inputs. (C) Kinetics of Rac1 nucleotide exchange measured in the presence of 20 nM Rac1(GTP) sensor (Cy3-p67/phox) and 50 nM P-Rex1. (D) Visualization of membrane conjugated RTK derived pY peptide, Rac1(GTP), and GβGγ by TIRF-M. Representative TIRF-M images showing the membrane localization of 20 nM nSH2-Cy3 in the absence and presence of membranes conjugated with a solution concentration of 10 µM pY peptide. Representative images showing the membrane localization of 20 nM Cy3-p67/phox Rac1(GTP) sensor before (GDP) and after (GTP) the addition of the guanine nucleotide exchange factor, P-Rex1. Equilibrium localization of 50 nM (low) or 200 nM (high) farnesyl GβGγ-SNAP-AF488. (E) Representative TIRF-M images showing the equilibrium membrane localization of 10 nM Dy647-PI3Kγ measured in the absence and presence of membranes equilibrated with 200 nM farnesyl GβGγ. (F) Representative TIRF-M images showing the equilibrium membrane localization of 5 pM and 10 nM Dy647-PI3Kβ measured in the presence of membranes containing either pY, Rac1(GTP), or GβGγ. The inset image (+GβGγ) shows low frequency single molecule binding events detected in the presence of 10 nM Dy647-PI3Kβ. (G) Bulk membrane absorption kinetics for 10 nM Dy647-PI3Kβ measured on membranes containing either pY, Rac1(GTP), or GβGγ. Membrane composition: 96% DOPC, 2% PI(4,5)P2, 2% MCC-PE. Cy3-p67/Phox Rac1(GTP) Dy647-PI3Kβ sensor intensity 10 nM 5 pM Dy647-PI3Kβ membrane intensity +GβGγ – GβGγ bioRxiv preprint doi: https://doi.org/10.1101/2023.05.01.538969; this version posted May 1, 2023. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Previous biochemical analysis of p110β-p85α, the presence of GβGγ alone, the binding frequency referred to as PI3Kβ, established that receptor tyrosine was reduced 2000-fold compared our measurements kinases (Zhang et al. 2011b), Rho-type GTPases on pY membranes (Figure 1 – figure supplement (Fritsch et al. 2013a), and heterotrimeric G-protein 2). In addition, localization of wild-type Dy647-PI3Kβ GβGγ (Hashem A. Dbouk et al. 2012) are capable of phenocopied the GβGγ binding mutant, Dy647-PI3Kβ binding and stimulating lipid kinase activity. To decipher (K532D, K533D), indicating that the low frequency how PI3Kβ prioritizes interactions between these three binding events we observed are mostly mediated by membrane-tethered proteins we established a method lipid interactions rather than direct binding to GβGγ to directly visualize PI3Kβ localization on supported (Figure 1 – figure supplement 2). lipid bilayers (SLBs) using Total Internal Reflection Fluorescence (TIRF) Microscopy (Figure 1A). For PI3Kβ cooperatively engages a single membrane this assay, we covalently attached either a doubly tethered pY peptide tyrosine phosphorylated platelet derived growth Previous biochemical analysis of PI3Kβ utilized pY factor (PDGF) peptide (pY) peptide or recombinantly peptides in solution to study the regulation of lipid purified Rac1 to supported membranes using kinase activity (Zhang et al. 2011b; Hashem A. Dbouk cysteine reactive maleimide lipids. We confirmed et al. 2012). Using membrane-tethered pY peptide, we membrane conjugation of the pY peptide and Rac1 quantitatively mapped the relationship between the pY by visualizing the localization of fluorescently labeled membrane surface density and the membrane binding nSH2-Cy5 or Cy3-p67/phox (Rac1(GTP) sensor), behavior of Dy647-PI3Kβ (Figure 2A). To calculate respectively (Figure 1B). Nucleotide exchange of the membrane surface density of conjugated pY, we membrane conjugated Rac1(GDP) was achieved by incorporated a defined concentration of Alexa488-pY the addition of a guanine nucleotide exchange factor, (Figure 2A). We measured the relationship between P-Rex1 (phosphatidylinositol 3,4,5-trisphosphate- the total solution concentration of pY peptide used for dependent Rac exchanger 1 protein) diluted in GTP the membrane conjugation step and the corresponding containing buffer (Figure 1C). As previously described final membrane surface density (pY per µm2). Over a (Rathinaswamy et al. 2021; 2023), AF488-SNAP dye range of pY peptide solution concentrations (0-10 µM), labeled farnesyl GβGγ was directly visualized following we observed a linear increase in the membrane passive absorption into supported membranes conjugation efficiency based on the incorporation (Figure 1D and Figure 1 – figure supplement 1). of fluorescent Alexa488-pY (Figure 2B). Bulk We confirmed that membrane bound GβGγ was membrane localization of a nSH2-Cy3 sensor showed functional by visualizing robust membrane recruitment a corresponding linear increase in fluorescence as of Dy647-PI3Kγ by TIRF-M (Figure 1E). Overall, this a function of pY peptide membrane density (Figure assay functions as a mimetic to the cellular plasma 2C). By quantifying the average number of Alexa488- membrane and allowed us to examine how different pY particles per unit area of supported membrane we membrane tethered signaling inputs regulate PI3Kβ calculated the absolute density of pY per µm2 (Figure membrane localization in vitro. 2D). We visualized both single molecule binding events To determine how the membrane binding behavior of and bulk membrane localization of Dy647-PI3Kβ by PI3Kβ is modulated by the membrane surface density TIRF-M to determine which inputs can autonomously of pY, we measured the bulk membrane absorption recruit autoinhibited Dy647-PI3Kβ from solution to kinetics of Dy647-PI3Kβ. When Dy647-PI3Kβ was a supported membrane (Figure 1F). Comparing flowed over a membrane containing a low density of ≤ membrane localization of Dy647-PI3Kβ in the presence 500 pY/µm2, we observed rapid equilibration kinetics of pY, Rac1(GTP), and GβGγ revealed that only the consistent with a 1:1 binding stoichiometry (Figure 2E). tyrosine phosphorylated peptide (pY) could robustly When the membrane surface density of pY crossed localize Dy647-PI3Kβ to supported membranes a threshold density of ~1000 pY/µm2, we observed (Figure 1F-1G). This prioritization of interactions was slower equilibration kinetics consistent with Dy647- consistently observed across a variety of membrane PI3Kβ either engaging two pY peptides or exhibiting lipid compositions (Figure 1 – figure supplement 2). membrane hopping. Single particle tracking of Dy647- Incorporation of up to 20% phosphatidylserine (PS) in PI3Kβ on membranes containing varying densities of supported membranes to increase the anionic charge pY peptide revealed that the dwell time was relatively did not facilitate complex formation between Dy647- insensitive to the pY peptide density (Figure 2F and PI3Kβ and Rac1(GTP) or GβGγ (Figure 1 – figure Table 1). Similarly, the displacement (or step size) of supplement 2). Although we could detect some pY-tethered Dy647-PI3Kβ was nearly identical across transient Dy647-PI3Kβ membrane binding events in a range of pY membrane densities examined (Figure 2G and Table 1). Together, these results suggest that bioRxiv preprint doi: https://doi.org/10.1101/2023.05.01.538969; this version posted May 1, 2023. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. A B D AF488-pY peptide C 3500 20 5000 AF488 3000 4000 2500 15 2000 3000 10 1500 2000 1000 5 1000 DOPC 500 MCC 0 0 0 Glass 0 2 4 6 8 10 0 2 4 6 8 10 5 µm solution coupling solution coupling conc. of pY, µM conc. of pY, µM E F G 10000 1 30.3µM 9000 0.3µM 0.3µM0.6µM 0.6µM 8000 0.6µM 7000 1.25µM 1.25µM 1.25µM 2.5µM 0.1 2 2.5µM 6000 2.5µM5µM 5µM 5000 5µM10µM 10µM 4000 10µM 3000 0.01 1 2000 1000 0 0.001 0 0 200 400 600 800 0 2 4 6 8 10 12 0 0.3 0.6 0.9 1.2 1.5 Time (seconds) Dwell Time (sec) step size (µm) H I J 12000 PI3Kβ 1 PI3K 3β PI3Kβ 10000 PI3Kβ nSH2* PI3Kβ nSH2* 8000 PI3Kβ cSH2*0.1 PI3Kβ cSH2*2 6000 PI3Kβ nSH2* 4000 0.01 1 2000 PI3Kβ cSH2* 0 0.001 0 0 200 400 600 800 0 2 4 6 8 10 12 0 0.2 0.4 0.6 0.8 1.0 1.2 Time (seconds) Dwell Time (sec) step size (µm) Figure 2 Density dependent membrane binding behavior of Dy647-PI3Kβ measured in the presence of RTK-derived pY peptides (A) Cartoon schematic showing conjugation of pY peptides (+/- Alexa488 label) using thiol reactive maleimide lipids (MCC-PE). (B) Representative image showing the single molecule localization of Alexa488-pY. Particle detection (purple circles) was used to quantify the number of pY peptides per µm2. (C) Relationship between the total pY solution concentration (x-axis) used for covalent conjugation, the bulk membrane intensity of covalently attached Alexa488-pY (left y-axis), and the final surface density of pY peptides per µm2 (right y-axis). (D) Relationship between the total pY solution conjugation concentration and bulk membrane intensity of measured in the presence of 50 nM nSH2-Cy3. (E-G) Membrane localization dynamics of Dy647-PI3Kβ measured on SLBs containing a range of pY surface densities (250–15,000 pY/µm2, based on Figure 1C). (E) Bulk membrane localization of 10 nM Dy647-PI3Kβ as a function of pY density. (F) Single molecule dwell time distributions measured in the presence of 5 pM Dy647-PI3Kβ. Data plotted as log10(1–CDF) (cumulative distribution frequency). (G) Step size distributions showing Dy647-PI3Kβ single molecule displacements from > 500 particles (>10,000 steps) per pY surface density. (H-J) Membrane localization dynamics of Dy647-PI3Kβ nSH2(R358A) and cSH2(R649A) mutants measured on SLBs containing ~15,000 pY/µm2 (10µM conjugation concentration). (H) Bulk membrane absorption kinetics of 10 nM Dy647-PI3Kβ (WT, nSH2*, and cSH2*). (I) Single molecule dwell time distributions measured in the presence of 5 pM Dy647-PI3Kβ (WT, nSH2*, and cSH2*). Data plotted as log10(1–CDF) (cumulative distribution frequency). (J) Step size distributions showing single molecule displacements of > 500 particles (>10,000 steps) in the presence of 5 pM Dy647- PI3Kβ (WT, nSH2*, and cSH2*). Membrane composition: 96% DOPC, 2% PI(4,5)P2, 2% MCC-PE. Dy647-PI3Kβ membrane intensity Dy647-PI3Kβmembrane intensity log10(1-CDF) log10(1-CDF) mean AF488-pY membrane intensity Probability Density Probability Density mean Cy3-SH2 membrane intensity pY per µm2 (x103) bioRxiv preprint doi: https://doi.org/10.1101/2023.05.01.538969; this version posted May 1, 2023. