Chester, N., Yu, I. J., Marshak, D. R. (March 1995) Identification and characterization of protein kinase CKII isoforms in HeLa cells. Isoform-specific differences in rates of assembly from catalytic and regulatory subunits. J Biol Chem, 270 (13). pp. 7501-14. ISSN 0021-9258 (Print)
Abstract
Protein kinase CKII (formerly casein kinase II) can be isolated as a heterotetramer, containing two catalytic (alpha or alpha') and two regulatory (beta) subunits. We have characterized the forms of CKII in HeLa cells using antibodies specific for the alpha or alpha' subunits. Following metabolic labeling with [35S]methionine, whole cell soluble extracts were analyzed by immunoprecipitation and gel electrophoresis. Both alpha and alpha' coprecipitate with beta and with each other. However, when extracts are depleted of alpha, a pool of CKII containing only alpha' and beta is identified. Similarly, depletion of alpha' revealed a pool exclusively of alpha and beta. Therefore, we propose that there are three distinct isoforms of CKII within HeLa cells with different catalytic subunit stoichiometries (alpha 2 beta 2, alpha alpha' beta 2, and alpha' 2 beta 2). With our immunodepletion procedure we have characterized the isoforms by activity analysis, turnover of pulse-labeled subunits, and by localization in subcellular fractions obtained from labeled cells. We have also analyzed complex formation between the catalytic and regulatory subunits by examining the differences in the rate of signal incorporation into subunits in immunoprecipitates obtained from continuously labeled and pulse-labeled cells. We have found that the alpha 2 beta 2 and alpha alpha' beta 2 isoforms assemble relatively slowly (12-16 h), whereas complex formation of the alpha' 2 beta 2 isoform occurs more rapidly (2-4 h). Analysis of isoform complex formation in subcellular fractions from pulse-labeled cells revealed that the majority of nuclear CKII is assembled in the nucleus from free catalytic and regulatory subunit polypeptides.
| Item Type: | Paper |
|---|---|
| Uncontrolled Keywords: | Amino Acid Sequence Antibodies Casein Kinase II Comparative Study Hela Cells Humans Immunoblotting Isoenzymes/chemistry/ isolation & purification/ metabolism Kinetics Macromolecular Substances Methionine/metabolism Molecular Sequence Data Molecular Weight Peptides/chemical synthesis/metabolism Protein Processing, Post-Translational Protein-Serine-Threonine Kinases/chemistry/ isolation & purification/ metabolism Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S. Substrate Specificity |
| Subjects: | bioinformatics > genomics and proteomics > design > amino acid design bioinformatics > genomics and proteomics > design > protein network design > peptide design bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > antibodies organism description > animal > mammal > primates > hominids > human |
| CSHL Authors: | |
| Communities: | CSHL labs |
| Depositing User: | Jessica Koos |
| Date: | 31 March 1995 |
| Date Deposited: | 14 Aug 2014 16:29 |
| Last Modified: | 14 Aug 2014 16:29 |
| URI: | https://repository.cshl.edu/id/eprint/30576 |
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