Activity of Atypical Protein Kinase C: From Regulation to Substrate Localization

Abstract

The phosphorylation activity of protein kinases is involved in virtually all biological processes of living organisms. As uncontrolled kinase cascades cause devastating defects such as cancer, cells employ complex regulatory networks to precisely control their activity. Atypical Protein Kinase C (aPKC) is a well-conserved protein kinase that plays a central role in the establishment of the Par complex-mediated cell polarity. The goal of my research is to understand how aPKC activity is regulated and how aPKC phosphorylates its substrates. The first part of my study focused on the mechanism by which intra- and intermolecular interactions regulate aPKC activity. aPKC contains a pseudosubstrate domain that acts as an internal inhibitor. Despite the presence of the cis-acting inhibitor, another Par complex member, Par-6, is thought to repress aPKC activity. To examine the precise mechanism by which the pseudosubstrate domain and Par-6 regulate aPKC activity, I reconstituted the system in vitro and performed a detailed kinetic analysis. We confirmed that the pseudosubstrate domain is responsible for the autoinhibition. Surprisingly, rather than acting as an inhibitor, Par-6 activates aPKC by displacing the pseudosubstrate from the kinase domain. Par-6 activation of aPKC is consistent with our observation that the Par-6/aPKC complex, but not aPKC alone, releases its substrate from the cell membrane in Drosophila S2 cells. The data support a model in which aPKC activity is coupled to localization via Par-6. In the second part, I investigated how the phosphorylation activity of aPKC is coupled to cortical displacement of fate determinants, which often contain multiple phospho-accepting residues. Using Lgl as a model substrate in S2 localization assays, I examined the role of multiple phosphorylations and found that multi-site phosphorylation is required for cortical release. Also, I examined how aPKC phosphorylates Lgl in an in vitro kinase assay and found that aPKC cooperatively phosphorylates Lgl in an ordered manner. These results provide new insights into how multiple phosphorylation and phosphorylation rates could regulate localization behaviors of fate determinants at the cortex.

This dissertation contains previously published coauthored materials as well as unpublished materials.