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Dy647-PI3Kβ engages one doubly phosphorylated in the presence of both pY and GβGγ (Figure 3C). peptide over a broad range of pY densities in our Consistent with Dy647-PI3Kβ forming a complex with bilayer assay. pY and GβGγ, we observed a 22% reduction in the The regulatory subunit of PI3Kβ (p85α) contains two average single particle displacement and a decrease SH2 domains that form inhibitory contacts with the in the diffusion coefficient due to synergistic binding catalytic domain (p110β) (Zhang et al. 2011b). The (Figure 3D). SH2 domains of class 1A PI3Ks have a conserved Parallel to our experiments using membrane peptide motif, FLVR, that mediates the interaction with conjugated pY, we tested whether solution pY tyrosine phosphorylated peptides (Bradshaw, Mitaxov, could promote Dy647-PI3Kβ localization to GβGγ- and Waksman 1999; Waksman et al. 1992; Rameh, containing membranes. Based on the bulk membrane Chen, and Cantley 1995). Mutating the arginine to recruitment, solution pY did not strongly enhance alanine (FLVA mutant) prevents the interaction with pY membrane binding of Dy647-PI3Kβ on GβGγ- peptides for both PI3Kα and PI3Kβ (Yu et al. 1998; containing membranes (Figure 3B). Single molecule Dornan et al. 2020; Nolte et al. 1996; Zhang et al. 2011b; dwell analysis revealed few transient Dy647-PI3Kβ Breeze et al. 1996). To determine how the membrane membrane interactions (inset Figure 3A) with a mean binding behavior of PI3Kβ is modulated by each SH2 dwell time of 116 ms in the presence of GβGγ alone domain, we individually mutated the FLVR amino acid (Figure 3 – figure supplement 1A). The presence of sequence to FLVA. Compared to wild type Dy647- 10 µM solution pY modestly increased the mean dwell PI3Kβ, the nSH2(R358A) and cSH2(R649A) mutants time of Dy647-PI3Kβ to 136 ms on GβGγ containing showed a 60% and 75% reduction in membrane membranes (Figure 3 – figure supplement 1B). This localization at equilibrium, respectively (Figure 2H). suggests that the affinity between PI3Kβ and GβGγ Single molecule dwell time analysis also showed a is relatively weak, which is consistent with previous significant reduction in membrane affinity for Dy647- structural biochemistry studies (Hashem A. Dbouk et PI3Kβ nSH2(R358A) and cSH2(R649A) compared al. 2012). to wild type PI3Kβ (Figure 2I and Table 1). Single For PI3Kβ to engage GβGγ, it is hypothesized molecule diffusion (or mobility) of membrane bound that the nSH2 domain must move out of the way nSH2(R358A) and cSH2(R649A) mutants, however, from sterically occluding the GβGγ binding site. This were nearly identical to wild type Dy647-PI3Kβ (Figure model is supported by previous hydrogen deuterium 2J and Table 1). Because the nSH2 and cSH2 mutants exchange mass spectrometry (HDX-MS) experiments can only interact with a single phosphorylated tyrosine that only detected interactions between GβGγ and residue on the doubly phosphorylated pY peptide, this PI3Kβ (p110β) when the nSH2 domain was either further supports a model in which the p85α regulatory absent or disengaged by activation using a soluble subunit of PI3Kβ cooperatively engages one doubly RTK pY peptide (H. A. Dbouk et al. 2012). We phosphorylated pY peptide under our experimental examined the putative interface of GβGγ bound to conditions. the p110β catalytic domain using AlphaFold multimer (Jumper et al. 2021; Evans et al. 2022; Varadi et al. GβGγ dependent enhancement in PI3Kβ 2022) which defined ha1 in the helical domain as the localization requires release of the nSH2 binding site. This result was consistent with previous Having established that PI3Kβ engagement with mutagenesis and HDX-MS analysis of GβGγ binding a membrane tethered pY peptide is the critical first to p110β (Hashem A. Dbouk et al. 2012). Comparing step for robust membrane localization, we examined our model to previous X-ray crystallographic data of the secondary role that GβGγ serves in controlling SH2 binding to either p110a and p110β (Zhang et al. membrane localization of PI3Kβ bound to pY. To 2011a; Mandelker et al. 2009) suggested that the nSH2 measure synergistic membrane localization mediated domain sterically obstructs the GβGγ binding interface by the combination of pY and GβGγ, we covalently (Figure 3E and Figure 3 – figure supplement 2), linked pY peptides to supported membrane at a surface with GβGγ activation only possible when the p110β- density of ~10,000 pY/µm2 and then allowed farnesyl nSH2 interface is disrupted. To test this hypothesis, GβGγ to equilibrate into the membrane. Comparing we measured the membrane binding dynamics the bulk membrane absorption of Dy647-PI3Kβ in of Dy647-PI3Kβ nSH2(R358A) on membranes the presence of pY alone, we observed a 2-fold containing pY and GβGγ. Comparing the bulk increase in membrane localization due to synergistic membrane recruitment of these constructs revealed association with pY and GβGγ (Figure 3A-3B). Single that the inability of the Dy647-PI3Kβ nSH2 domain molecule imaging experiments also showed a 1.9-fold to bind to pY peptides, made the kinase insensitive increase in the membrane dwell time of Dy647-PI3Kβ to synergistic membrane recruitment mediated by bioRxiv preprint doi: https://doi.org/10.1101/2023.05.01.538969; this version posted May 1, 2023. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. A pY GβGγ pY, GβGγ pY(soln), GβGγ B 14000 + pY, GβGγ 12000 10000 8000 + pY 6000 4000 2000 + pY (soln), GβGγ 0 0 200 400 600 800 5 µm Time (seconds) C D E 4 1 clash PIK3R1(p85α) + pY + pY, GβGγ nSH2 + pY, GβGγ 3 0.1 2 iSH2 + pY 0.01 1 0.001 0 GβGγ PIK3CB (p110β) 0 4 8 12 16 0.0 0.3 0.6 0.9 1.2 1.5 Dwell Time (sec) step size (µm) F nSH2* G GβGγ* pY (RK->AA) 8000 8000 7000 7000 6000 6000 5000 5000 4000 4000 3000 3000 2000 + pY 2000 + pY 1000 + pY, GβGγ 1000 + pY, GβGγ 0 0 0 200 400 600 800 0 200 400 600 800 Time (seconds) Time (seconds) Figure 3 Mechanism controlling synergistic Dy647-PI3Kβ membrane binding by pY and GβGγ (A) Representative TIRF-M images showing the equilibrium membrane localization of 5 pM and 10 nM Dy647-PI3Kβ on membranes containing either pY, GβGγ, pY/GβGγ, or pY(solution)/GβGγ. The inset image (+GβGγ and +pY/GβGγ) shows low frequency single molecule binding events detected in the presence of 10 nM Dy647-PI3Kβ. Supported membranes were conjugated with 10 µM pY peptide (final surface density of ~15,000 pY/µm2) and equilibrated with 200 nM farnesyl-GβGγ before adding Dy647-PI3Kβ. pY(solution) = 10 µM. (B) Bulk membrane recruitment dynamics of 10 nM Dy647-PI3Kβ measured in the presence of either pY alone, pY/GβGγ, or pY(solution)/GβGγ. pY(solution) = 10 µM. (C) Single molecule dwell time distributions measured in the presence of 5 pM Dy647-PI3Kβ on supported membranes containing pY alone (t1=0.55±0.11s, t2=1.44±0.56s, α=0.54, N=4698 particles, n=5 technical replicates) or pY/GβGγ (t1=0.61±0.13s, t2=3.09±0.27s, α=0.58, N=3421 particles, n=4 technical replicates). (D) Step size distributions showing single molecule displacements measured in the presence of either pY alone (D1=0.34±0.04 µm2/sec, D2=1.02±0.07 µm2/sec, α=0.45) or pY/ GβGγ (D1=0.23±0.03 µm2/sec, D2=0.88±0.08 µm2/sec, α=0.6); n=3-4 technical replicates from > 3000 tracked particles with 10,000- 30,000 total displacements measured. (E) Combined model of the putative nSH2 and GβGγ binding sites on p110β. The p110β-GβGγ binding site is based on an Alphafold multimer model supported by previous HDX-MS and mutagenesis experiments. The orientation of the nSH2 is based on previous X-ray crystallographic data on PI3Kα (p110α-p85α, niSH2, PDB:3HHM) aligned to the structure of PI3Kβ (p110β-p85α, icSH2, PDB:2Y3A). (F) Bulk membrane recruitment dynamics of 10 nM Dy647-PI3Kβ, WT and nSH2(R358A), measured on membranes containing either pY or pY/GβGγ. (F) Bulk membrane recruitment dynamics of 10 nM Dy647-PI3Kβ, WT and GβGγ binding mutant, measured on membranes containing either pY or pY/GβGγ. (A-G) Membrane composition: 96% DOPC, 2% PI(4,5)P2, 2% MCC- PE. Dy647-PI3Kβ membrane intensity log10(1-CDF) Dy647-PI3Kβ 10 nM 5 pM Probability Density Dy647-PI3Kβ membrane intensity Dy647-PIK3CB membrane intensity bioRxiv preprint doi: https://doi.org/10.1101/2023.05.01.538969; this version posted May 1, 2023. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. A B C pY, Rac1(GDP) pY, Rac1(GTP) Cy3 AF488 Cy3-Rac1 AF488-pY Rac1 GTPase pY GDP GDP DOPC MCC PIP2 Glass 5 µm 5 µm D E F 8000 1 5+pY,Rac1(GDP) +pY,Rac1(GTP) +pY,Rac1(GTP) +pY,Rac1(GTP) 4 6000 +pY,Rac1(GDP) 0.1 3 4000 +pY,Rac1(GDP) 2 0.01 2000 1 0 0.001 0 0 100 200 300 400 0 5 10 15 20 25 0.0 0.2 0.4 0.6 0.8 1.0 Time (seconds) Dwell Time (sec) step size (µm) Figure 4 Membrane anchored pY peptides synergistically enhance Dy647-PI3Kβ membrane binding in the presence of Rac1(GTP) (A) Cartoon schematic showing membrane conjugation of Cy3-Rac1 and AF488-pY on membranes containing unlabeled Rac1 and pY. (B) Representative TIRF-M images showing localization of Cy3-Rac1 (1:10,000 dilution) and AF488-pY (1:30,000 dilution) after membrane conjugation in the presence of 30 µM Rac1 and 10 µM pY. Membrane surface density equals ~4,000 Rac1/µm2 and ~5,000 pY/µm2. (C) Representative TIRF-M images showing the equilibrium membrane localization of 5 pM and 10 nM Dy647-PI3Kβ measured in the presence of membranes containing either pY/Rac1(GDP) or pY/Rac1(GTP). (D) Bulk membrane recruitment dynamics of 10 nM Dy647-PI3Kβ measured in the presence of pY/Rac1(GDP) or pY/Rac1(GTP). (E) Single molecule dwell time distributions measured in the presence of 5 pM Dy647-PI3Kβ on supported membranes containing pY/Rac1(GDP) or pY/Rac1(GTP). (F) Step size distributions showing single molecule displacements from > 500 Dy647-PI3Kβ particles (>10,000 steps) in the presence of either pY/Rac1(GDP) or pY/Rac1(GTP). Membrane composition: 96% DOPC, 2% PI(4,5)P2, 2% MCC-PE. pY and GβGγ (Figure 3F). Similarly, the membrane Alexa488-pY into our Rac1-pY membrane coupling association dynamics of Dy647-PI3Kβ nSH2(R358A), reaction we were able to visualize single membrane phenocopied a PI3Kβ (K532D, K533D) mutant that anchored proteins and calculate the membrane lacks the ability to engage GβGγ (Figure 3G). surface density of ~4,000 Rac1/µm2 and ~5,000 pY/ µm2 (Figure 4A-4B). Bulk localization to membranes Rac1(GTP) and pY synergistically enhance PI3Kβ containing either pY-Rac1(GDP) or pY-Rac1(GTP), membrane localization revealed that active Rac1 could enhance Dy647- PI3Kβ is the only class IA PI3K that has been shown PI3Kβ localization by 1.4-fold (Figure 4C-4D). to interact with Rho-family GTPases, Rac1 and Cdc42 Similarly, single molecule analysis revealed a 1.5-fold (Fritsch et al. 2013b). Our membrane localization increase in the mean dwell time of Dy647-PI3Kβ in the studies indicate however that Dy647-PI3Kβ does not presence of pY-Rac1(GTP) (Figure 4E). The average strongly localize to membranes containing Rac1(GTP) displacement of Dy647-PI3Kβ per frame (i.e. 52 ms) alone (Figure 1C-1D and Figure 1 – figure also decreased by 28% in the presence of pY and supplement 2B). To determine whether membrane Rac1(GTP) (Figure 4F), consistent with the formation anchored pY peptides can facilitate interactions with of a membrane bound PI3Kβ-pY-Rac1(GTP) complex. Rac1(GTP), we visualized the localization of Dy647- PI3Kβ on membranes containing pY-Rac1(GDP) or Rac1(GTP) and GβGγ stimulate PI3Kβ activity pY-Rac1(GTP). Our experiments were designed to beyond enhancing membrane localization have the same pY surface density across conditions. Previous in vitro measurements of PI3Kβ activity By incorporating a small fraction of Cy3-Rac1 and have shown that solution pY stimulates lipid kinase Dy647-PIK3CB membrane intensity log10(1-CDF) Probability Density Dy647-PI3Kβ 10 nM 5 pM bioRxiv preprint doi: https://doi.org/10.1101/2023.05.01.538969; this version posted May 1, 2023. