Protein kinase B/Akt is a novel cysteine string protein kinase that regulates exocytosis release kinetics and quantal size.
Protein kinase B/Akt has been implicated in the insulin-dependent exocytosis of GLUT4-containing vesicles, and, more recently, insulin secretion. To determine if Akt also regulates insulin-independent exocytosis, we used adrenal chromaffin cells, a popular neuronal model. Akt1 was the predominant isoform expressed in chromaffin cells, although lower levels of Akt2 and Akt3 were also found. Secretory stimuli in both intact and permeabilized cells induced Akt phosphorylation on serine 473, and the time course of Ca2+-induced Akt phosphorylation was similar to that of exocytosis in permeabilized cells. To determine if Akt modulated exocytosis, we transfected chromaffin cells with Akt constructs and monitored catecholamine release by amperometry. Wild-type Akt had no effect on the overall number of exocytotic events, but slowed the kinetics of catecholamine release from individual vesicles, resulting in an increased quantal size. This effect was due to phosphorylation by Akt, because it was not seen in cells transfected with kinase-dead mutant Akt. As overexpression of cysteine string protein (CSP) results in a similar alteration in release kinetics and quantal size, we determined if CSP was an Akt substrate. In vitro 32P-phosphorylation studies revealed that Akt phosphorylates CSP on serine 10. Using phospho-Ser10-specific antisera, we found that both transfected and endogenous cellular CSP is phosphorylated by Akt on this residue. Taken together, these findings reveal a novel role for Akt phosphorylation in regulating the late stages of exocytosis and suggest that this is achieved via the phosphorylation of CSP on serine 10.
Evans
Gareth J O
GJ
The Physiological Laboratory, School of Biomedical Sciences, University of Liverpool, Liverpool L69 3BX, United Kingdom.
Barclay
Jeff W
JW
Prescott
Gerald R
GR
Jo
Sung-Ro
SR
Burgoyne
Robert D
RD
Birnbaum
Morris J
MJ
Morgan
Alan
A
D000311:Adrenal Glands / Q000166:cytology / Q000502:physiology; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002460:Cell Line; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000502:physiology; D003553:Cystine; D004594:Electrophysiology / Q000379:methods; D005089:Exocytosis / Q000502:physiology*; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D007668:Kidney; D016296:Mutagenesis; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011789:Quantum Theory; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection
D007527:Isoenzymes; D010446:Peptide Fragments; D011994:Recombinant Proteins; D003553:Cystine; D051057:Proto-Oncogene Proteins c-akt