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The product of the KIN1 locus in Saccharomyces cerevisiae is a serine/threonine‐specific protein kinase
Author(s) -
Lamb Allen,
Tibbetts Michael,
Hammond Charlotte I.
Publication year - 1991
Publication title -
yeast
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.923
H-Index - 102
eISSN - 1097-0061
pISSN - 0749-503X
DOI - 10.1002/yea.320070304
Subject(s) - autophosphorylation , biology , biochemistry , saccharomyces cerevisiae , casein kinase 2 , serine , protein kinase domain , threonine , protein kinase a , kinase , phosphorylation , microbiology and biotechnology , yeast , gene , mitogen activated protein kinase kinase , mutant
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 (p145 KIN1 ). 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 detrmined by the transfer of label from [γ‐ 32 P]ATP to either pp145 KIN1 or to an exogenously added substrate, α‐casein. The optimal metal ion concentration in this assay was 20 m M ‐MnCl 2 . Subsequent phosphoamino acid analysis of the radiolabelled product, pp145 KIN1 , 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 detrmining the role of phosphorylation in cellular growth and physiology.