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. activity (Zhang et al. 2011b; Hashem A. Dbouk et lipid kinase activity is synergistically modulated by al. 2012). Similar mechanisms of activation have either GβGγ or Rho-family GTPases, in the presence been reported for other class IA kinases, including of membrane tethered pY peptides, we used TIRF-M PI3Kα and PI3Kδ (Buckles et al. 2017; Burke et al. to simultaneously visualize Dy647-PI3Kβ membrane 2011; Dornan et al. 2017). Functioning in concert binding and monitor the production of PI(3,4,5)P3 with pY peptides, GβGγ (Hashem A. Dbouk et al. lipids. To measure the kinetics of PI(3,4,5)P3 formation, 2012) or Rho-family GTPase (Fritsch et al. 2013b) we purified and fluorescently labeled the pleckstrin synergistically enhance PI3Kβ activity by a mechanism homology and Tec homology (PH-TH) domain derived that remains unclear. Similarly, RTK derived peptides from Bruton’s tyrosine kinase (Btk). We used a form of and H-Ras(GTP) have been shown to synergistically Btk containing a mutation that disrupts the peripheral activate PI3Kα (Buckles et al. 2017; Siempelkamp PI(3,4,5)P3 lipid binding domain (Wang et al. 2015). et al. 2017; Yang et al. 2012). In the case of PI3Kβ, This Btk mutant was previously shown to associate previous experiments have not determined whether with a single PI(3,4,5)P3 head group and exhibits synergistic activation by multiple signaling inputs rapid membrane equilibration kinetics in vitro (Chung results from an increase in membrane affinity (KD) or et al. 2019). Consistent with previous observations, direct modulation of lipid kinase activity (kcat) through Btk fused to SNAP-AF488 displayed high specificity an allosteric mechanism. To determine how PI3Kβ and rapid membrane equilibration kinetics on SLBs A C Btk-SNAP-AF488 INPUT = pY + Rac1(GDP) D [ [ [ [ 30000 30000 PI(4,5)P2 PI(3,4,5)P3 1 membrane equilibation (no ATP) 2 +ATP +pY, Rac1(GDP)25000 25000 pY SLOW KINETICS 20000 20000 Rac1 PI3Kβ 15000 15000 GDP PIP PIP2 3 ATP 10000 10000 5 µm B 5000 5000 0 0 8000 -500 0 500 1000 1500 INPUT = pY + Rac1(GTP) 30000 30000 6000 +pY, Rac1(GTP) 1 membrane equilibation (no ATP) 2 +ATP 25000 25000 4000 20000 20000 pY 2000 Rac1 FAST KINETICS 15000 15000PI3K ATP GTP GTP PIP PIP3 10000 100002 0 0 20 40 60 80 100 5000 5000 Time (seconds) 0 0 -500 0 500 1000 1500 Time (seconds) E F + pY + pY, GβGγKINETICS? G 30000 + pY +20pM PI3Kβ 25000 PIP PIP + pY, GβGγ2 3 20000 pY Gβ Gγ 15000 +10pM PI3KβPI3K 10000 ATP 5000 +20pM PI3Kβ 0 5 µm -500 0 500 1000 1500 Time (seconds) Figure 5 GβGγ and Rac1(GTP) stimulate PI3Kβ activity beyond enhancing localization on pY membranes (A) Representative TIRF-M images showing localization of 20nM Btk-SNAP-AF488 on SLBs containing either 2% PI(4,5)P2 or 2% PI(3,4,5)P3, plus 98% DOPC. (B) Bulk membrane recruitment kinetics of 50nM Btk-SNAP-AF488 on a SLB containing 98% DOPC, 2% PI(3,4,5)P3. (C) Cartoon schematic illustrating method for measuring Dy647-PI3Kβ activity in the presence of either pY/Rac1(GDP) or pY/ Rac1(GTP). Phase 1 of the reconstitution involves membrane equilibration of Dy647-PI3Kβ in the absence of ATP. During phase 2, 1 mM ATP is added to stimulate lipid kinase activity of Dy647-PI3Kβ. (D) Dual color TIRF-M imaging showing 2 nM Dy647-PI3Kβ localization and catalysis measured in the presence of 20nM Btk-SNAP-AF488. Dashed line represents the addition of 1 mM ATP to the reaction chamber. (E) Cartoon schematic showing experimental design for measuring synergistic binding and activation of Dy647-PI3Kβ in the presence of pY and GβGγ. (F) Representative single molecule TIRF-M images showing the localization of 20 pM Dy647-PI3Kβ in (G). (G) Kinetics of PI(3,4,5)P3 production monitored in the presence of 20nM Btk-SNAP-AF488 and 20 pM Dy647-PI3Kβ. Membrane contained either pY or pY/GβGγ. (D,F,G) Membrane composition: 96% DOPC, 2% PI(4,5)P2, 2% MCC-PE. Btk1-SNAP-Ax488 membrane intensity Dy647-PIK3CB PI(3,4,5)P3 lipids/μm2 PI(3,4,5)P3 lipids/μm2 PI(3,4,5)P3 lipids/μm2 Dy647-PI3Kβ Dy647-PI3Kβ membrane intensity membrane intensity bioRxiv preprint doi: https://doi.org/10.1101/2023.05.01.538969; this version posted May 1, 2023. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. containing PI(3,4,5)P3 (Figure 5A-5B). Compared to in Dy647-PI3Kβ membrane localization comparing the PI(3,4,5)P lipid sensor derived from the Cytohesin/ pY-Rac1(GDP) and pY-Rac1(GTP) membranes, we 3 Grp1 PH domain (He et al. 2008; J. D. Knight et al. calculated a 4.3-fold increase in PI3Kβ activity that 2010), the Btk mutant sensor had a faster association was Rac1(GTP) dependent. rate constant (k ) and a more transient dwell time We next examined how pY and GβGγ synergistically ON (1/kOFF) making it ideal for kinetic analysis of PI3Kβ activate PI3Kβ using the two-phase kinase assay lipid kinase activity (Figure 5 – figure supplement 1). described above (Figure 5E). In our pilot experiments, we immediately observed more robust PI3Kβ Using Btk-SNAP-AF488, we measured the production activation in the presence of pY-GβGγ, compared of PI(3,4,5)P3 lipids on SLBs by quantifying the time to pY-Rac1(GTP). To accurately measure the rapid dependent recruitment in the presence of PI3Kβ. The kinetics of PI(3,4,5)P3 generation on SLBs we had to change in Btk-SNAP-AF488 membrane fluorescence use a 100-fold lower concentration of Dy647-PI3Kβ. could be converted to the absolute number of PI(3,4,5) Under these conditions, single membrane bound P lipids produced per µm2 to determine the catalytic Dy647-PI3Kβ molecules could be spatially resolved, 3 efficiency per membrane bound Dy647-PI3Kβ. which allowed us to measure the catalytic efficiency While our findings provide a mechanism for enhanced per PI3Kβ (Figure 5F). Comparing the activity of PI3Kβ membrane localization in the presence of either Dy647-PI3Kβ on membranes with either pY or pY- pY-Rac1(GTP) or pY-GβGγ, these results did not GβGγ, we observed a 22-fold increase in catalytic reveal the mechanism controlling synergistic activation efficiency comparing the PI3Kβ-pY and PI3Kβ-pY- of lipid kinase activity. To probe if synergistic activation GβGγ complexes (Figure 5G). Based on membrane results from enhanced membrane localization or bound density of ~0.2 Dy647-PI3Kβ per µm2, we allosteric modulation of PI3Kβ, we first examined calculate a kcat of 57 PI(3,4,5)P3 lipids/sec•PI3Kβ on how well the pY peptide stimulates PI3Kβ lipid kinase pY-GβGγ containing membranes. By contrast, the activity on SLBs. In the absence of pY peptides, PI3Kβ Dy647-PI3Kβ-pY complex had a kcat of ~3 PI(3,4,5)P3 did not catalyze the production of PI(3,4,5)P3 lipids, lipids/sec•PI3Kβ. while the addition of 10 µM pY in solution resulted in a subtle but detectable increase in PI3Kβ lipid kinase DISCUSSION activity (Figure 5 – figure supplement 2). By contrast, covalent conjugation of pY peptides to supported lipid Prioritization of signaling inputs bilayers increased the rate of PI(3,4,5)P production The exact mechanisms that regulate how PI3Kβ 3 by 207-fold (Figure 5 – figure supplement 2). The prioritizes interactions with signaling input, such as pY, observed difference in kinetics were consistent Rac1(GTP), and GβGγ remains unclear. To fill this gap with robust membrane recruitment of Dy647-PI3Kβ in knowledge, we directly visualized the membrane requiring membrane tethered pY peptides. association and dissociation dynamics of fluorescently Next, we sought to assess if the combination of labeled PI3Kβ on supported lipid bilayers using single pY and Rac1(GTP) could synergistically stimulate molecule TIRF microscopy. This is the first study to PI3Kβ activity beyond the expected increase due to reconstitute membrane localization and activation the enhanced membrane localization of the PI3Kβ- of a class 1A PI3K using multiple signaling inputs pY-Rac1(GTP) complex. To decipher the mechanism that are all membrane tethered in a physiologically of synergistic activation, we performed two-phase experiments that accounted for both the total amount relevant configuration. Previous experiments have of membrane localized Dy647-PI3Kβ and the relied exclusively on phosphotyrosine peptides (pY) corresponding kinetics of PI(3,4,5)P3 generation. In in solution to activate PI3Kα, PI3Kβ, or PI3Kδ (Zhang phase 1 of our experiments, Dy647-PI3Kβ was flowed et al. 2011a; Dornan et al. 2017; Hashem A. Dbouk over SLBs and allowed to equilibrate with either pY- et al. 2012). However, pY peptides are derived from Rac1(GDP) or pY-Rac1(GTP) in the absence of ATP the cytoplasmic domain of transmembrane receptors, (Figure 5C-5D). This resulted in a 1.8-fold increase such as receptor tyrosine kinases (RTKs), which reside in Dy647-PI3Kβ localization mediated by Rac1(GTP) in the plasma membrane (Lemmon and Schlessinger on pY containing membranes. Following membrane 2010). Although pY peptides in solution can disrupt equilibration of Dy647-PI3Kβ, phase 2 was initiated by the inhibitory contacts between the regulatory and adding 1 mM ATP to the reaction chamber to stimulate catalytic subunits of class 1A PI3Ks (Zhang et al. lipid kinase activity. We found that the addition of ATP did not alter the bulk localization of Dy647-PI3Kβ, 2011a; Yu et al. 1998), they do not robustly localize though the kinase was in dynamic equilibrium between PI3Ks to membranes. When conjugated to a SLB we the solution and membrane. Conducting experiments find that pY peptides strongly localize auto-inhibited in this manner allowed us to measure activation by PI3Kβ, while membranes containing only Rac1(GTP) inputs while removing uncertainty from differential or GβGγ are unable to localize PI3Kβ. We observed Dy647-PI3Kβ association with various signaling this prioritization of signaling input interactions over inputs. After accounting for the 1.8-fold difference a range of membrane compositions that contained bioRxiv preprint doi: https://doi.org/10.1101/2023.05.01.538969; this version posted May 1, 2023. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. physiologically relevant densities of anionic lipids, et al. 2012). Using AlphaFold Multimer (Evans et al. such as phosphatidylserine and PI(4,5)P2. Although 2022; Jumper et al. 2021), we created a model that a small fraction of PI3Kβ may transiently adopt a illustrates how the p85α(nSH2) domain is predicted conformation that is compatible with direct Rac1(GTP) to sterically block GβGγ binding to p110β. This model or GβGγ association in the absence of pY, these was validated by comparing the AlphaFold Multimer events are rare and do not represent a probable path model to previous reported HDX-MS (H. A. Dbouk et for initial membrane docking of PI3Kβ. al. 2010) and X-ray crystallography data (Zhang et Based on our single molecule dwell time and al. 2011a). Further supporting this model, we found diffusion analysis, Dy647-PI3Kβ can cooperatively that the Dy647-PI3Kβ nSH2(R358A) mutant tethered bind to one doubly phosphorylated peptide derived to membrane conjugated pY peptide was unable from the PDGF receptor. Supporting this model, to engage membrane anchored GβGγ. Membrane Dy647-PI3Kβ with a mutated nSH2 or cSH2 domain targeting of PI3Kβ by pY was required to relieve that eliminates pY binding, still displayed membrane nSH2 mediated autoinhibition and expose the GβGγ diffusivity indistinguishable from wild-type PI3Kβ. The binding site. Recruitment by membrane tethered pY diffusion coefficient of membrane bound pY-PI3Kβ also reduces the translational and rotational entropy complexes also did not significantly change over a of PI3Kβ, which facilitates PI3Kβ-pY-GβGγ complex broad range of pY membrane surface densities that formation. We observed a similar mechanism of span 3 orders of magnitude. Given that diffusivity synergistic PI3Kβ localization on SLBs containing pY of peripheral membrane binding proteins is strongly and Rac1(GTP). However, we did not determine the correlated with the valency of membrane interactions role p85α inhibition serves in regulating the association (Ziemba and Falke 2013; Hansen et al. 2022), we between PI3Kβ and Rac1(GTP). In the case of PI3Kα, expected to observe a decrease in Dy647-PI3Kβ interactions with Ras(GTP) on vesicles or in solution diffusion with increasing membrane surface densities have previously been shown to require pY peptide to of pY. Instead, our data suggests that the vast majority relieve autoinhibition. of PI3Kβ molecules engage a single pY peptide, rather than binding one tyrosine phosphorylated residue on Mechanism of synergistic activation two separate pY peptides. While no structural studies Previous characterization of PI3Kβ lipid kinase activity have shown how exactly the tandem SH2 domains has utilized solution-based assays to measure P(3,4,5) of PI3K (p85α) simultaneously bind to a doubly P3 production. These solution-based measurements phosphorylated pY peptide, the interactions likely lack spatial information concerning the mechanism resemble the mechanism reported for ZAP-70 (z-chain of PI3Kβ membrane recruitment and activation. Our of T-cell Receptor Associated Protein Kinase 70). The ability to simultaneously visualize PI3Kβ membrane tandem SH2 domains of ZAP-70 can bind to a doubly localization and P(3,4,5)P3 production is critical for phosphorylated z-chain derived from the TCR with determining which regulatory factors directly modulate only 11 amino acids spacing between the two tyrosine the catalytic efficiency of PI3Kβ. In the case of PI3Kα, phosphorylation sites (Hatada et al. 1995). In the case the enhanced membrane recruitment model has been of our PDGFR derived pY peptide that binds p85α, 10 used to explain the synergistic activation mediated by amino acids separate the two tyrosine phosphorylated pY and Ras(GTP) (Buckles et al. 2017). In other words, residues. the PI3Kα-pY-Ras complex is more robustly localized Following the engagement of a pY peptide, we to membranes compared to the PI3Kα-pY and PI3Kα- find that PI3Kβ can then associate with membrane Ras complexes, which results in a larger total catalytic anchored Rac1(GTP) or GβGγ. We detected the output for the system. Although the Ras binding formation of PI3Kβ-pY-Rac1(GTP) and PI3Kβ-pY- domain (RBD) of PI3Kα and PI3Kβ are conserved, GβGγ complexes based on the following criteria: (1) these kinases interact with distinct Ras superfamily increase in Dy647-PI3Kβ bulk membrane recruitment, GTPases (Fritsch et al. 2013b). Therefore, it’s possible (2) increase in single molecule dwell time, and (3) a that PI3Kα and PI3Kβ display different mechanisms of decrease in membrane diffusivity. Consistent with synergistic activation, which could explain their non- Dy647-PI3Kβ having a weak affinity for GβGγ, pY overlapping roles in cell signaling. peptides in solution were unable to strongly localize Studies of PI3Kβ mouse knock-in mutations in primary Dy647-PI3Kβ to SLBs containing membrane anchored macrophages and neutrophils have shown that robust GβGγ. This is in agreement with HDX-MS data showing PI3Kβ activation requires coincident activation through that the p110β-GβGγ interaction can only be detected the RTK and GPCR signaling pathways (Houslay using a GβGγ-p85α(icSH2) chimeric fusion or pre- et al. 2016). This response most strongly depends activating PI3Kβ with solution pY (Hashem A. Dbouk on the ability of PI3Kβ to bind GβGγ and to a lesser bioRxiv preprint doi: https://doi.org/10.1101/2023.05.01.538969; this version posted May 1, 2023. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. extent Rac1/Cdc42 (Houslay et al. 2016). Although receptors (Backer et al. 1992; Fantl, Martin, and these mutational studies have nicely demonstrated Turck 1992). As a result, we still have not defined the the modes of synergistic PI3Kβ activation in cells, broad specificity p85α has for tyrosine phosphorylated signaling network crosstalk and redundancy limits our peptides. Biochemistry studies indicate that the nSH2 mechanistic understanding of how PI3Kβ prioritizes and cSH2 domains of p85α robustly bind pY residues signaling inputs and the exact mechanism for driving with a methionine in the +3 position (pYXXM) (Breeze PI(3,4,5)P3 production. Based on our single molecule et al. 1996; Nolte et al. 1996; Backer et al. 1992; membrane binding experiments, auto-inhibited PI3Kβ Fantl, Martin, and Turck 1992). The p85α subunit is is unable to bind directly to either Rac1(GTP) or also predicted to interact with the broad repertoire of GβGγ in the absence of pY peptides. We found that receptors that contain immunoreceptor tyrosine-based PI3Kβ kinase activity is also relatively insensitive to activation motifs (ITAMs) baring the pYXX(L/I) motif either Rac1(GTP) or GβGγ alone. This is in contrast to (Reth 1989; Osman et al. 1996; Zenner et al. 1996; previous reports that showed Rho-GTPases (Fritsch Love and Hayes 2010). Based on RNA seq data, human et al. 2013a) and GβGγ (Katada et al. 1999; Hashem neutrophils express at least six different Fc receptors A. Dbouk et al. 2012; Maier, Babich, and Nürnberg (FcRs) that all contain phosphorylated ITAMs that can 1999) can activate PI3Kβ, albeit modest, compared to potentially facilitate membrane localization of class 1A synergistic activation with pY peptides plus Rac1(GTP) PI3Ks (Rincón, Rocha-Gregg, and Collins 2018). or GβGγ. A variety of human diseases result from the In our single molecule TIRF experiments, we find overexpression of RTKs, especially the epithelial that the pY peptide is the only factor that robustly growth factor receptor (EGFR) (Sauter et al. 1996). localizes PI3Kβ to supported membranes in an When the cellular plasma membrane contains autonomous manner. However, the pY-PI3Kβ complex densities of EGFR greater than 2000 receptors/µm2, displays weak lipid kinase activity (kcat = ~3 PI(3,4,5) trans-autophosphorylation and activation can occur P3 lipids/sec•PI3Kβ). This is consistent with cellular in a EGF-independent manner (Endres et al. 2013). measurements showing that RTK activation by Receptor membrane surface densities above the insulin (Z. A. Knight et al. 2006), PDGF (Guillermet- threshold needed for spontaneous receptor trans- Guibert et al. 2008), or EGF (Ciraolo et al. 2008) show autophosphorylation have been observed in many little PI3Kβ dependence for PI(3,4,5)P3 production. cancer cells (Haigler et al. 1978). In these disease Although the dominant role of PI3Kα in controlling states, PI3K is expected to localize to the plasma PI(3,4,5)P3 production downstream of RTKs can mask membrane in the absence of ligand induced RTK the contribution from PI3Kβ in some cell types, these or GPCR signaling. The slow rate of PI(3,4,5)P3 results highlight the need for PI3Kβ to be synergistically production we measured for the membrane tethered activated. When we measured the kinetics of lipid pY-PI3Kβ complex suggests that PI(3,4,5)P3 levels are phosphorylation for PI3Kβ-pY-Rac1(GTP) and not likely to rise above the global inhibition imposed by PI3Kβ-pY-GβGγ complexes we observed synergistic lipid phosphatases until synergistic activation of PI3Kβ activation beyond simply enhancing PI3Kβ membrane by RTKs and GPCRs. However, loss of PTEN in some localization. After accounting for the ~1.8-fold cancers (Jia et al. 2008) could produce an elevated increase in membrane localization between PI3Kβ-pY- level of PI(3,4,5)P3 due to PI3Kβ being constitutively Rac1(GTP) and PI3Kβ-pY-Rac1(GDP), we calculated membrane localized via ligand independent trans- a 4.3-fold increase in kcat that was dependent on autophosphorylation of RTKs. engaging Rac1(GTP). Comparing the kinase activity of PI3Kβ-pY and PI3Kβ-pY-GβGγ complexes that are present at the same membrane surface density (~0.2 PI3Kβ/µm2) revealed a 22-fold increase in kcat mediated by the GβGγ interaction. Together, these results indicate that PI3Kβ-pY complex association with either Rac1(GTP) or GβGγ allosterically modulates PI3Kβ, making it more catalytically efficient. Mechanisms controlling cellular activation of PI3Kβ Studies of PI3K activation by pY peptides have mostly been performed using peptides derived from the IRS- 1 (Insulin Receptor Substrate 1) and the EGFR/PDGF bioRxiv preprint doi: https://doi.org/10.1101/2023.05.01.538969; this version posted May 1, 2023. