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: 4.6). 02/2006; 281(3):1564-72. DOI: 10.1074/jbc.M503628200
Source: PubMed

ABSTRACT 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.

Download full-text


Available from: Gareth J O Evans, Jul 06, 2015
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Exocytosis of neurotransmitter containing vesicles supports neuronal communication. The importance of molecular interactions involving specific lipids has become progressively more evident and the lipid composition of both the synaptic vesicle and the pre-synaptic plasma membrane at the active zone has significant functional consequences for neurotransmitter release. Several classes of lipids have been implicated in exocytosis including polyunsaturated fatty acids and phosphoinositides. This minireview will focus on recent developments regarding the role of phosphoinositides in neurosecretion.
    Journal of Neurochemistry 08/2006; 98(2):336-42. DOI:10.1111/j.1471-4159.2006.03892.x · 4.24 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: In cultured bovine adrenal chromaffin cells, where Akt1 is the predominant isoform over Akt2 and Akt3, chronic (≥12 h) treatment with 1–20 mM LiCl, an inhibitor of glycogen synthase kinase-3, decreased Akt1 level by ~ 52% (EC 50 = 3.7 mM; t 1/2 = l2 h); it was associated with LiCl-induced increased levels of Ser 9 -phosphorylated glycogen synthase kinase-3β (~ 37%) and β-catenin (~ 59%), two hallmarks of glycogen synthase kinase-3β 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 ~67% (EC 50 = 2 μM; t 1/2 = l2 h), when SB216763 caused concentration-and time-dependent increase of β-catenin level by ~ 76%. LiCl-or SB216763-induced Akt1 decrease, as well as increases of Ser 9 -phosphorylated glycogen synthase kinase-3β and β-catenin were restored to the control levels of nontreated cells after the washout of LiCl (20 mM for 24 h)-or SB216763 (30 μM for 24 h)-treated cells. LiCl-induced Akt1 reduction was not prevented by β-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-3β inhibition caused down-regulation of Akt1 mRNA and Akt1 protein levels; conversely, constitutive activity of glycogen synthase kinase-3β maintains steady-state level of Akt1 in quiescent adrenal chromaffin cells.
  • The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 09/2006; 26(31):8021-2. DOI:10.1523/JNEUROSCI.2497-06.2006 · 6.75 Impact Factor