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

The Physiological Laboratory, School of Biomedical Sciences, University of Liverpool, Liverpool L69 3BX, United Kingdom.
Journal of Biological Chemistry (Impact Factor: 4.57). 02/2006; 281(3):1564-72. DOI: 10.1074/jbc.M503628200
Source: PubMed


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.

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    • "The latter event resulted in increased synaptic strength via a direct phosphorylation of the GABAA receptor by Akt in response to insulin. Akt can also regulate the docking or fusion of vesicles in several secretory cells (27–30). However, our work highlights the absence of a role for Akt in SV exocytosis at the presynapse, with overexpression of constitutively active Akt leaving both SV turnover and release kinetics unaltered. "
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    Traffic 04/2012; 13(7):1004-11. DOI:10.1111/j.1600-0854.2012.01365.x · 4.35 Impact Factor
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    • "In adrenal chromaffin cells, Evans et al. (2006) documented that nicotinic receptor-induced Ca 2+ influx via voltage-dependent calcium channel caused Akt phosphorylation, increasing catecholamine exocytosis. In pancreatic β-cells, glucose-induced insulin exocytosis required Akt (Bernal-Mizrachi et al., 2004). "
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    ABSTRACT: In cultured bovine adrenal chromaffin cells, where Akt1 is the predominant isoform over Akt2 and Akt3, chronic (> or =12 h) treatment with 1-20 mM LiCl, an inhibitor of glycogen synthase kinase-3, decreased Akt1 level by approximately 52% (EC50=3.7 mM; t1/2=l2 h); it was associated with LiCl-induced increased levels of Ser9-phosphorylated glycogen synthase kinase-3beta (approximately 37%) and beta-catenin (approximately 59%), two hallmarks of glycogen synthase kinase-3beta inhibition. The same LiCl treatment did not change phosphoinositide 3-kinase, phosphoinositide-dependent kinase 1, and extracellular signal-regulated kinase-1/2 levels. Treatment with SB216763 [3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione], a selective inhibitor of glycogen synthase kinase-3, lowered Akt1 level by approximately 67% (EC50=2 microM; t1/2=l2 h), when SB216763 caused concentration- and time-dependent increase of beta-catenin level by approximately 76%. LiCl- or SB216763-induced Akt1 decrease, as well as increases of Ser9-phosphorylated glycogen synthase kinase-3beta and beta-catenin were restored to the control levels of nontreated cells after the washout of LiCl (20 mM for 24 h)- or SB216763 (30 microM for 24 h)-treated cells. LiCl-induced Akt1 reduction was not prevented by beta-lactone, lactacystin (two inhibitors of proteasome), calpastatin (an inhibitor of calpain), or leupeptin (an inhibitor of lysosome). LiCl decreased Akt1 mRNA level by 20% at 6 h, with no effect on Akt1 mRNA stability. These results suggest that glycogen synthase kinase-3beta inhibition caused down-regulation of Akt1 mRNA and Akt1 protein levels; conversely, constitutive activity of glycogen synthase kinase-3beta maintains steady-state level of Akt1 in quiescent adrenal chromaffin cells.
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    • "PKB/Akt acts downstream of PI3K in a number of signalling events. Interestingly, PI3K inhibitors only partially affected PKB/Akt activity in this system, which might suggest a role for PI3K-C2a (Evans et al. 2006). PtdIns(3)P may also play a part in another exocytotic process, the trafficking of glucose transporter 4 (GLUT4)- containing vesicles to the plasma membrane (Maffucci et al. 2003; Ishiki et al. 2005). "
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