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

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, this novel kinase activity phosphorylated 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 (carboxyl-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.

• Authors: P R Vulliet, F L Hall, J P Mitchell, D G Hardie
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 264 Issue 27 Pg. 16292-8 (Sep 25 1989) ISSN: 0021-9258 [Print] United States
• PMID: 2570779 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, Non-P.H.S.)

Chemical References

• Nerve Growth Factors
• Phosphopeptides
• Proline
• Tyrosine 3-Monooxygenase
• Protein Kinases
• Calcium-Calmodulin-Dependent Protein Kinases

Topics

• Adrenal Gland Neoplasms (enzymology)
• Amino Acid Sequence
• Animals
• Calcium-Calmodulin-Dependent Protein Kinases
• Centrifugation, Density Gradient
• Chromatography, Affinity
• Chromatography, High Pressure Liquid
• Molecular Sequence Data
• Molecular Weight
• Nerve Growth Factors (pharmacology)
• Pheochromocytoma (enzymology)
• Phosphopeptides (analysis)
• Phosphorylation
• Proline
• Protein Kinases (isolation & purification, metabolism)
• Rats
• Substrate Specificity
• Tyrosine 3-Monooxygenase (isolation & purification, metabolism)