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-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.
Highlights
Cyclic AMP-dependent protein kinase (PKA) enhances regulated exocytosis in neurons and most other secretory cells
We found that ␣-SNAP is phosphorylated by PKA, in agreement with Ref. 24, and that cysteine string protein (CSP) is a novel PKA substrate
Under conditions optimized for maximal phosphorylation, it was found that phosphorylation of CSP by PKA plateaued after 60 min at 30 °C at a stoichiometry of ϳ1.0 mol of phosphate/mol of protein (Fig. 1B). ␣-SNAP by comparison was phosphorylated to a lesser extent by PKA with a stoichiometry of only ϳ0.6 mol of phosphate/mol of protein (Fig. 1B)
Summary
To explore the molecular basis of this effect, known exocytotic proteins were screened for PKA substrates. As regulated exocytosis is the basis of chemical transmission in the brain, much research has been devoted to uncovering its molecular mechanism This has revealed the involvement of a large number of proteins [2, 3], which can be classified into three groups. The third class can be defined as proteins whose role in regulated exocytosis is cell type-specific An example from this group is the synapsins, which are important modulators of the synaptic vesicle cycle in neurons [4]. Identification of PKA or PKC exocytotic substrates will reveal fundamental mechanisms for the direct regulation of exocytosis by phosphorylation To address this issue, our approach was to screen known exocytotic proteins for in vitro kinase substrates. PKA may enhance exocytosis by changing the protein-protein interactions of CSP
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