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. ACKNOWLEDGMENTS We thank John Burke (University of Victoria) for assistance generating the AlphaFold2 Multimer model of PI3Kβ bound to GβGγ. We thank Grace Waddell for preliminary characterization of PI3Kβ. We thank Colin Hawkinson for assistance with protein purification. We thank Jean Chung (Colorado State, Fort Collins) and Orion Weiner (University of California at San Francisco) for plasmids encoding Btk and P-Rex1 plasmids, respectively. AUTHOR CONTRIBUTIONS Resources: B.R.D, G.M.B, S.E.P, S.D.H. Experiments and investigation: B.R.D, N.E.W., G.M.B, S.E.P, S.D.H. Data Analysis: B.R.D, N.E.W., S.E.P., S.D.H. Conceptualization: B.R.D, N.E.W., S.D.H. Interpretation: B.R.D, N.E.W., S.D.H. Data curation: B.R.D, N.E.W., S.D.H. Writing – Review and editing: B.R.D, N.E.W., G.M.B, S.E.P, S.D.H. Writing – Original draft: S.D.H. Supervision: S.D.H. Project administration: S.D.H. Funding acquisition: S.D.H. FUNDING Research was supported by the University of Oregon Start-up funds (S.D.H.), National Science Foundation CAREER Award (S.D.H., MCB-2048060), Molecular Biology and Biophysics Training Program (B.R.D, N.E.W., NIH T32 GM007759), and the Summer Program for Undergraduate Research (SPUR) at the University of Oregon (G.M.B.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Science Foundation. DATA AVAILABILITY All the information needed for interpretation of the data is presented in the manuscript or the supplemental material. Plasmids related to this work are available upon request. CONFLICT OF INTEREST The authors declare that they have no conflicts of interest with the contents of this article. bioRxiv preprint doi: https://doi.org/10.1101/2023.05.01.538969; this version posted May 1, 2023. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. MATERIALS & METHODS Molecular Biology The following genes were used as templates for PCR to clone plasmids used for recombinant protein expression: PIK3CB (human 1-1070aa; Uniprot Accession #P42338), PIK3R1 (human 1-724aa; Uniprot Accession #P27986), PIK3CG (mouse 1-1102aa; Uniprot Accession #Q9JHG7), PIK3R5 (mouse 1-871aa; Uniprot Accession #Q5SW28), RAC1 (human 1-192aa; Uniprot Accession #P63000), CYTH3/Grp1 (human 1-400aa; Uniprot Accession #O43739), BTK (bovine 1-659aa; Uniprot Accession #Q3ZC95), neutrophil cytosol factor 2 (NCF2, human 1-526aa; Uniprot Accession #P19878, referred to as p67/phox), PREX1 (human 1-1659aa; Uniprot Accession #Q8TCU6), GNB1/GBB1 (Gβ1, bovine 1-340aa; Uniprot Accession #P62871), GNG2/GBG2 (Gγ2, bovine 1-71aa; Uniprot Accession #P63212). The following plasmids were purchased as cDNA clones from Horizon (PerkinElmer), formerly known as Open Biosystems and Dharmacon: mouse PIK3CG (clone #BC051246, cat #MMM1013-202770664) and mouse PIK3R5 (clone #BC128076, cat #MMM1013-211693360), human PIK3R1 (clone #30528412, cat #MHS6278-202806334), human CYTH3/Grp1 (clone #4811560, cat #MHS6278-202806616). Genes encoding bovine Gβ1 and Gγ2 were derived from the following plasmids: YFP-Gβ1 (Addgene plasmid # 36397) and YFP-Gγ2 (Addgene plasmid # 36102). These Gβ1 and Gγ2 containing plasmids were kindly provided to Addgene by Narasimhan Gautam (Saini et al. 2007). In this study, we used a previously described mutant form of Btk with mutations in the peripheral PI(3,4,5)P3 binding site (R49S/K52S) (Chung et al. 2019; Wang et al. 2015). The Btk peripheral site mutant was PCR amplified using a plasmid provided by Jean Chung (Colorado State, Fort Collins) that contained the following coding sequence: his6-SUMO-Btk(PH- TH, R49S/K52S)-EGFP. The nSH2 biosensor was derived from human PIK3R1. The gene encoding human PREX1 was provided by Orion Weiner (University of California San Francisco). Refer to supplemental text to see exact peptide sequence of every protein purified in this study. The following mutations were introduced into either the PIK3CB (p110β) or PIK3R1 (p85α) genes using site-directed mutagenesis: p85α nSH2 (R358A, FLVR- >FLVA), p85α cSH2 (R649A, FLVR->FLVA), p110β GβG𝛾 mutant (K532D/K533D). For cloning, genes were PCR amplified using AccuPrime Pfx master mix (ThermoFisher, Cat#12344040) and combined with a restriction digested plasmids using Gibson assembly (Gibson et al. 2009). Refer to supplemental text for a complete list of plasmids used in this study. Information about the specific peptide sequences for recombinantly expressed and purified proteins is organized in the supplemental information document. The complete open reading frame of all vectors used in this study were sequenced to ensure the plasmids lacked deleterious mutations. BACMID and baculovirus production We generated BACMID DNA as previously described (Hansen et al. 2019). FASTBac1 plasmids containing our gene of interested were transformed into DH10 Bac cells and plated on LB agar media containing 50 µg/ mL kanamycin, 10 µg/mL tetracycline, 7 µg/mL gentamycin, 40 µg/mL X-GAL, and 40 µg/mL IPTG. Plated cells were incubated for 2-3 days at 37ºC before positive clones were isolated based on blue-white colony selection. White colonies were inoculated into 5mL of TPM containing 50 µg/mL kanamycin, 10 µg/mL tetracycline, 7 µg/ mL gentamycin and grown overnight at 37ºC. To purify the BACMID DNA, we first pelleted the cultures via centrifugation, then re-suspended the pellet in 300 µL of buffer containing 50 mM Tris [pH 8.0], 10 mM EDTA, 100 µg/mL RNase A. We lysed bacteria via addition of 300 µL of buffer containing 200 mM NaOH, 1% SDS before neutralization with 300 µL of 4.2 M Guanidine HCl, 0.9 M KOAc [pH 4.8]. We then centrifuged the sample at 23ºC for 10 minutes at 14,000 x g. Supernatant containing the BACMID DNA was combined with 700 µL 100% isopropanol and spun for 10-minute at 14,000 x g. The DNA pellets were washed twice with 70% ethanol (200 µL and 50 µL) and centrifuged. The ethanol was removed by vacuum aspiration and the final DNA pellet was dried in a biosafety hood. Finally, we solubilized the BACMID DNA in 50-100 µL of sterile filtered MilliQ water. A Nanodrop was used to quantify the total DNA concentration. BACMID DNA was be stored at -20ºC or used immediately for higher transfection efficiency. Baculovirus was generated as previously described. In brief, we incubated 5-7 µg of BACMID DNA with 4 µL Fugene (Thermo Fisher, Cat# 10362100) in 250 µL of Opti-MEM serum free media for 30 minutes at 23ºC. The DNA-Fugene mixture was then added to a Corning 6-well plastic dish (Cat# 07-200-80) containing 1 x 106 Spodoptera frugiperda (Sf9) insect cells in 2 mL of ESF 921 Serum- Free Insect Cell Culture media (Expression Systems, Cat# 96-001, Davis, CA.). 4-5 days following the initial transfection, we harvested and centrifuged the viral supernatant (called “P0”). P0 was used to generate a P1 titer by infecting 7 x 106 Sf9 cells plated in a 10 cm tissue culture grade petri dish containing 10 mL of ESF 921 media and 2% Fetal Bovine serum (Seradigm, Cat# 1500-500, Lot# 176B14). We harvested and centrifuged the P1 bioRxiv preprint doi: https://doi.org/10.1101/2023.05.01.538969; this version posted May 1, 2023. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. titer after 4 days of transfection. The P1 titer was expanded at a concentration of 1% vol/vol of P1 titer into a 100 mL Sf9 cell culture grown to a density of 1.25-1.5 x 106 cells/mL in a sterile 250 mL polycarbonate Erlenmeyer flask with vented cap (Corning, #431144). The P2 titer (viral supernatant) was harvested, centrifuged, and 0.22 µm filtered in 150 mL filter-top bottle (Corning, polyethersulfone (PES), Cat#431153). We used this P2 titer for protein expression in High 5 cells grown in ESF 921 Serum-Free Insect Cell Culture media (0% FBS) at a final baculovirus concentration of ~2% vol/vol. All our media contained 1x concentration of Antibiotic-Antimycotic (Gibco/Invitrogen, Cat#15240-062). Protein purification PI3Kβ and PI3Kγ. Genes encoding human his6-TEV-PIK3CB (1-1070aa) and ybbr-PIK3R1 (1-724aa) were cloned into a modified FastBac1 dual expression vector containing tandem polyhedrin (pH) promoters. Genes encoding mouse his6-TEV-PIK3CG (1-1102aa) and mouse his6-TEV-ybbr-PIK3R5 (1-871aa) were expressed from separate FastBac1 vectors under the polyhedrin (pH) promoters. For protein expression, high titer baculovirus was generated by transfecting 1 x 106 Spodoptera frugiperda (Sf9) with 0.75-1µg of BACMID DNA as previously described (Hansen et al. 2019). After two rounds of baculovirus amplification and protein test expression, 2 x 106 cells/mL High 5 cells were infected with 2% vol/vol PI3Kβ (PIK3CB/PIK3R1) or 2% vol/vol PI3Kγ (PIK3CG/PIK3R5) baculovirus and grown at 27ºC in ESF 921 Serum-Free Insect Cell Culture media (Expression Systems, Cat# 96-001) for 48 hours. High 5 cells were harvested by centrifugation and washed with 1x PBS [pH 7.2] and centrifuged again. Final cell pellets were resuspending in an equal volume of 1x PBS [pH 7.2] buffer containing 10% glycerol and 2x protease inhibitor cocktail (Sigma, Cat# P5726) before being stored in the -80ºC freezer. For protein purification, frozen cell pellets from 4 liters of cell culture were lysed by Dounce homogenization into buffer containing 50 mM Na2HPO4 [pH 8.0], 10 mM imidazole, 400 mM NaCl, 5% glycerol, 2 mM PMSF, 5 mM BME, 100 µg/mL DNase, 1x protease inhibitor cocktail (Sigma, Cat# P5726). Lysate was centrifuged at 35,000 rpm (140,000 x g) for 60 minutes under vacuum in a Beckman centrifuge using a Ti-45 rotor at 4ºC. Lysate was batch bound to 5 mL of Ni-NTA Agarose (Qiagen, Cat# 30230) resin for 90 minutes stirring in a beaker at 4ºC. Resin was washed with buffer containing 50 mM Na2HPO4 [pH 8.0], 30 mM imidazole, 400 mM NaCl, and 5 mM BME. Protein was eluted from NiNTA resin with wash buffer containing 500 mM imidazole. The his6-TEV-PIK3CB/ybbr-PIK3R1 complex was then desalted on a G25 Sephadex column in buffer containing 20 mM Tris [pH 8.0], 100 mM NaCl, 1 mM DTT. Peak fractions were pooled and loaded onto a Heparin anion exchange column equilibrated in 20 mM Tris [pH 8.0], 100 mM NaCl, 1 mM DTT buffer. Proteins were resolved over a 10-100% linear gradient (0.1-1 M NaCl) at 2 mL/min flow rate over 20 minutes. Peak fractions were pooled and supplemented with 10% glycerol, 0.05% CHAPS, and 200 µg/mL his6-TEV(S291V) protease. The his6-TEV- PIK3CB/ybbr-PIK3R1 complex was incubated overnight at 4ºC with TEV protease to remove the affinity tag. The TEV protease cleaved PIK3CB/ybbr-PIK3R1 complex was separated on a Superdex 200 size exclusion column (GE Healthcare, Cat# 17-5174-01) equilibrated with 20 mM Tris [pH 8.0], 150 mM NaCl, 10% glycerol, 1 mM TCEP, 0.05% CHAPS. Peak fractions were concentrated in a 50 kDa MWCO Amicon centrifuge tube and snap frozen at a final concentration of 10 µM using liquid nitrogen. This protein is referred to as PI3Kβ throughout the manuscript. The same protocol was followed to purify mouse PI3Kγ (PIK3CG/ybbr-PIK3R5) and the various PI3Kβ mutants reported in this study. Rac1. The gene encoding human Rac1 were expressed in BL21 (DE3) bacteria as his10-SUMO3-(Gly)5 fusion proteins. Bacteria were grown at 37°C in 4L of Terrific Broth for two hours or until OD600 = 0.8. Cultures were shifted to 18°C for 1 hour then induced with 0.1 mM IPTG. Expression was allowed to continue for 20 hours before harvesting. Cells were lysed into 50 mM Na2HPO4 [pH 8.0], 400 mM NaCl, 0.4 mM BME, 1 mM PMSF, 100 μg/mL DNase using a microfluidizer. Lysate was centrifuged at 16,000 rpm (35000 x g) for 60 minutes in a Beckman JA-20 rotor at 4ºC. Lysate was circulated over 5 mL HiTrap Chelating column (GE Healthcare, Cat# 17-0409-01) loaded with CoCl2. Bound protein was eluted at a flow rate of 4mL/min into 50 mM Na2HPO4 [pH 8.0], 400 mM NaCl, 500mM imidazole. Peak fractions were pooled and combined with SUMO protease (SenP2) at a final concentration of 50µg/mL and dialyzed against 4 liters of buffer containing 20 mM Tris [pH 8.0], 250 mM NaCl, 10% Glycerol, 1 mM MgCl2, 0.4mM BME. Dialysate containing SUMO cleaved protein was recirculated for 2 hours over a 5 mL HiTrap Chelating column. Flowthrough containing (Gly)5 -Rac1 was concentrated in a 5 MWCO Vivaspin 20 before being loaded on a 124 mL Superdex 75 column equilibrated in 20 mM Tris [pH 8.0], 150 mM NaCl, 10% Glycerol, 1 mM TCEP, 1 mM MgCl2. Peak fractions containing (Gly)5 -Rac1 were pooled and bioRxiv preprint doi: https://doi.org/10.1101/2023.05.01.538969; this version posted May 1, 2023. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. concentrated to a concentration of 400-500 µM (~10 mg/mL) and snap frozen with liquid nitrogen and stored at -80ºC. Grp1 and nSH2. The gene encoding the Grp1 PH domain derived from human CYTH3 was expressed in BL21 (DE3) bacteria as a his6-MBP-N10-TEV-GGGG-Grp1-Cys fusion protein. The gene encoding the N-terminal SH2 (nSH2, 322-440aa) domain derived from the PIK3R1 gene was cloned and expressed as a his6-GST-TEV- nSH2 fusion protein. A single cysteine was added to the C-terminus of the nSH2 domain to allow for chemical labeling with maleimide dyes. For both recombinant proteins, bacteria were grown at 37°C in 4 L of Terrific Broth for two hours or until OD600 = 0.8 and then shifted to 18°C for 1 hour. Cells were then induced to express either the Grp1 or nSH2 fusion by adding 0.1 mM IPTG. Cells were harvested 20 hours post-induction. We lysed bacteria into 50 mM Na2HPO4 [pH 8.0], 400 mM NaCl, 0.4 mM BME, 1 mM PMSF, 100 μg/mL DNase using a microfluidizer. Next, lysate was centrifuged at 16,000 rpm (35000 x g) for 60 minutes in a Beckman JA-20 rotor at 4ºC. Supernatant was circulated over 5 mL HiTrap chelating column (GE Healthcare, Cat# 17-0409-01) that was pre-incubated with 100 mM CoCl2 for 10 minutes, wash with MilliQ water, and equilibrated into lysis buffer lacking PMSF and DNase. Clarified cell lysate containing his6-MBP-N10-TEV-GGGG-Grp1-Cys was circulated over the HiTrap column and washed with 20 column volumes of 50 mM Na2HPO4 [pH 8.0], 300 mM NaCl, 0.4 mM BME containing buffer. Protein was eluted with buffer containing 50 mM Na2HPO4 [pH 8.0], 300 mM NaCl, and 500 mM imidazole at a flow rate of 4mL/min. Peak HiTrap elutant fractions were combined with 750 µL of 2 mg/mL TEV protease and dialyzed overnight against 4L of buffer containing 20 mM Tris [pH 8.0], 200 mM NaCl, and 0.4 mM BME. The next day, we recirculated cleaved proteins over two HiTrap (Co+2) columns (2 x 5 mL) that were equilibrated in 50 mM Na2HPO4 [pH 8.0], 300 mM NaCl, and 0.4 mM BME containing buffer for 1 hour. We concentrated the proteins via 10 kDa MWCO Vivaspin 20 to a volume of 5mL. The concentrated Grp1 protein was then loaded on a 124 mL Superdex 75 column equilibrated in in 20 mM Tris [pH 8], 200 mM NaCl, 10% glycerol, and 1 mM TCEP. Protein was eluted at a flow rate of 1mL/min. Peak fractions containing Grp1 were pooled and concentrated to 500-600 µM (~8mg/mL). Peak fractions containing nSH2 were pooled and concentrated to 200-250 µM (~3mg/mL). Proteins were frozen with liquid nitrogen and stored at -80ºC. P-Rex1 (DH-PH) domain. The DH-PH domain of human P-Rex1 was expressed as a fusion protein, his6-MBP- N10-TEV-PRex1(40-405aa), in BL21(DE3) Star bacteria. Bacteria were grown at 37°C in 2L of Terrific Broth for two hours or until OD600 = 0.8. Cultures were shifted to 18°C for 1 hour then induced with 0.1 mM IPTG. Expression was allowed to continue for 20 hours before harvesting. Cells were lysed into buffer containing 50 mM NaHPO4 [pH 8.0], 400 mM NaCl, 5% glycerol, 1 mM PMSF, 0.4 mM BME, 100 μg/mL DNase using microtip sonication. Cell lysate was clarified by centrifugation at 16,000 rpm (35000 x g) for 60 minutes in a Beckman JA- 20 rotor at 4ºC. To capture his6-tagged P-Rex1, cell lysate was circulated over a 5 mL HiTrap Chelating column (GE Healthcare, Cat# 17-0409-01) charged CoCl2. The column was washed with 100 mL of 50 mM NaHPO4 [pH 8.0], 400 mM NaCl, 5% glycerol, 0.4 mM BME buffer. Protein was eluted into 15 mL with buffer containing 50 mM NaHPO4 [pH 8.0], 400 mM NaCl, 500 mM imidazole, 5% glycerol, 0.4 mM BME. Peak fractions were pooled and combined with his6-TEV protease and dialyzed against 4 liters of buffer containing 50 mM NaHPO4 [pH 8.0], 400 mM NaCl, 5% glycerol, 0.4 mM BME. The next day, dialysate containing TEV protease cleaved protein was recirculated for 2 hours over a 5 mL HiTrap chelating column. Flowthrough containing P-Rex1 (40-405aa) was desalted into 20 mM Tris [pH 8.0], 50 mM NaCl, 1 mM DTT using a G25 Sephadex column. Note that some of the protein precipitated during the desalting step. Desalted protein was clarified using centrifugation followed by a 0.22µm syringe filter. P-Rex1(40-405aa, pI = 8.68) was further purified by cation exchange chromatography (i.e. MonoS) using a 20 mM Tris [pH 8.0], 0 – 1000 mM NaCl, 1 mM DTT. P-Rex1(40-405aa) bound eluted broadly in the presence of 100-260 mM NaCl. Pure fractions as determined by SDS-PAGE were pooled, concentration, and loaded onto a 120 mL Superdex 75 column equilibrated in 20 mM Tris [pH 8], 150 mM NaCl, 10% glycerol, 1 mM TCEP. Peak fractions containing P-Rex1(40-405aa) were pooled and concentrated to a concentration of 114 µM, aliquoted, frozen with liquid nitrogen, and stored at -80ºC. Btk. The mutant Btk PI(3,4,5)P3 fluorescent biosensor was recombinantly expressed in BL21 Star E. coli as a his6-SUMO-Btk(1-171aa PH-TH domain; R49S/K52S)-SNAP fusion. Bacteria were grown at 37°C in Terrific Broth to an OD600 of 0.8. These cultures were then shifted to 18°C for 1 hr, induced with 0.1 mM IPTG, and allowed to express protein for 20 hr at 18°C before being harvested. Cells were lysed into 50 mM NaPO4 (pH 8.0), 400 mM NaCl, 0.5 mM BME, 10 mM Imidazole, and 5% glycerol. Lysate was then centrifuged at 16,000 bioRxiv preprint doi: https://doi.org/10.1101/2023.05.01.538969; this version posted May 1, 2023. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. rpm (35,172 × g) for 60 min in a Beckman JA-20 rotor chilled to 4°C. Lysate was circulated over 5 mL HiTrap Chelating column (GE Healthcare, Cat# 17-0409-01) charged with 100 mM CoCl2 for 2 hrs. Bound protein was then eluted with a linear gradient of imidazole (0–500 mM, 8 CV, 40 mL total, 2 mL/min flow rate). Peak fractions were pooled, combined with SUMO protease Ulp1 (50 µg/mL final concentration), and dialyzed against 4 L of buffer containing 20 mM Tris [pH 8.0], 200 mM NaCl, and 0.5 mM BME for 16–18 hr at 4°C. SUMO protease cleaved Btk was recirculated for 1 hr over a 5 mL HiTrap Chelating column. Flow-through containing Btk-SNAP was then concentrated in a 5 kDa MWCO Vivaspin 20 before being loaded on a Superdex 75 size- exclusion column equilibrated in 20 mM Tris [pH 8.0], 200 mM NaCl, 10% glycerol, 1 mM TCEP. Peak fractions containing Btk-SNAP were pooled and concentrated to a concentration of 30 µM before snap-freezing with liquid nitrogen and storage at –80°C. For labeling, Btk-SNAP was combined with a 1.5x molar excess of SNAP- Surface Alexa488 dye (NEB, Cat# S9129S) and incubated overnight at 4ºC. The next day, Btk-SNAP-AF488 was desalted into buffer containing 20 mM Tris [pH 8.0], 200 mM NaCl, 10% glycerol, 1 mM TCEP using a PD10 column. The protein was then spin concentrated using a Amicon filter and loaded onto a Superdex 75 column to isolate dye free monodispersed Btk-SNAP-AF488. The peak elution was pooled, concentrated, aliquoted, and flash frozen with liquid nitrogen. p67/phox. Genes encoding the Rac1(GTP) biosensor, p67/phox, were cloned into a his10-TEV-SUMO plasmid and expressed in Rosetta2 (DE3) pLysS bacteria. We grew bacteria in 3L of Terrific Broth 37°C for two hours or until OD600 =0.8 before shifting temperature to 18°C for 1 hour. We induced protein expression in cells via addition of 50 µM IPTG. Cells expressed overnight for 20 hours at 18ºC before harvesting. We lysted cells into buffer containing 50 mM Na2HPO4 [pH 8.0], 400 mM NaCl, 0.4 mM BME, 1 mM PMSF, and 100 μg/mL DNase using a microfluidizer. The lysate was centrifuged at 16,000 rpm (35000 x g) for 60 minutes in a Beckman JA-20 rotor at 4ºC. Supernatant was then circulated over 5 mL HiTrap Chelating column (GE Healthcare, Cat# 17- 0409-01) that was inoculated with 100mM CoCl2 for ten minutes. The HiTrap column was washed with 20 column volumes (100mL) of 50 mM