Structure-function analysis of casein kinase 2 with synthetic peptides and anti-peptide antibodies.
Casein kinase 2 (CK2) is a ubiquitous, multifunctional protein-seryl/threonyl kinase that has been implicated in cellular regulation. Synthetic peptides were patterned after three highly conserved regions in CK2: the N terminus (CK2-NT); the lysine-rich, kinase subdomain III segment (CK2-III) (nomenclature of Hanks et al. (Hanks, S. K., Quinn, A. M., and Hunter, T. (1988) Science 241, 42-52)); and a 10-residue segment located near kinase subdomain X that is shared between CK2 and p34cdc2 (CK2/cdc2). The CK2-III and CK2/cdc2 peptides markedly stimulated the autophosphorylation of the alpha- and alpha'-subunits of purified CK2 from sea star oocytes, and they elicited up to 2-fold increases in its casein or phosvitin phosphotransferase activity. These peptides completely reversed nearly total inhibition of CK2 phosphotransferase activity toward itself, casein, and phosvitin by either heparin or poly(Glu,Tyr; 4:1), whereas CK2-NT was ineffective. Elution of CK2 from heparin-agarose with the CK2-III peptide indicated that this region of CK2 might mediate heparin binding to CK2. Affinity-purified rabbit polyclonal antibodies developed against both CK2-III and CK2/cdc2, but not CK2-NT, also produced up to 1.8-fold enhancements of the casein and phosvitin phosphotransferase activities of purified CK2. All three of the antipeptide antibody preparations immunoreacted with the alpha- and alpha'-subunits of CK2 on Western blots. These studies indicate that kinase subdomains III and X are involved in the modulation of CK2 phosphotransferase activity.
Charlton
L A
LA
Biomedical Research Centre, University of British Columbia, Vancouver, Canada.
Sanghera
J S
JS
Clark-Lewis
I
I
Pelech
S L
SL
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D015153:Blotting, Western; D047388:Casein Kinases; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006493:Heparin / Q000494:pharmacology; D036341:Intercellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D010455:Peptides / Q000276:immunology / Q000378:metabolism* / Q000494:pharmacology; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D012615:Sea Anemones; D013329:Structure-Activity Relationship
D000906:Antibodies; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D047428:Protein Kinase Inhibitors; C027606:L-glutamic acid-L-tyrosine copolymer; D006493:Heparin; D010770:Phosphotransferases; D011494:Protein Kinases; D047388:Casein Kinases