Characterization of the KIN2 gene product in Saccharomyces cerevisiae and comparison between the kinase activities of p145KIN1 and p145KIN2.
We have isolated two yeast genes, KIN1 and KIN2, by their homology to the protein kinase family of viral oncogenes. Previous studies have identified the yeast KIN1 gene product (pp145KIN1) as a 145 kilodalton (kDa) phosphoprotein with serine/threonine-specific protein kinase activity. To identify and biochemically characterize the KIN2 gene product, antibodies were raised against a bacterial beta-galactosidase/KIN2 fusion polypeptide. In vivo, the KIN2 gene product is a 145 kDa phosphoprotein, pp145KIN2. In immune complexes, pp145KIN2 demonstrates serine/threonine protein kinase activity, transferring phosphate from [gamma-32P]ATP to either itself or the exogenously added substrates alpha-casein, acid-denatured enolase, or phosvitin. In vitro, kinase activity is dependent on either Mn2+ or Mg2+ ions. Both enzymes, pp145KIN1 and pp145KIN2, prefer ATP over GTP as their phosphoryl donor. Since a new class of yeast protein kinases has been identified which are serine/tyrosine-specific, we analysed a wide range of substrates to see if any could be phosphorylated by pp145KIN1 or pp145KIN2 on tyrosine residues. Both enzymes phosphorylate alpha-casein, acid-denatured enolase, and phosvitin on serine and threonine residues. Neither enzyme could phosphorylate tyrosine residues even though good substrates for tyrosine-specific kinases such as enolase, angiotensin II, and the synthetic polymer GLU80TYR20 were used. The biochemical analysis of KIN2 kinase activity shows remarkable similarity to that of its most closely related yeast kinase, KIN1. It remains to be seen if these two yeast protein kinases share any functional relationships or substrates in vivo.
Donovan
M
M
Department of Molecular Biology and Biochemistry, Wesleyan University, Middletown, Connecticut 06459-0175.
Romano
P
P
Tibbetts
M
M
Hammond
C I
CI
D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D005800:Genes, Fungal; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D015688:Oncogene Protein pp60(v-src) / Q000737:chemistry; D010750:Phosphoproteins / Q000235:genetics* / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D005656:Fungal Proteins; D008565:Membrane Proteins; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D015688:Oncogene Protein pp60(v-src); C490209:KIN1 protein, S cerevisiae; C087572:KIN2 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases