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 Xenopus laevis cDNA library. The derived amino acid sequence of the protein contains 337 amino acids and has a calculated molecular mass of 38874 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 DNA with EcoR1 and Hind3, suggesting that X. laevis possesses a family of related CK1 genes. CK1 alpha was expressed in Escherichia coli as a glutathione transferase fusion protein (GT-CK1 alpha) and certain of its characteristics were determined. The recombinant GT-CK1 alpha fusion protein was found to have apparent Km values for ATP (12 microM), casein (1.5 mg/ml) and the specific peptide substrate RRKDLHDDEEDEAMSITA (180 microM) which are similar to those of the rat liver CK1 enzyme. The recombinant CK1 alpha activity is weakly inhibited by heparin, but strongly inhibited by poly(Glu80:Tyr20). This inhibition is competitive and shows an approximate K1 of 5 microM. CK1 alpha can phosphorylate the tyrosine residues of poly(Glu80:Tyr20) and the tyrosine residue in the synthetic peptide RRREEEYEEEE. This kinase preparation also autophosphorylates in serine, threonine and weakly in tyrosine.
Pulgar
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V
Departamento de Bioquímica, Facultad de Medicina, Universidad de Chile, Chile.
Tapia
C
C
Vignolo
P
P
Santos
J
J
Sunkel
C E
CE
Allende
C C
CC
Allende
J E
JE
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D047388:Casein Kinases; D002417:Cattle; D003001:Cloning, Molecular; D005796:Genes; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D019000:Phosphotyrosine / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins; D016415:Sequence Alignment; D013379:Substrate Specificity; D014982:Xenopus laevis / Q000378:metabolism*
D010455:Peptides; D011994:Recombinant Proteins; D019000:Phosphotyrosine; D006493:Heparin; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases; D047388:Casein Kinases