The product of the KIN1 locus in Saccharomyces cerevisiae is a serine/threonine-specific protein kinase.
The catalytic domain (30 kDa) of all protein kinases can be aligned for maximum homology, thereby revealing both invariant and highly conserved residues. The KIN1 locus from Saccharomyces cerevisiae was isolated by hybridization to a degenerate oligonucleotide encoding the conserved protein kinase domain, DVWSFG. The predicted amino acid sequence revealed significant homology to the catalytic domain of protein kinases. Using antibodies raised against a bacterial LacZ/KIN1 fusion protein, we have identified by immunoprecipitation the yeast KIN1 gene product as a 145,000 dalton protein (p145KIN1). In exponentially growing yeast cells, the KIN1 protein is phosphorylated primarily on serine residues. The gene product of KIN1 was shown to be a serine/threonine-specific protein kinase in immune complexes, as determined by the transfer of label from [gamma-32P]ATP to either pp145KIN1 or to an exogenously added substrate, alpha-casein. The optimal metal ion concentration in this assay was 20 mM-MnCl2. Subsequent phosphoamino acid analysis of the radiolabelled product, pp145KIN1, demonstrated that this autophosphorylation was specific for serine/threonine residues. There is no apparent difference between wild-type cells and cells containing a disrupted KIN1 gene. The biochemical characterization of protein kinases in simple eukaryotes such as yeast will aid us in determining the role of phosphorylation in cellular growth and physiology.
Lamb
A
A
Department of Molecular Biology and Biochemistry, Wesleyan University, Middletown, Connecticut 06457.
Tibbetts
M
M
Hammond
C I
CI
D000595:Amino Acid Sequence; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D003720:Densitometry; D004591:Electrophoresis, Polyacrylamide Gel; D007106:Immune Sera / Q000276:immunology; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011233:Precipitin Tests; D011494:Protein Kinases / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*
D007106:Immune Sera; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases