Akt regulates the subcellular localization of the Rab27a-binding protein JFC1 by phosphorylation.
Here, we show that the Rab27a-binding protein JFC1/Slp1 (synaptotagmin-like protein) is regulated by Akt-mediated phosphorylation. Using the phosphatase and tensin homolog-null LNCaP cells and the phosphatidylinositol 3-kinase inhibitor LY294002, we show that the phosphorylation of endogenous JFC1 is dependent on the phosphatidylinositol 3-kinase/Akt pathway. JFC1 was phosphorylated in cells expressing a constitutively active Akt, confirming that it is an Akt substrate in vivo. Direct phosphorylation of JFC1 by Akt was confirmed in vitro. Using microcapillary high-performance liquid chromatography tandem mass spectrometry, we identified five Akt-phosphorylation sites in JFC1. By mutagenesis analysis and subsequent immunoprecipitation (IP), we established that Akt phosphorylates JFC1 at serine 241. JFC1 and Rab27a colocalize in the proximity of the plasma membrane in LNCaP cells. The interaction was confirmed by IP analysis and was abolished by the point mutation W83S in JFC1. Phosphorylation did not alter the ability of JFC1 to bind to Rab27a. Instead, phosphorylation by Akt dramatically decreased when JFC1 was bound to Rab27a. Finally, we show that as a consequence of in vivo phosphorylation, JFC1 dissociates from the membrane, promoting JFC1 redistribution to the cytosol. Our results suggest that Akt regulates JFC1/Slp1 function by phosphorylation and may have implications on Rab27a-containing vesicle secretion.
Division of Biochemistry, Department of Molecular and Experimental Medicine, The Scripps Research Institute,10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
Jennifer L
JL
Johnson
Sandrine
S
Pacquelet
William S
WS
Lane
Boreth
B
Eam
Sergio D
SD
Catz
D045744:Cell Line, Tumor; D002867:Chromones / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D015850:Interleukin-6 / Q000494:pharmacology; D008387:Marine Toxins; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009025:Morpholines / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D010080:Oxazoles / Q000494:pharmacology; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000494:pharmacology / Q000502:physiology*; D011518:Proto-Oncogene Proteins / Q000494:pharmacology / Q000502:physiology*; D051057:Proto-Oncogene Proteins c-akt; D011993:Recombinant Fusion Proteins; D022142:Secretory Vesicles / Q000502:physiology; D014162:Transfection; D033921:Vesicular Transport Proteins; D020691:rab GTP-Binding Proteins / Q000378:metabolism*; D000075745:rab27 GTP-Binding Proteins
D002867:Chromones; D004791:Enzyme Inhibitors; D015850:Interleukin-6; D008387:Marine Toxins; D008565:Membrane Proteins; D009025:Morpholines; D010080:Oxazoles; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; C431146:SYTL1 protein, human; C424133:Sytl1 protein, mouse; D033921:Vesicular Transport Proteins; D000075745:rab27 GTP-Binding Proteins; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C059041:calyculin A; C494918:AKT1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D010749:Phosphoprotein Phosphatases; C066694:RAB27A protein, human; C512962:Rab27a protein, mouse; D020691:rab GTP-Binding Proteins