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

Gareth J.O Evans,Jeff W Barclay,Gerald R Prescott,Sung-Ro Jo,Robert D Burgoyne,Morris J Birnbaum,Alan Morgan

doi:10.1074/jbc.m503628200

Gareth J.O Evans, Jeff W Barclay + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m503628200

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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.

Highlights

Exocytosis is the fusion of secretory vesicles with the plasma membrane
To determine which Akt isoforms are expressed in bovine adrenal chromaffin cells, cell lysates were run on SDS-PAGE alongside recombinant Akt protein standards and Western blotted with isoform-specific Akt antibodies (Fig. 1A)
The existing evidence linking Akt to the regulation of exocytosis is mainly based on studies of insulin-stimulated glucose transporter 4 (GLUT4) translocation

Summary

EXPERIMENTAL PROCEDURES

Materials—Plasmids encoding wild-type and mutant constructs of CSP (pQE30-CSP1, pQE30-CSP1 (S10A), pcDNA3-myc-CSP1, and pcDNA3-myc-CSP1(S10A)) and Akt (pLNCX1-HA-AKT1, pLNCX1HA-myr-AKT1, and pcDNA3-HA-AKT AAA) have been previously described [17, 18, 23, 26, 30]. Recombinant Protein Purification and in Vitro Phosphorylation Assays—Recombinant His6-CSP was expressed and purified as previously described [32]. In vitro phosphorylation of His6-CSP was performed in 50 mM Tris, pH 7.5, 20 mM MgCl2, 1 mM EGTA, 15 mM dithiothreitol, 0.25 mM NaVO43Ϫ, 0.03% Brij-35 for Akt, and in 50 mM MES, pH 6.9, 10 mM MgCl2, 0.5 mM EDTA, 1 mM dithiothreitol for PKA In both cases, 5 ␮g/ml purified kinase was added, and the reaction was initiated by the addition of 2 ␮Ci of [␥-32P]ATP and unlabeled ATP to a final concentration of 100 ␮M. Primary antibodies (mouse anti-HA-tag, sheep anti-CSP, or rabbit antiphospho-CSP) were applied for 2 h at room temperature, and the cells were washed three times in phosphate-buffered saline. Secondary antibodies were incubated for 1 h followed by washing three times in phosphate-buffered saline; streptavidin-fluorophore was added for 30 min. Catecholamine release was assayed using a fluorometric assay and expressed as the percentage of total cellular catecholamine

RESULTS

Quantal size

DISCUSSION

No Akt