Characterization of the KIN2 gene product in Saccharomyces cerevisiae and comparison between the kinase activities of p145 KIN1 and p145 KIN2

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 (pp145 KIN1 ) 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 β‐galactosidase/KIN2 fusion polypeptide. In vivo , the KIN2 gene product is a 145 kDa phosphoprotein, pp145 KIN2 . In immune complexes, pp145 KIN2 demonstrates serine/threonine protein kinase activity, transferring phosphate from [γ‐ 32 P]ATP to either itself or the exogenously added substrates α‐casein, acid‐denatured enolase, or phosvitin. In vitro , kinase activity is dependent on either Mn 2+ or Mg 2+ ions. Both enzymes, pp145 KIN1 and pp145 KIN2 , 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 pp145 KIN1 or pp145 KIN2 on tyrosine residues. Both enzymes phosphorylate α‐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 GLU 80 TYR 20 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 .