Rab2B requires atypical PKCι to mediate MAPK signaling in the early secretory pathway.

Rab2B is required for membrane trafficking in the secretory pathway where Rab2B function is linked to signaling molecules: Rab2B requires Src phosphorylation of atypical PKC iota (aPKCι). aPKCι is necessary for β-COP recruitment. Our most recent studies showed Rab2B overexpressing NRK cells have significantly increased MAPK activity. Interestingly, aPKCι is a candidate molecule to stimulate MAPK activity. Therefore, we performed experiments to determine if aPKCι, which interacts with Rab2B residues 1-19 contributed to Rab2B-ERK1/2 activation. NRK cells were transfected with Rab2B cDNA or with Rab2B N'Δ19 cDNA, and the cell lysates analyzed. Rab2B overexpressing cells had increased activated ERK1/2 that was reduced in mutant lysate to control level. Cell lysate from co-overexpression of Rab2B and aPKCι had the highest level of activated ERK1/2 indicating the importance of both Rab2B-aPKCι interaction and concentration for increased MAPK activity. Since GRASP65 is a substrate of Rab2B-ERK1/2 activation, we determined that aPKCι played a role in GRASP65 phosphorylation. pGRASP65 on the ERGIC contributes to Golgi homeostasis by regulating ERGIC membrane input to the cis Golgi. Because the ERGIC binds β-COP and aPKCι kinase activity is required for Rab2B-aPKCι to generate COPI vesicles, we evaluated the role of aPKCι-MAPK in a membrane binding/vesicle budding assay. Membranes treated with a MEK1 inhibitor or kinase dead aPKCι bound β-COP but produced significantly fewer COPI vesicles than Rab2-aPKCι treated membranes. These combined results suggest that Rab2B requires aPKCι for MAPK activation and GRASP65 phosphorylation, which delays ERGIC redistribution to the cis Golgi until COPI vesicle formation.