Phosphorylation of synapsin I at a novel site by proline-directed protein kinase.

Abstract

Previous studies identified synapsin I as a potential substrate for a newly discovered growth factor-sensitive, proline-directed protein kinase originally isolated from rat pheochromocytoma. The present study describes the site-specific phosphorylation of synapsin I by highly purified preparations of proline-directed protein kinase. The incorporation of [32P]phosphate into bovine brain synapsin I was dependent upon both the amount of kinase present and the time of incubation. The maximum stoichiometry of phosphorylation approached 1 mol of phosphate/mol of synapsin I protein. When analyzed by sodium dodecyl sulfate-gel electrophoresis and autoradiography, [32P]phosphate was found to be incorporated into both synapsin Ia and Ib. Phosphoamino acid analysis demonstrated that serine residues were phosphorylated exclusively. Digestion of phosphorylated synapsin I with trypsin followed by high performance liquid chromatography (HPLC) phosphopeptide analysis indicated that the tryptic peptide containing the major phosphorylation site eluted as a single peak at approximately 17% acetonitrile. The primary structure of this phosphopeptide, determined by gas-phase sequencing, was found to be Gln-Ser-Arg-Pro-Val-Ala-Gly-Gly-Pro-Gly-Ala-Pro-Pro-Ala-Thr-Arg-Pro-Pro- Ala-Ser-Pro-Ser-Pro-Gln-Arg. Sequential Edman degradation of this HPLC-purified tryptic phosphopeptide revealed that serine 20 of this peptide was the major phosphorylated residue. This phosphoacceptor site is immediately flanked by a carboxyl-terminal proline residue, an observation that further verifies the proline-directed nature of this protein kinase. The tryptic phosphopeptide corresponds exactly to a sequence in the collagenase-sensitive, proline-rich "tail" region of bovine synapsin I. This novel phosphorylation site is close to but distinct from phosphorylation sites 2 and 3, which are known to be phosphorylated by calcium/calmodulin-dependent protein kinase II and are considered to be of regulatory importance.