Identification and characterization of protein kinase CKII isoforms in HeLa cells. Isoform-specific differences in rates of assembly from catalytic and regulatory subunits.
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.
Chester
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W. M. Keck Structural Biology Laboratory, Arnold and Mabel Beckman Neuroscience Center, Cold Spring Harbor Laboratory, New York 11724, USA.
Yu
I J
IJ
Marshak
D R
DR
D000595:Amino Acid Sequence; D000906:Antibodies; D047390:Casein Kinase II; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D013379:Substrate Specificity
D000906:Antibodies; D007527:Isoenzymes; D046911:Macromolecular Substances; D010455:Peptides; D008715:Methionine; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases