Identification of a novel proline-directed serine/threonine protein kinase in rat pheochromocytoma

Philip R Vulliet, F. L. Hall, J. P. Mitchell, D. G. Hardie

Research output: Contribution to journalArticle

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Abstract

During investigations of the regulation of tyrosine hydroxylase (TH) by protein phosphorylation, a novel protein kinase activity has been discovered in rat pheochromocytoma. Originally detected as a trace contaminant in preparations of highly purified TH at serine 8 in the proline-rich amino-terminal region of the enzyme. This particular site is not phosphorylated by, nor is the amino acid sequence surrounding this site selective for, any of the classical (i.e. well characterized) protein kinases. In this report, we describe the identification, characterization, and partial purification of this novel protein kinase. By utilizing a synthetic peptide corresponding to the amino-terminal region of TH, a selective assay for this protein kinase was developed. The kinase activity utilized ATP and magnesium, although GTP could also be utilized as a phosphate donor. The kinase activity was found to co-purify with TH activity through ammonium sulfate precipitation and DEAE-cellulose chromatography and could be only partially resolved from TH by heparin-agarose affinity chromatography. Substantial kinase activity could be resolved from TH by phosphocellulose chromatography. The novel kinase migrates as a protein with a molecular mass of approximately 45 kDa on gel permeation chromatography as well as sucrose density gradient centrifugation. Studies of site specificity indicate that this Ser/Thr kinase activity appears to be directed by an adjacent (carboxy-terminal) proline residue, exhibiting a minimal recognition sequence of -X-Ser/Thr-Pro-X. In addition to TH, this proline-directed protein kinase will also phosphorylate synapsin, I, histone H1, and glycogen synthase, suggesting that this kinase may have multiple substrates in vivo. Additional findings indicate that the activity of proline-directed protein kinase is increased transiently in PC12 pheochromocytoma cells following treatment with nerve growth factor. Distinctions between this novel kinase and other well characterized protein kinases can be made on the basis of phosphorylation site specificity, chromatographic behavior, and physical characteristics.

Original languageEnglish (US)
Pages (from-to)16292-16298
Number of pages7
JournalJournal of Biological Chemistry
Volume264
Issue number27
StatePublished - 1989

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Proline-Directed Protein Kinases
Protein-Serine-Threonine Kinases
Tyrosine 3-Monooxygenase
Pheochromocytoma
Proline
Rats
Phosphotransferases
Protein Kinases
Phosphorylation
Chromatography
Synapsins
Affinity chromatography
Glycogen Synthase Kinases
DEAE-Cellulose Chromatography
DEAE-Cellulose
Agarose Chromatography
Density Gradient Centrifugation
Centrifugation
PC12 Cells
Ammonium Sulfate

ASJC Scopus subject areas

  • Biochemistry

Cite this

Identification of a novel proline-directed serine/threonine protein kinase in rat pheochromocytoma. / Vulliet, Philip R; Hall, F. L.; Mitchell, J. P.; Hardie, D. G.

In: Journal of Biological Chemistry, Vol. 264, No. 27, 1989, p. 16292-16298.

Research output: Contribution to journalArticle

Vulliet, Philip R ; Hall, F. L. ; Mitchell, J. P. ; Hardie, D. G. / Identification of a novel proline-directed serine/threonine protein kinase in rat pheochromocytoma. In: Journal of Biological Chemistry. 1989 ; Vol. 264, No. 27. pp. 16292-16298.
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