THE RECOMBINANT ALPHA-ISOFORM OF PROTEIN-KINASE CK1 FROM XENOPUS-LAEVIS CAN PHOSPHORYLATE TYROSINE IN SYNTHETIC SUBSTRATES

The cDNA coding for protein kinase CK1 alpha has been cloned from a Xe nopus laevis cDNA library. The derived amino acid sequence of the prot ein contains 337 amino acids and has a calculated molecular mass of 38 874 Da. The sequence is identical to that of the human CK1 alpha and to the bovine CK1 alpha, except that it is 12 amino acids longer than the latter protein. Southern blotting with a 264-bp probe demonstrates that four or more fragments are obtained upon digestion of genomic DN A with EcoR1 and Hind3, suggesting that X. laevis possesses a family o f related CK1 genes. CK1 alpha was expressed in Escherichia coli as a glutathione transferase fusion protein (GT-CK1 alpha) and certain of i ts characteristics were determined. The recombinant GT-CK1 alpha fusio n protein was found to have apparent K-m values for ATP (12 mu M), cas ein (1.5 mg/ml) and the specific peptide substrate RRKDLHDDEEDEAMSITA (180 mu M) which are similar to those of the rat liver CK1 enzyme. The recombinant CK1 alpha activity is weakly inhibited by heparin, but st rongly inhibited by poly(Glu(80):Tyr(20)). This inhibition is competit ive and shows an approximate K-i of 5 mu M. CK1 alpha can phosphorylat e the tyrosine residues of poly(Glu(80):Tyr(20)) and the tyrosine resi due in the synthetic peptide RRREEEYEEEE. This kinase preparation also autophosphorylates in serine, threonine and weakly in tyrosine.