Phosphorylation of cysteine string protein by protein kinase A. Implications for the modulation of exocytosis.
Cyclic AMP-dependent protein kinase (PKA) enhances regulated exocytosis in neurons and most other secretory cells. To explore the molecular basis of this effect, known exocytotic proteins were screened for PKA substrates. Both cysteine string protein (CSP) and soluble NSF attachment protein-alpha (alpha-SNAP) were phosphorylated by PKA in vitro, but immunoprecipitation of cellular alpha-SNAP failed to detect (32)P incorporation. In contrast, endogenous CSP was phosphorylated in synaptosomes, PC12 cells, and chromaffin cells. In-gel kinase assays confirmed PKA to be a cellular CSP kinase, with phosphorylation occurring on Ser(10). PKA phosphorylation of CSP reduced its binding to syntaxin by 10-fold but had little effect on its interaction with HSC70 or G-protein subunits. Furthermore, an in vivo role for Ser(10) phosphorylation at a late stage of exocytosis is suggested by analysis of chromaffin cells transfected with wild type or non-phosphorylatable mutant CSP. We propose that PKA phosphorylation of CSP could modulate the exocytotic machinery, by selectively altering its availability for protein-protein interactions.
Evans
G J
GJ
Physiological Laboratory and School of Biological Sciences, University of Liverpool, Crown Street, Liverpool L69 3BX, United Kingdom.
Wilkinson
M C
MC
Graham
M E
ME
Turner
K M
KM
Chamberlain
L H
LH
Burgoyne
R D
RD
Morgan
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D017931:DNA Primers; D005089:Exocytosis; D050956:HSP40 Heat-Shock Proteins; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D016716:PC12 Cells; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D050765:Qa-SNARE Proteins; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000378:metabolism
D017931:DNA Primers; D050956:HSP40 Heat-Shock Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D050765:Qa-SNARE Proteins; D011994:Recombinant Proteins; C088460:cysteine string protein; D012694:Serine; D017868:Cyclic AMP-Dependent Protein Kinases