Phosphorylation of cysteine string protein by protein kinase A. Implications for the modulation of exocytosis

G.J.O. Evans; M.C. Wilkinson; M.E. Graham; K.M. Turner; L.H. Chamberlain; A. Morgan; R.D. Burgoyne

doi:10.1074/jbc.M108186200

Phosphorylation of cysteine string protein by protein kinase A. Implications for the modulation of exocytosis

G.J.O. Evans, M.C. Wilkinson, M.E. Graham, K.M. Turner, L.H. Chamberlain, A. Morgan, R.D. Burgoyne

Research output: Contribution to journal › Article › peer-review

Abstract

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- (-SNAP) were phosphorylated by PKA in vitro, but immunoprecipitation of cellular -SNAP failed to detect 32P 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 Ser10. 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 Ser10 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.

Original language	English
Pages (from-to)	47877-47885
Number of pages	8
Journal	Journal of Biological Chemistry
Volume	276
Issue number	51
DOIs	https://doi.org/10.1074/jbc.M108186200
Publication status	Published - Dec 2001

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10.1074/jbc.M108186200

http://dx.doi.org/10.1074/jbc.M108186200

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@article{f79ce528241b4d7982b84f978ad20199,

title = "Phosphorylation of cysteine string protein by protein kinase A. Implications for the modulation of exocytosis",

abstract = "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- (-SNAP) were phosphorylated by PKA in vitro, but immunoprecipitation of cellular -SNAP failed to detect 32P 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 Ser10. 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 Ser10 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.",

author = "G.J.O. Evans and M.C. Wilkinson and M.E. Graham and K.M. Turner and L.H. Chamberlain and A. Morgan and R.D. Burgoyne",

year = "2001",

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journal = "Journal of Biological Chemistry",

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publisher = "American Society for Biochemistry and Molecular Biology Inc.",

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TY - JOUR

T1 - Phosphorylation of cysteine string protein by protein kinase A. Implications for the modulation of exocytosis

AU - Evans, G.J.O.

AU - Wilkinson, M.C.

AU - Graham, M.E.

AU - Turner, K.M.

AU - Chamberlain, L.H.

AU - Morgan, A.

AU - Burgoyne, R.D.

PY - 2001/12

Y1 - 2001/12

N2 - 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- (-SNAP) were phosphorylated by PKA in vitro, but immunoprecipitation of cellular -SNAP failed to detect 32P 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 Ser10. 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 Ser10 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.

AB - 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- (-SNAP) were phosphorylated by PKA in vitro, but immunoprecipitation of cellular -SNAP failed to detect 32P 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 Ser10. 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 Ser10 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.

U2 - 10.1074/jbc.M108186200

DO - 10.1074/jbc.M108186200

M3 - Article

SN - 0021-9258

VL - 276

SP - 47877

EP - 47885

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

IS - 51

ER -