Rab2 Interacts Directly with Atypical Protein Kinase C (aPKC) ι/λ and Inhibits aPKCι/λ-dependent Glyceraldehyde-3-phosphate Dehydrogenase Phosphorylation

Abstract Atypical protein kinase C ι/λ (PKCι/λ) is essential for protein transport in the early secretory pathway. The small GTPase Rab2 selectively recruits the kinase to vesicular tubular clusters (VTCs) where PKCι/λ phosphorylates glyceraldehyde-3-phosphate dehydrogenase (GAPDH). VTCs are composed of small vesicles and tubules and serve as transport intermediates that shuttle cargo from the endoplasmic reticulum to the Golgi complex. These structures are the first site of segregation of the anterograde and retrograde pathways. When Rab2 binds to a VTC subcompartment, the subsequent recruitment of PKCι/λ and soluble components, including COPI (coatomer and ADP-ribosylation factor), results in the release of retrograde-directed vesicles. Because Rab2 stimulates PKCι/λ membrane association in a dose-dependent manner, we investigated whether the two proteins physically interact. Using a combination of in vivo and in vitro assays, we found that Rab2 interacts directly with PKCι/λ and that this interaction occurs through the Rab2 amino terminus (residues 1–19) and the PKCι/λ regulatory domain. A mutant lacking the PKCι/λ binding domain (Rab2N′Δ19) was functionally characterized. In contrast to Rab2, Rab2N′Δ19 failed to recruit PKCι/λ to normal rat kidney microsomes in a quantitative binding assay. To determine whether Rab2 modulates the ability of PKCι/λ to phosphorylate GAPDH, an in vitro kinase assay was supplemented with Rab2 or Rab2N′Δ19. Rab2 inhibited PKCι/λ-dependent GAPDH phosphorylation, whereas no effect was observed when the assay was performed with the aminoterminal truncation mutant. These results suggest that a downstream effector recruited to the VTC stimulates PKCι/λ-mediated GAPDH phosphorylation by alleviating the inhibition imposed by Rab2-PKCι/λ interaction.