Chen, Jing

Rab32 is a small GTPase and molecular switch implicated in vesicular trafficking. Rab32 is also an A-Kinase Anchoring Protein (AKAP), which anchors cAMP-dependent Protein Kinase (PKA) to specific subcellular locations and specifies PKA phosphorylation of nearby substrates. Surprisingly, we found that a form of Rab32 deficient in PKA binding (Rab32 L188P) relocalized away from the Golgi apparatus and induced a marked disruption in Golgi organization, assembly, and dynamics. Although Rab32 L188P did not cause a global defect in PKA activity, our data indicate that Rab32 facilitates the phosphorylation of a specific PKA substrate. We uncovered a direct interaction between Rab32 and the adaptor protein optineurin (OPTN), which regulates Golgi dynamics. Further, our data indicate that optineurin is phosphorylated by PKA at Ser342 in a Rab32-dependent manner. Critically, blocking phosphorylation at OPTN Ser342 leads to Golgi fragmentation, and a phospho-mimetic version of OPTN rescues Golgi defects induced by Rab32 L188P. Finally, Rab32 AKAP function and OPTN phosphorylation are required for Golgi repositioning during cell migration, contributing to tumor cell invasion. Together, these data reveal a role for Rab32 in regulating Golgi dynamics through PKA-mediated phosphorylation of OPTN.

Eps15 (EGFR pathway substrate clone 15) is well known for its role in clathrin-coated vesicle formation at the plasma membrane through interactions with other clathrin adaptor proteins such as AP-2. Interestingly, we observed that in addition to its plasma membrane localization, Eps15 is also present at the trans-Golgi network (TGN). Therefore, we predicted that Eps15 might associate with clathrin adaptor proteins at the TGN and thereby mediate the formation of Golgi-derived vesicles. Indeed, we have found that Eps15 and the TGN clathrin adaptor AP-1 coimmunoprecipitate from rat liver Golgi fractions. Furthermore, we have identified a 14-amino acid motif near the AP-2–binding domain of Eps15 that is required for binding to AP-1, but not AP-2. Disruption of the Eps15–AP-1 interaction via siRNA knockdown of AP-1 or expression of mutant Eps15 protein, which lacks a 14-amino acid motif representing the AP-1 binding site of Eps15, significantly reduced the exit of secretory proteins from the TGN. Together, these findings indicate that Eps15 plays an important role in clathrin-coated vesicle formation not only at the plasma membrane but also at the TGN during the secretory process.

It is well-known that dynamin 2 (Dyn2) participates in clathrin- and caveolae-mediated endocytosis; however, the role of Dyn2 in coat-independent endocytic processes remains controversial. Here we demonstrate a role for specific spliced variants of Dyn2 in the micropinocytosis of fluid in epithelial cells, independent of coat-mediated endocytic pathways. A general inhibition of Dyn2 was first performed using either microinjection of anti-dynamin antibodies or Dyn2-siRNA treatment. Both of these methods resulted in reduced uptake of transferrin, a marker for clathrin-mediated endocytosis, and, under unstimulated conditions, reduced the uptake of the fluid-phase markers dextran and horseradish peroxidase (HRP). By contrast, cells treated similarly but stimulated with serum or EGF internalized substantial amounts of dextran or HRP, indicating that Dyn2 is not required for stimulated fluid uptake via macropinocytosis. We next tested whether a specific spliced variant might selectively affect fluid-phase endocytosis. Mutation of specific Dyn2 spliced variants resulted in a differential attenuation of transferrin and dextran internalization. Furthermore, the reduction in fluid uptake in Dyn2-siRNA-treated cells was only rescued upon re-expression of select spliced variants. These findings suggest that Dyn2 function is required for the coat-independent internalization of fluid through endocytic pathways distinct from macropinocytosis and, in addition, implicate different Dyn2 spliced variants in specific endocytic functions.