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J. Biol. Chem., Vol. 281, Issue 3, 1564-1572, January 20, 2006

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Protein Kinase B/Akt Is a Novel Cysteine String Protein Kinase That Regulates Exocytosis Release Kinetics and Quantal Size^*

Gareth J. O. Evans12, Jeff W. Barclay1, Gerald R. Prescott13, Sung-Ro Jo, Robert D. Burgoyne, Morris J. Birnbaum, and Alan Morgan4

From the The Physiological Laboratory, School of Biomedical Sciences, University of Liverpool, Liverpool L69 3BX, United Kingdom and the Howard Hughes Medical Institute, The Cox Institute, Department of Medicine, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104

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.

Received for publication, April 4, 2005 , and in revised form, October 19, 2005.

^* This work was funded by research grants from the UK Medical Research Council (to A. M.), by Grant R01-DK56886 from National Institutes of Health (to M. J. B.), and by the Wellcome Trust (to R. D. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ These authors contributed equally to this work.

² Present address: Membrane Biology Group, School of Biomedical & Clinical Laboratory Sciences, Hugh Robson Building, George Square, Edinburgh, EH8 9XD, United Kingdom.

³ GRP is supported by a Wellcome Trust Prize Studentship.

⁴ To whom correspondence should be addressed. Tel.: 44-151-794-5333; Fax: 44-151-794-5337; E-mail: amorgan{at}liv.ac.uk.

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