Rab4 is phosphorylated by the insulin-activated extracellular-signal-regulated kinase ERK1.

Rab4, a low-molecular-mass GTP-binding protein, is associated with vesicles containing Glut 4 in adipocytes. Following insulin stimulation, the translocation of Glut 4 to the plasma membrane is associated with the movement of Rab4 to the cytosol. The same modifications are induced by the phosphatase inhibitor, okadaic acid. To establish a possible role for phosphorylation in Rab4 cycling, we searched for insulin-stimulated cytosolic kinase(s) which could phosphorylate Rab4. In 3T3-L1 adipocytes, insulin induced a rapid and transient activation of cytosolic kinase(s), which phosphorylated Rab4 in vitro. At least part of the Rab4 phosphorylation can be accounted for by ERK (extracellular-signal-regulated kinases) since immunopurified ERK1 from insulin-stimulated cells phosphorylated Rab4 with a comparable time-course. Both with cytosolic extracts and immunopurified ERK1, only serine residues were phosphorylated on Rab4. The phosphorylation site was localized in the C-terminus of the molecule, and occurred very probably on Ser196. These results indicate that Rab4 is an in vitro substrate for ERK, and suggest that the insulin-induced movement of Rab4 from the Glut-4-containing vesicles to the cytosol could result from phosphorylation of Rab4 by ERK.