Atypical protein kinase C (aPKC) isozymes are unique in the protein kinase C (PKC) superfamily in that they are not regulated by the lipid second messenger diacylglycerol. Whether a different second messenger acutely controls their function is unknown. Here we show that the lipid mediator, sphingosine 1-phosphate (S1P), controls the cellular activity of aPKC. Using a genetically-encoded reporter we designed, aPKC-specific C Kinase Activity Reporter (aCKAR), we demonstrate that intracellular S1P activates aPKC. Biochemical studies reveal that S1P directly binds to the kinase domain of aPKC to relieve autoinhibitory constraints. In silico studies identify potential binding sites on the kinase domain, one of which was validated biochemically. Lastly, functional studies reveal that S1P-dependent activation of aPKC suppresses apoptosis in HeLa cells. Taken together, our data reveal a previously undescribed molecular mechanism for controlling the cellular activity of atypical PKC and identify a new molecular target for S1P.
The first genetically-encoded biosensor for atypical protein kinase C isozymes is used to identify intracellular sphingosine 1-phosphate as a novel activator of this class of protein kinase C isozymes, with computational, biochemical, and cellular studies identifying the mechanism and function of this activation.