Purification and characterization of echinoderm casein kinase II. Regulation by protein kinase C.
Casein kinase II (CKII) is one of several protein kinases that become activated before germinal-vesicle breakdown in maturing sea-star oocytes. Echinoderm CKII was purified over 11,000-fold with a recovery of approximately 10% by sequential fractionation of the oocyte cytosol on tyrosine-agarose, heparin-agarose, casein-agarose and MonoQ. The purified enzyme contained 45, 38 and 28 kDa polypeptides, which corresponded to its alpha, alpha' and beta subunits respectively. The beta-subunit was autophosphorylated on one major tryptic peptide on serine residues, whereas the alpha'-subunit incorporated phosphate into at least two tryptic peptides primarily on threonine residues. Western-blotting analysis of sea-star oocyte extracts with two different anti-peptide antibodies that recognized conserved regions of the alpha-subunit indicated that the protein levels of the alpha- and alpha'-subunits of CKII were unchanged during oocyte maturation. The purified CKII was partly inactivated (by 25%) by preincubation with protein-serine/threonine phosphatase 2A, but protein-tyrosine phosphatases had no effect. The beta-subunit of CKII was phosphorylated on a serine residue(s) up to 0.54 mol of P/mol of beta-subunit by purified protein kinase C, and this correlated with a 1.5-fold enhancement of its phosphotransferase activity with phosvitin as a substrate. CKII was not a substrate for the maturation-activated myelin basic protein kinase p44mpk from sea-star oocytes, nor for cyclic-AMP-dependent protein kinase. These studies point to possible regulation of CKII by protein phosphorylation.
Sanghera
J S
JS
Department of Medicine, University of British Columbia, Vancouver, Canada.
Charlton
L A
LA
Paddon
H B
HB
Pelech
S L
SL
D000818:Animals; D015153:Blotting, Western; D047390:Casein Kinase II; D002845:Chromatography; D003600:Cytosol / Q000201:enzymology; D004447:Echinodermata / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D007700:Kinetics; D009865:Oocytes / Q000201:enzymology* / Q000502:physiology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity
D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D011493:Protein Kinase C