Na2HPO4 [pH 8.0], 400 mM NaCl, 10 mM imidazole, and 0.4 mM BME containing buffer. Bound protein was eluted at a flow rate of 4mL/min with 15-20 mL of 50 mM Na2HPO4 [pH 8.0], 400 mM NaCl, and 500 mM imidazole containing buffer. Peak fractions were pooled and combined with his6-SenP2 (SUMO protease) at a final concentration of 50 µg/mL and dialyzed against 4 liters of buffer containing 25 mM Tris [pH 8.0], 400 mM NaCl, and 0.4 mM BME. Dialysate containing SUMO cleaved protein was recirculated for 2 hours over two 5 mL HiTrap Chelating (Co2+) columns that were equilibrated in buffer containing 25 mM Tris [pH 8.0], 400 mM NaCl, and 0.4 mM BME. Recirculated protein was concentrated to a volume of 5 mL using a 5 kDa MWCO Vivaspin 20 before loading on a 124 mL Superdex 75 column at a flow rate of 1mL/min. The column was equilibrated in buffer containing 20 mM HEPES [pH 7], 200 mM NaCl, 10% glycerol, and 1 mM TCEP. Peak fractions off the Superdex 75 column were concentrated in a 5 kDa MWCO Vivaspin 20 to a concentration between 200-500 µM (5-12mg/mL). Protein was frozen with liquid nitrogen and stored at -80ºC. Farnesylated Gβ1/Gγ2 and SNAP-Gβ1/Gγ2. The native eukaryotic farnesyl Gβ1/Gγ2 and SNAP-Gβ1/Gγ2 complexes were expressed and purified from insect cells as previously described (Rathinaswamy et al. 2021; Kozasa and Gilman 1995; Hashem A. Dbouk et al. 2012). The Gβ1 and Gγ2 genes were cloned into dual expression vectors containing tandem polyhedron promoters. A single baculovirus expressing either Gβ1/his6-TEV-Gγ2 or SNAP-Gβ1/ his6-TEV-Gγ2 were used to infect 2-4 liters of High Five cells (2 x 10 6 cells/mL) with 2% vol/vol of baculovirus. Cultures were then grown in shaker flasks (120 rpm) for 48 hours at 27ºC before harvesting cells by centrifugation. Insect cells pellets were stored as 10 g pellets in the -80ºC before purification. To isolate farnesylated Gβ1/his6- TEV-Gγ2 or SNAP-Gβ1/his6-TEV-Gγ2 complexes, insect cells were lysed by Dounce homogenization into 50 mM HEPES-NaOH [pH 8], 100 mM NaCl, 3 mM MgCl2, 0.1 mM EDTA, 10 µM GDP, 10 mM BME, Sigma PI tablets (Cat #P5726), 1 mM PMSF, DNase (GoldBio, Cat# D-303-1). We centrifuged homogenized lysate for 10 minutes at 800 x g to remove nuclei and large cell debris. We then centrifuged remaining lysate using a Beckman Ti45 rotor 100,000 x g for 30 minutes at 4 ºC. The post-centrifugation pellet containing plasma membranes was the resuspended in a buffer containing 50 mM HEPES-NaOH [pH 8], 50 mM NaCl, 3 mM MgCl2, 1% sodium deoxycholate (wt/vol, Sigma D6750), 10µM GDP (Sigma, cat# G7127), 10 mM BME, and a Sigma Protease Inhibitor tablet (Cat #P5726) to a concentration of 5 mg/mL total protein. We Dounce homogenized the sample to break apart membranes and then allowed the homogenized solution to stir for 1 hour at 4ºC. We centrifuged the solubilized extracted membrane solution in a Beckman Ti45 rotor 100,000 x g for 45 minutes at 4ºC. We diluted the supernatant containing solubilized Gβ1/his6-TEV-Gγ2 or SNAP-Gβ1/his6-TEV-Gγ2 in buffer composed of 20 bioRxiv preprint doi: https://doi.org/10.1101/2023.05.01.538969; this version posted May 1, 2023. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. mM HEPES-NaOH [pH 7.7], 100 mM NaCl, 0.1 % C12E10 (Polyoxyethylene (10) lauryl ether; Sigma, P9769), 25 mM imidazole, and 2 mM BME. We affinity purified the soluble membrane extracted Gβ1/his6-TEV-Gγ2 or SNAP-Gβ1/his6-TEV-Gγ2 using Qiagen NiNTA resin. After adding NiNTA resin to the diluted solubilized extracted membrane solution, we allowed the resin to incubate and stir in a beaker at 4ºC for 2 hours. We packed our protein-bound resin beads into a gravity flow column and washed with 20 column volumes of buffer containing 20 mM HEPES-NaOH [pH 7.7], 100 mM NaCl, 0.1 % C12E10, 20 mM imidazole, and 2 mM BME. We eluted and discarded the G alpha subunit of the heterotrimeric G-protein complex by washing with warm buffer (30ºC) containing 20 mM HEPES-NaOH [pH 7.7], 100 mM NaCl, 0.1 % C12E10, 20 mM imidazole, 2 mM BME, 50 mM MgCl2, 10µM GDP, 30 µM AlCl3 (J.T. Baker 5-0660), and 10 mM NaF. We eluted Gβ1/his6-TEV-Gγ2 or SNAP-Gβ1/his6-TEV-Gγ2 form the NiNTA resin using buffer containing 20 mM Tris-HCl (pH 8.0), 25 mM NaCl, 0.1 % C12E10, 200 mM imidazole, and 2 mM BME. The eluted protein was incubated overnight at 4ºC with TEV protease to cleave off the his6 affinity tag. The next day, the cleaved protein was desalted on a G25 Sephadex column into buffer containing 20 mM Tris- HCl [pH 8.0], 25mM NaCl, 8 mM CHAPS, and 2 mM TCEP. Next, we performed anion exchange chromatography using a MonoQ column equilibrated with the desalting column buffer. We eluted Gβ1/Gγ2 or SNAP-Gβ1/Gγ2 from the MonoQ column in the presence of 175-200 mM NaCl. Peak-containing fractions were combined and concentrated using a Millipore Amicon Ultra-4 (10 kDa MWCO) centrifuge filter. Concentrated samples of Gβ1/ Gγ2 or SNAP-Gβ1/Gγ2, respectively, were loaded on either Superdex 75 or Superdex 200 gel filtration columns equilibrated 20 mM Tris [pH 8.0], 100 mM NaCl, 8 mM CHAPS, and 2 mM TCEP. Peak fractions were combined and concentrated in a Millipore Amicon Ultra-4 (10 kDa MWCO) centrifuge tube. Finally, we aliquoted the concentrated Gβ1/Gγ2 or SNAP-Gβ1/Gγ2 and flash frozen with liquid nitrogen before storing at -80ºC. Fluorescent labeling of SNAP-Gβ1/Gγ2. To fluorescently label SNAP-Gβ1/Gγ2, protein was combined with 1.5x molar excess of SNAP-Surface Alexa488 dye (NEB, Cat# S9129S). SNAP dye labeling was performed in buffer containing 20 mM Tris [pH 8.0], 100 mM NaCl, 8 mM CHAPS, and 2 mM TCEP overnight at 4ºC. Labeled protein was then separated from free Alexa488-SNAP surface dye using a 10 kDa MWCO Amicon spin concentrator followed by size exclusion chromatography (Superdex 75 10/300 GL) in buffer containing 20 mM Tris [pH 8.0], 100 mM NaCl, 8 mM CHAPS, 1 mM TCEP. Peak SEC fractions containing Alexa488-SNAP-Gβ1/Gγ2 were pooled and centrifuged in a 10 kDa MWCO Amicon spin concentrator to reach a final concentration of 15-20 µM before snap freezing in liquid nitrogen and storing in the -80ºC. To calculate the SNAP dye labeling efficiency, we determined that Alexa488 contributes 11% of the peak A494 signal to the measured A280. Note that Alexa488 non-intuitively has a peak absorbance at 494 nm. We calculate the final concentration of Alexa488-SNAP-Gβ1/Gγ2 using an adjusted A280 (i.e. A280(protein) = A280(observed) – A494(dye)*0.11) and the following extinction coefficients: e280(SNAP-Gβ1/Gγ2) = 78380 M-1•cm-1, e -1 -1494(Alexa488) = 71,000 M •cm . Fluorescent labeling of PI3K using Sfp transferase As previously described (Rathinaswamy et al. 2021), we generated a Dyomics647-CoA derivative by incubating a mixture of 15 mM Dyomics647 maleimide (Dyomics, Cat #647P1-03) in DMSO with 10 mM CoA (Sigma, #C3019, MW = 785.33 g/mole) overnight at 23ºC. To quench excess unreacted Dyomics647 maleimide, we added 5 mM DTT. We thawed purified PIK3CB/ybbr-PIK3R1 (referred to as PI3Kβ or p110β-p85α in manuscript) and chemically labeled with Dyomics647-CoA using Sfp-his6. The ybbrR13 motif fused to PIK3R1 contained the following peptide sequence: DSLEFIASKLA (Yin et al. 2006). In a total reaction volume of 2mL we combined 5µM PI3Kβ, 4µM Sfp-his6, and 10µM DY647-CoA in buffer containing 20 mM Tris [pH 8], 150 mM NaCl, 10 mM MgCl2, 10% Glycerol, 1 mM TCEP, and 0.05% CHAPS. The ybbr labeling reaction was allowed to proceed for 4 hours on ice. Excess Dyomics647-CoA was removed via a using a gravity flow PD-10 column. We concentrated labeled Dy647-PI3Kβ in a 50 kDa MWCO Amicon centrifuge tube before loading on a Superdex 200 gel filtration column equilibrated in 20 mM Tris [pH 8], 150 mM NaCl, 10% glycerol, 1 mM TCEP, and 0.05% CHAPS (GoldBio, Cat# C-080-100). We pooled and concentrated peak fractions to 5-10µM before we aliquoted and flash froze with liquid nitrogen. Labeled protein was stored at -80ºC. Preparation of supported lipid bilayers We generated small unilamellar vesicles (SUVs) for this study using the following lipids:: 1,2-dioleoyl-sn-glycero- 3-phosphocholine (18:1 DOPC, Avanti # 850375C) and 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (18:1 DOPS, Avanti # 840035C), L-α-phosphatidylinositol-4,5-bisphosphate (Brain PI(4,5)P2, Avanti # 840046X), synthetic bioRxiv preprint doi: https://doi.org/10.1101/2023.05.01.538969; this version posted May 1, 2023. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. phosphatidylinositol 4,5-bisphosphate 18:0/20:4 (PI(4,5)P2, Echelon, P-4524), and 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide] (18:1 MCC-PE, Avanti # 780201C). We report lipid mixtures as percentages equivalent to molar fractions. We dried a total of 2 µmoles lipids were combined with 2mL of chloroform in a 35 mL glass round bottom flask containing. This mixture was dried to a thin film via rotary evaporation where the glass round-bottom flask was kept in a 42ºC water bath. Following evaporation, we either flushed the lipid-containing flask with nitrogen gas or placed in it a vacuum desiccator for a minimum of 30 minutes. We obtained a concentration of 1 mM of lipids by resuspending the dried film in 2 mL of 1x PBS [pH 7.2]. We generated 30-50 nm SUVs from this 1 mM total lipid mixture via extrusion of the resuspended lipid mixture through 0.03 µm pore size 19 mm polycarbonate membrane (Avanti #610002) with filter supports (Avanti #610014) on both sides of the PC membrane. We prepared coverglass (25x75 mm, IBIDI, cat #10812) for depositing of SUV’s by first cleaning with heated (60-70ºC) 2% Hellmanex III (Fisher, Cat#14-385- 864) in a glass coplin jar. We incubated hot Hellmanex III and coverglass for at least 30 minutes before rinsing with MilliQ water. The cleaned glass was then etched with Piranha solution (1:3, hydrogen peroxide:sulfuric acid) for 5-10 minutes. We rinsed and stored the etched coverglass in MilliQ. We rapidly dried our MilliQ-rinsed etched coverglass slides with nitrogen gas before adhering to a 6-well sticky-side chamber (IBIDI, Cat# 80608). We created SLBs by flowing 100-150µL of SUVs with a total lipid concentration of 0.25 mM in 1x PBS [pH 7.2] into the IBIDI chamber. Following 30 minutes of incubation, supported membranes were washed with 4 mL of 1x PBS [pH 7.2] to remove non-absorbed SUVs. To block membrane defects, we prepared 1 mg/mL beta casein (Thermo FisherSci, Cat# 37528) by clarifying with a centrifugation step at 4°C for 30 minutes