Calcium Stimulates Self-Assembly of Protein Kinase C ? In Vitro.

ABSTRACT: Protein kinase C ? (PKC?) is a nodal regulator in several intracellular signaling networks. PKC? is composed of modular domains that interact with each other to dynamically regulate spatial-temporal function. We find that PKC? specifically, rapidly and reversibly self-assembles in the presence of calcium in vitro. This phenomenon is dependent on, and can be modulated by an intramolecular interaction between the C1a and C2 protein domains of PKC?. Next, we monitor self-assembly of PKC-mCitrine fusion proteins using time-resolved and steady-state homoFRET. HomoFRET between full-length PKC? molecules is observed when in solution with both calcium and liposomes containing either diacylglycerol (DAG) or phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). Surprisingly, the C2 domain is sufficient to cluster on liposomes containing PI(4,5)P2, indicating the C1a domain is not required for self-assembly in this context. We conclude that three distinct clustered states of PKC? can be formed depending on what combination of cofactors are bound, but Ca2+ is minimally required and sufficient for clustering.

SUBMITTER: Swanson CJ 

PROVIDER: S-EPMC5051681 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature