Characterization of synthetic peptide substrates for p34cdc2 protein kinase

Marshak, D. R., Vandenberg, M. T., Bae, Y. S., Yu, I. J. (1991) Characterization of synthetic peptide substrates for p34cdc2 protein kinase. J Cell Biochem, 45 (4). pp. 391-400. ISSN 0730-2312 (Print)0730-2312 (Linking)

URL: http://www.ncbi.nlm.nih.gov/pubmed/2045431
DOI: 10.1002/jcb.240450413

Abstract

Synthetic peptide substrates for the cell division cycle regulated protein kinase, p34cdc2, have been developed and characterized. These peptides are based on the sequences of two known substrates of the enzyme, Simian Virus 40 Large T antigen and the human cellular recessive oncogene product, p53. The peptide sequences are H-A-D-A-Q-H-A-T-P-P-K-K-K-R-K-V-E-D-P-K-D-F-OH (T antigen) and H-K-R-A-L-P-N-N-T-S-S-S-P-Q-P-K-K-K-P-L-D-G-E-Y-NH2 (p53), and they have been employed in a rapid assay of phosphorylation in vitro. Both peptides show linear kinetics and an apparent Km of 74 and 120 microM, respectively, for the purified human enzyme. The T antigen peptide is specifically phosphorylated by p34cdc2 and not by seven other protein serine/threonine kinases, chosen because they represent major classes of such enzymes. The peptides have been used in whole cell lysates to detect protein kinase activity, and the cell cycle variation of this activity is comparable to that measured with specific immune and affinity complexes of p34cdc2. In addition, the peptide phosphorylation detected in mitotic cells is depleted by affinity adsorption of p34cdc2 using either antibodies to p34cdc2 or by immobilized p13, a p34cdc2-binding protein. Purification of peptide kinase activity from mitotic HeLa cells yields an enzyme indistinguishable from p34cdc2. These peptides should be useful in the investigation of p34cdc2 protein kinase and their regulation throughout the cell division cycle.

Item Type: Paper
Uncontrolled Keywords: Amino Acid Sequence CDC2 Protein Kinase/isolation & purification/*metabolism Cell Cycle Cell Separation HeLa Cells Humans Immunoblotting Molecular Sequence Data Peptides/chemical synthesis/*metabolism Phosphorylation Precipitin Tests Substrate Specificity
Subjects: bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > cdc2
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > enzymes > kinase
CSHL Authors:
Communities: CSHL labs
Depositing User: Matt Covey
Date Deposited: 14 Jan 2016 15:40
Last Modified: 14 Jan 2016 15:40
Related URLs:
URI: http://repository.cshl.edu/id/eprint/32055

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