at 21370 x g before passing through 0.22 µm syringe filtration unit (0.22 µm PES syringe filter (Foxx Life Sciences, Cat#381-2116-OEM). We then blocked membrane defects with 1 mg/mL beta casein (Thermo FisherSci, Cat# 37528) for 5-10 minutes. Protein conjugation of maleimide lipid After blocking SLBs with beta casein, membranes were washed with 2mL of 1x PBS and stored at room temperature for up to 2 hours before mounting on the TIRF microscope. Prior to single molecule imaging experiments, supported membranes were washed into TIRF imaging buffer. Supported membrane containing with MCC-PE lipids were used to covalently couple either H-Ras(GDP) or phosphotyrosine peptide (pY). For the pY peptide experiments we used a doubly phosphorylated peptide derived from the mouse platelet derived growth factor receptor (PDGFR) with the following sequence: CSDGG(pY)MDMSKDESID(pY)VPMLDMKGDIKYADIE (33aa). The Alexa488-pY contained the same sequence with the dye conjugated to the C-terminus of the peptide. For these SLBs, 100 µL of 30 µM H-Ras diluted in a 1x PBS [pH 7.2] and 0.1 mM TCEP buffer was added to the IBIDI chamber and incubated for 2 hours at 23ºC. Importantly, the addition of 0.1 mM TCEP significantly increases the coupling efficiency. SLBs with MCC-PE lipids were then washed with 2 mL of 1x PBS [pH 7.2] containing 5 mM beta-mercaptoethanol (BME) and incubated for 15 minutes to neutralize the unreacted maleimide headgroups. SLBs were washed with 1mL of 1x PBS, followed by 1 mL of kinase buffer before starting smTIRF-M experiments. Nucleotide exchange of Rac1 Membrane conjugated Rac1(GDP) was converted to Rac1(GTP) using either chemical activation (i.e. EDTA/ GTP/MgCl2) or the guanine nucleotide exchange factor (GEF), P-Rex1. Chemical activation was accomplished by washing supported membranes containing maleimide linked Rac1(GDP) with 1x PBS [pH 7.2] containing 1 mM EDTA and 1 mM GTP. Following a 15-minute incubation to exchange GDP for GTP, chambers were washed 1x PBS [pH 7.2] containing 1 mM MgCl2 and 50 µM GTP. A complementary approach that utilizes GEF-mediated activation of Rac1 was achieved by flowing 50 nM P-Rex1 DH-PH domain over Rac1(GDP) conjugated membranes (Figure 1C). Nucleotide exchange was carried out in buffer containing 1x PBS, 1 mM MgCl2, 50 µM GTP. Both methods of activation yielded the same density of Rac1(GTP). Nucleotide exchange of membrane tethered Rac1 was assessed by visualizing the localization of the Cy3-p67/phox Rac1(GTP) sensor using TIRF-M. Single molecule TIRF microscopy We preformed all supported membrane TIRF-M experiments in buffer containing 20 mM HEPES [pH 7.0], 150 mM NaCl, 1 mM ATP, 5 mM MgCl2, 0.5 mM EGTA, 20 mM glucose, 200 µg/mL beta casein (ThermoScientific, Cat# 37528), 20 mM BME, 320 µg/mL glucose oxidase (Biophoretics, Cat #B01357.02 Aspergillus niger), 50 µg/mL catalase (Sigma, #C40-100MG Bovine Liver), and 2 mM Trolox (Cayman Chemicals, Cat# 10011659). bioRxiv preprint doi: https://doi.org/10.1101/2023.05.01.538969; this version posted May 1, 2023. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Perishable reagents (i.e. glucose oxidase, catalase, and Trolox) were added 5-10 minutes before starting image acquisition. Microscope hardware and imaging acquisition Single molecule imaging experiments were performed at room temperature (23ºC) using an inverted Nikon Ti2 microscope using a 100x oil immersion Nikon TIRF objective (1.49 NA). We controlled the x-axis and y-axis position using a Nikon motorized stage, joystick, and Nikon’s NIS element software. We also controlled microscope hardware using Nikon NIS elements. Fluorescently labelled proteins were excited with one of three diode lasers: a 488 nm, a 561nm, or 637 nm (OBIS laser diode, Coherent Inc. Santa Clara, CA). The lasers were controlled with a Vortran laser launch and acousto-optic tuneable filters (AOTF) control. Excitation and emission light was transmitted through a multi-bandpass quad filter cube (C-TIRF ULTRA HI S/N QUAD 405/488/561/638; Semrock) containing a dichroic mirror. The laser power measured through the objective for single particle visualized was 1-3 mW. Fluorescence emission was captured on an iXion Life 897 EMCCD camera (Andor Technology Ltd., UK) after passing through one of the following 25 mm a Nikon Ti2 emission filters mounted in a Nikon emission filter wheel: ET525/50M, ET600/50M, and ET700/75M (Semrock). Kinetics measurements of PI(3,4,5)P3 lipid production The phosphorylation of PI(3,4,5)P3 was measured on SLB’s formed in IBIDI chambers visualized via TIRF microscopy. We monitored the production of PI(3,4,5)P3 by solution-based PI3K at membrane surfaces using solution concentrations of 50 nM Btk-SNAP-AF488. Reaction buffer for experiments contained 20mM HEPES (pH 7.0), 150 mM NaCl, 5 mM MgCl2, 1 mM ATP, 0.1mM GTP, 0.5 mM EGTA, 20 mM glucose, 200 µg/mL beta- casein (Thermo Scientific, Cat# 37528), 20 mM BME, 320 µg/mL glucose oxidase (Serva, #22780.01 Aspergillus niger), 50 µg/mL catalase (Sigma, #C40-100MG Bovine Liver), and 2 mM Trolox (Cayman Chemicals, Cat# 10011659). In experiments where inactive GTPases were coupled to membranes, no ATP was present in the reaction buffer and the 0.1 mM of GTP was replaced with 0.1 mM of GDP. 5-10 minutes before image acquisition, chemicals and enzymes needed the oxygen scavenging system were added to the TIRF imaging buffer. Surface density calibration The density of membrane-tethered proteins attached to supported lipid bilayers was determined by coupling a defined ratio of either fluorescently labeled Cy3-Rac1 (e.g. 1:10,000) or Alexa488-pY (1:30,000) in the presence of either 10 µM pY or 30 µM Rac1. Single spatially resolved fluorescent proteins were visualize by TIRF microscopy. We calculated the density of fluorescent particles using ImageJ/Fiji Trackmake Plugin. The total surface density was calculated based on the dilution factor. Alphafold2 Multimer modelling We utilized the AlphaFold2 using Mmseqs2 notebook of ColabFold at colab.research.google.com/github/ sokrypton/ColabFold/blob/main/AlphaFold2.ipynb to make structural predictions of PI3Kβ (p110β/p85α) bound to Gbg. The pLDDT confidence values consistently scored above 90% for all models, with the predicted aligned error and pLDDT scores for all models are shown in Figure 3 – figure Supplement 1. Single particle tracking Single fluorescent Dy647-PI3Kβ complexes bound to supported lipid bilayers were identified and tracked using the ImageJ/Fiji TrackMate plugin (Jaqaman et al. 2008). Data was loaded into ImageJ/Fiji as .nd2 files. We used the LoG detector to identify particles based on their size (~6 pixel diameter), brightness, and signal-to-noise ratio. We then used the LAP tracker to generate trajectories that followed particle displacement as a function of time. Particle trajectories were then filtered based on Track Start (remove particles at start of movie), Track End (remove particles at end of movie), Duration (particles track ≥ 2 frames), Track displacement, and X - Y location (removed particles near the edge of the movie). The output files from TrackMate were then analyzed using Prism 9 graphing software to calculate the dwell times. To calculate the dwell times of membrane bound proteins we generated cumulative distribution frequency (CDF) plots with the bin size set to image acquisition frame interval (e.g. 52 ms). The log10(1-CDF) was plotted as a function dwell time and fit to a single or double exponential curve. For the double exponential curve fits, the alpha value is the percentage of the fast-dissociating molecules characterized by the time constant, t1. A typical data set contained dwell times measured for n ≥ 1000 trajectories repeated as n = 3 technical replicates. bioRxiv preprint doi: https://doi.org/10.1101/2023.05.01.538969; this version posted May 1, 2023. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Single exponential curve fit: Two exponential curve fit: To calculate the diffusion coefficient (µm2/sec), we plotted probability density (i.e. frequency divided by bin size of 0.01 µm) versus step size (µm). The step size distribution was fit to the following models: Single species model: Two species model: Image processing, statistics, and data analysis Image analysis was performed on ImageJ/Fiji and MatLab. Curve fitting was performed using Prism 9 GraphPad. The X-fold change in dwell time we report in the main text was calculated by comparing the mean single particle dwell time for different experimental conditions (e.g. Figure 3C). Note that this is different from directly comparing the calculated dwell time (or exponential decay time constant, t1). The X% reduction in diffusion or mobility (e.g. Figure 3D) we report in the main text was calculated by comparing the mean single particle displacement (or step size) measured under different experimental conditions. bioRxiv preprint doi: https://doi.org/10.1101/2023.05.01.538969; this version posted May 1, 2023. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. TABLE 1 protein pY/µm2 t1 ± SD t 2 ± SD a ± SD AVE D1 ± SD D2 ± SD a ± SD MEDIAN visualized (sec) (sec) DT (sec) (µm2/sec) (µm2/sec) step (µm) PI3Kβ (WT) 250 0.58±0.28 1.78±0.58 0.60±0.37 1.00±0.09 0.39±0.07 1.45±0.09 0.29±0.08 0.37±0.02 PI3Kβ (WT) 573 0.39±0.06 1.37±0.14 0.27±0.02 1.12±0.09 0.28±0.06 1.15±0.14 0.22±0.04 0.35±0.01 PI3Kβ (WT) 1226 0.36±0.13 1.29±0.06 0.30±0.09 1.05±0.11 0.20±0.02 1.18±0.09 0.16±0.02 0.36±0.02 PI3Kβ (WT) 2935 0.44±0.11 1.53±0.38 0.47±0.25 1.00±0.07 0.28±0.09 1.09±0.12 0.26±0.09 0.33±0.01 PI3Kβ (WT) 6661 0.46±0.08 1.28±0.16 0.61±0.13 0.82±0.11 0.35±0.17 1.28±0.34 0.35±0.18 0.34±0.01 PI3Kβ (WT) 14944 0.55±0.11 1.44±0.56 0.54±0.22 0.91±0.09 0.45±0.15 1.40±0.54 0.48±0.06 0.33±0.05 PI3Kβ (WT) 14944 0.49±0.17 1.38±0.19 0.35±0.17 1.10±0.04 0.32±0.04 0.99±0.12 0.40±0.11 0.30±0.02 PI3Kβ (nSH2*) 14944 0.23±0.02 1.48±0.23 0.86±0.03 0.45±0.06 0.38±0.09 1.45±0.25 0.41±0.18 0.34±0.01 PI3Kβ (cSH2*) 14944 0.38±0.08 1.54±0.55 0.76±0.1 0.65±0.08 0.34±0.13 1.12±0.29 0.43±0.15 0.30±0.04 SD = standard deviation from 3-5 technical replicates N = 331 – 1909 total particles for each technical replicate steps = 4277 – 39378 total particle displacements measured for each technical replicate alpha (α) = fraction of molecules with characteristic dwell time (τ1) or diffusion coefficient (D1). membrane composition: 96% DOPC, 2% PI(4,5)P2, 2% MCC-PE. 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