Protein kinase D 3 is localized in vesicular structures and interacts with vesicle-associated membrane protein 2.
Protein kinase D localizes in the Golgi and regulates protein transport from the Golgi to the plasma membrane. In the present study, we found that PKD3, a novel member of the PKD family, and its fluorescent protein fusions localized in the Golgi and in the vesicular structures that are in part marked by endosome markers. Fluorescent recovery after photobleaching (FRAP) showed that the PKD3-associated vesicular structures were constantly forming and dissolving, reflecting active subcellular structures. FRAP on plasma membrane-located PKD3 indicated a slower recovery of PKD3 fluorescent signal compared to those of PKC isoforms, implying a different targeting mechanism at the plasma membrane. VAMP2, the vesicle-localized v-SNARE, was later identified as a novel binding partner of PKD3 through yeast two-hybrid screening. PKD3 directly interacted with VAMP2 in vitro and in vivo, and colocalized in part with VAMP2 vesicles in cells. PKD3 did not phosphorylate VAMP-GFP and the purified GST-VAMP2 protein in in vitro phosphorylation assays. Rather, PKD3 was found to promote the recruitment of VAMP2 vesicles to the plasma membrane in response to PMA, while the kinase dead PKD3 abolished this effect. Thus, the kinase activity of PKD3 was required for PMA-induced plasma membrane trafficking of VAMP2. In summary, our findings suggest that PKD3 localizes to vesicular structures that are part of the endocytic compartment. The vesicular distribution may be attributed in part to the direct interaction between PKD3 and vesicle-associated membrane protein VAMP2, through which PKD3 may regulate VAMP2 vesicle trafficking by facilitating its recruitment to the target membrane.
Lu
Ganwei
G
Department of Pharmacology, University of Pittsburgh, Pittsburgh, PA 15261, USA.
Chen
Jun
J
Espinoza
Luis A
LA
Garfield
Susan
S
Toshiyuki
Saito
S
Akiko
Hayashi
H
Huppler
Anna
A
Wang
Q Jane
QJ
D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000201:enzymology; D006224:Cricetinae; D003412:Cricetulus; D022162:Cytoplasmic Vesicles / Q000201:enzymology*; D003600:Cytosol / Q000201:enzymology; D011992:Endosomes / Q000201:enzymology; D006056:Golgi Apparatus / Q000201:enzymology; D006801:Humans; D010766:Phosphorylation; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism*; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D050988:Vesicle-Associated Membrane Protein 2 / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D050988:Vesicle-Associated Membrane Protein 2; C089212:protein kinase D; D011493:Protein Kinase C