Molecular cloning and enzymatic analysis of the rat homolog of "PhK-gamma T," an isoform of phosphorylase kinase catalytic subunit.
Messenger RNA encoding a protein kinase closely related to the catalytic subunit of skeletal muscle phosphorylase kinase has previously been isolated from a human HeLa cell cDNA library, and cross-species Northern hybridization analysis has shown that the rat homolog of this transcript is abundant in the adult testis (Hanks, S.K. (1989) Mol. Endocrinol. 3, 110-116). We now propose that the protein encoded by this transcript be designated as "PhK-gamma T." In this article, the primary structure of the rat homolog of PhK-gamma T is described, as deduced from nucleotide sequences of cDNA and genomic clones. RNase protection analysis reveals that PhK-gamma T transcripts are actually present in a wide variety of adult rat tissues, but at levels 20-100-fold less than what is observed in the testis. In the testis, transcription of the PhK-gamma T gene is initiated at multiple sites as shown by RNase protection and primer extension. Enzymatic activity of PhK-gamma T was demonstrated using renatured bacterially expressed protein. In the presence of Ca2+/calmodulin, PhK-gamma T is able to efficiently phosphorylate glycogen phosphorylase and convert it from an inactive to an active form. We conclude that PhK-gamma T represents a true isoform of phosphorylase kinase catalytic subunit.
Calalb
M B
MB
Department of Cell Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
Fox
D T
DT
Hanks
S K
SK
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002467:Cell Nucleus / Q000502:physiology; D003001:Cloning, Molecular / Q000379:methods; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D015723:Gene Library; D006801:Humans; D007527:Isoenzymes / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D008297:Male; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology; D009838:Oligodeoxyribonucleotides; D010764:Phosphorylase Kinase / Q000235:genetics* / Q000378:metabolism*; D011061:Poly A / Q000235:genetics / Q000302:isolation & purification; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D015347:RNA Probes; D012333:RNA, Messenger; D051381:Rats; D011919:Rats, Inbred Strains; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D013737:Testis / Q000201:enzymology*; D014158:Transcription, Genetic
D007527:Isoenzymes; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D015347:RNA Probes; D012333:RNA, Messenger; D011994:Recombinant Proteins; D011061:Poly A; D012313:RNA; D004247:DNA; D010764:Phosphorylase Kinase