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

Frederick L. Hall, Jeffrey P. Mitchell, Philip R Vulliet

Research output: Contribution to journalArticle

40 Citations (Scopus)

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 la 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.

Original languageEnglish (US)
Pages (from-to)6944-6948
Number of pages5
JournalJournal of Biological Chemistry
Volume265
Issue number12
StatePublished - 1990

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Proline-Directed Protein Kinases
Synapsins
Phosphorylation
Phosphopeptides
Calcium-Calmodulin-Dependent Protein Kinase Type 2
Phosphates
High performance liquid chromatography
Proline
Serine
High Pressure Liquid Chromatography
Phosphoamino Acids
Calcium-Calmodulin-Dependent Protein Kinases
Peptides
Pheochromocytoma
Collagenases
Electrophoresis
Autoradiography
Sodium Dodecyl Sulfate
Stoichiometry
Trypsin

ASJC Scopus subject areas

  • Biochemistry

Cite this

Phosphorylation of synapsin I at a novel site by proline-directed protein kinase. / Hall, Frederick L.; Mitchell, Jeffrey P.; Vulliet, Philip R.

In: Journal of Biological Chemistry, Vol. 265, No. 12, 1990, p. 6944-6948.

Research output: Contribution to journalArticle

Hall, Frederick L. ; Mitchell, Jeffrey P. ; Vulliet, Philip R. / Phosphorylation of synapsin I at a novel site by proline-directed protein kinase. In: Journal of Biological Chemistry. 1990 ; Vol. 265, No. 12. pp. 6944-6948.
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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 la 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.",
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