Cloning of the pka1 gene encoding the catalytic subunit of the cAMP-dependent protein kinase in Schizosaccharomyces pombe

T Maeda,Y Watanabe,H Kunitomo,M Yamamoto

doi:10.1016/s0021-9258(17)36928-4

Abstract

We have isolated Schizosaccharomyces pombe genes that confer sterility to the fission yeast cell when expressed from a multicopy plasmid. One of these genes strongly hybridized to a probe carrying the open reading frame of Saccharomyces cerevisiae TPK1, which encodes a catalytic subunit of the cAMP-dependent protein kinase (protein kinase A). This S. pombe gene, named pka1, has a coding potential of 512 amino acids, and the deduced gene product is 60% identical with the S. cerevisiae Tpk1 protein in the C-terminal 320 amino acids. Disruption of pka1 slows cell growth but is not lethal. The resultant cells, however, are highly derepressed for sexual development, readily undergoing conjugation and sporulation in the absence of nitrogen starvation. They are, thus, phenotypically indistinguishable from the adenylyl cyclase-defective (cyr1-) cells previously characterized, except that the pka1- spores are retarded in germination, whereas the cyr1- spores are not. Disruption of pka1 is epistatic to a defect in cgs1, which encodes the regulatory subunit of protein kinase A. These results strongly suggest that the product of pka1 is a catalytic subunit of protein kinase A and, furthermore, that S. pombe has only one gene encoding it. This situation contrasts with the case of S. cerevisiae, in which three genes encode the catalytic subunits.

Highlights

Theaboveresultsleadto a conclusion that activation of proteinkinase A isinhibitory,whereasinactivation of it is namedp k a l, has a coding potentiaolf 512 amino acids, stimulatory for theinitiation of sexualdevelopmentin S
These results strongly suggest that the product of control it according to the nutritional conditions [10].We s. p k a l is a catalytic subunitof protein kinaseA and, fur- showed that a decrease in the intracellular cAMPlevel, and thermore, that pombe has only one gene encodingit. in the protein kinaseA activity, results in indoufctthioen
The TPKl probe generated several faint hybridization bands in a long exposure, which may represent only distantly related kinasegenes (Fig.4B').All of these observations lead to the notion that p k a l is the only gene encoding the catalytic subunit of protein kinase A in S. pombe

Summary

RESULTS

TPKl ORF was thusamplified and recovered from an agarose gel after separation by electrophoresis. S. pombe genomic DNA was digestedwith either EcoRI, HindIII, or BamHI and analyzed by Southern blotting under low stringency conditions using part of the pkal ORF sequence as a probe. The TPKl probe generated several faint hybridization bands in a long exposure, which may represent only distantly related kinasegenes (Fig.4B').All of these observations lead to the notion that p k a l is the only gene encoding the catalytic subunit of protein kinase A in S. pombe. Mutants defective in cgsl are sterile because of the constitutive activation of protein kinase A.We constructed a strain inwhich both cgsl andpkal are disrupted The phenotype of this strain is exactly the same as the pkal disruptant (data not shownE)x.istence of the null cgsl allele in this strainwas proven by genetic and Southern analysis.

DISCUSSION

This study predicts that the Pkal protein bears an extra

Whether this

We also thank Takako Isshiki and Yukako Iwahara for assistance in