Abstract
The small GTPase Rab2 is required for membrane transport between the endoplasmic reticulum (ER) and the Golgi complex. Rab2 associates with pre-Golgi intermediates (also termed vesicular tubular clusters; VTCs) that sort cargo to the anterograde pathway from recycling proteins retrieved to the ER. Our previous studies have shown that Rab2 stimulates atypical protein kinase C iota/lambda (aPKCiota/lambda) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) recruitment to VTCs. Both aPKCiota/lambda and GAPDH bind directly to Rab2 and aPKCiota/lambda and GAPDH interact. Based on the reports demonstrating aPKCiota-Src interaction and Src activity in the retrograde pathway (Golgi-ER), studies were initiated to learn whether Rab2 also promoted Src recruitment to VTCs. Using a quantitative membrane binding assay, we found that Rab2-stimulated Src membrane association in a dose-dependent manner. The recruited Src binds to aPKCiota/lambda and GAPDH on the membrane; however, Src does not interact with Rab2. The membrane-associated Src tyrosine phosphorylates aPKCiota/lambda on the VTC. To determine the consequence of aPKCiota/lambda tyrosine phosphorylation, the membrane binding assay was supplemented with the Src-specific tyrosine kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo(3,4-d)pyrimidine (PP2). Although Rab2, Src, and GAPDH recruitment was not affected, the Rab2-PP2-treated membranes contained a negligible amount of aPKCiota/lambda. Since Rab2 requires aPKCiota/lambda for the downstream recruitment of beta-coat protein (beta-COP) to VTCs, the Rab2-PP2-treated membranes were evaluated for the presence of beta-COP. Like aPKCiota/lambda, the membranes contained a negligible amount of beta-COP that was reflected by the drastic reduction in Rab2-dependent vesicle formation. These data suggest that Src-mediated tyrosine phosphorylation of aPKCiota/lambda facilitates aPKCiota/lambda association with Rab2-Src-GAPDH on VTCs, which is ultimately necessary for the downstream recruitment of beta-COP and release of Rab2-mediated retrograde-directed vesicles.
Highlights
PKC/ is a member of the atypical subgroup of protein kinase C
We have shown that Rab2 bound to VTCs stimulates the recruitment of atypical protein kinase C /, which binds directly to the Rab2 amino terminus [9, 10]. aPKC/ kinase activity is required for Rab2-mediated vesicle formation
The nonreceptor tyrosine kinase Src was shown to interact directly with aPKC, and this interaction resulted in tyrosine phosphorylation of multiple residues within the aPKC catalytic and regulatory domains [20, 27]
Summary
PKC/ is a member of the atypical subgroup of protein kinase C. Src phosphorylates aPKC on multiple tyrosine residues located in both the regulatory and the catalytic domain, which influences aPKC/ association with specific binding proteins [20]. The Rab2-recruited Src is activated and tyrosine phosphorylates the membrane-associated aPKC/.
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