Characterization of the Schizosaccharomyces pombe Cdk9/Pch1 protein kinase: Spt5 phosphorylation, autophosphorylation, and mutational analysis Journal Article

  

Authors:	Pei, Y.; Shuman, S.
Article Title:	Characterization of the Schizosaccharomyces pombe Cdk9/Pch1 protein kinase: Spt5 phosphorylation, autophosphorylation, and mutational analysis
Abstract:	Schizosaccharomyces pombe Cdk9/Pch1 protein kinase is a functional ortholog of the essential Saccharomyces cerevisiae Bur1/Bur2 kinase and a putative ortholog of metazoan P-TEFb (Cdk9/cyclin T). SpCdk9/Pch1 phosphorylates of the carboxyl-terminal domain (CTD) of the S. pombe transcription elongation factor Spt5, which consists of 18 tandem repeats of a nonapeptide of consensus sequence TPAWNSGSK. We document the divalent cation dependence and specificity of SpCdk9/Pch1, its NTP dependence and specificity, the dependence of Spt5-CTD phosphorylation on the number of tandem nonamer repeats, and the specificity for phosphorylation of the Spt5-CTD on threonine at position 1 within the nonamer element. SpCdk9/Pch1 also phosphorylates the CTD heptaptide repeat array of the largest subunit of S. pombe RNA polymerase II (consensus sequence YSPTSPS) and does so exclusively on serine. SpCdk9/Pch1 catalyzes autophosphorylation of the kinase and cyclin subunits of the kinase complex. The distribution of phosphorylation sites on SpCdk9 (86% Ser(P), 11% Thr(P), 3% Tyr(P)) is distinct from that on Pch1 (2% Ser(P), 98% Thr(P)). We conducted a structure-guided mutational analysis of SpCdk9, whereby a total of 29 new mutations of 12 conserved residues were tested for in vivo function by complementation of a yeast bur1Δ mutant. We identified many lethal and conditional mutations of side chains implicated in binding ATP and the divalent cation cofactor, phosphoacceptor substrate recognition, and T-loop dynamics. We surmise that the lethality of the of T212A mutation in the T-loop reflects an essential phosphorylation event, insofar as the conservative T212S change rescued wild-type growth; the phosphomimetic T212E change rescued growth at 30 °C; and the effects of mutating the T-loop threonine were phenocopied by mutations in the three conserved arginines predicted to chelate the phosphate on the T-loop threonine.
Keywords:	unclassified drug; mutation; nonhuman; protein analysis; chromosomal proteins, non-histone; cyclin dependent kinase 9; serine; carboxy terminal sequence; protein binding; dose-response relationship, drug; enzyme substrate; tyrosine; autophosphorylation; phosphorylation; time factors; rna; enzyme phosphorylation; amino acid sequence; molecular sequence data; sequence homology, amino acid; saccharomyces cerevisiae; rna, messenger; peptides; glutathione transferase; models, genetic; models, molecular; mutagenesis, site-directed; protein structure, tertiary; threonine; binding sites; dna mutational analysis; yeast; adenosine triphosphate; enzyme specificity; cyclins; chemical reactions; enzyme subunit; biochemistry; protein kinase; rna polymerase ii; enzymes; hydrogen-ion concentration; arginine; chelation; schizosaccharomyces; schizosaccharomyces pombe proteins; metazoa; schizosaccharomyces pombe; transcriptional elongation factors; elongation factor; cyclin-dependent kinase 9; saccharomyces; positive ions; cations; adenosinetriphosphate; human; priority journal; article; elongation factor spt5
Journal Title:	Journal of Biological Chemistry
Volume:	278
Issue:	44
ISSN:	0021-9258
Publisher:	American Society for Biochemistry and Molecular Biology
Date Published:	2003-10-31
Start Page:	43346
End Page:	43356
Language:	English
DOI:	10.1074/jbc.M307319200
PUBMED:	12904290
PROVIDER:	scopus
DOI/URL:	http://www.scopus.com/inward/record.url?eid=2-s2.0-0242321980&partnerID=40&md5=f1055e1f5debdae21b139d5926f76ce9
Notes:	Export Date: 12 September 2014 -- Source: Scopus

https://synapse.mskcc.org/synapse/works/has_related_data_chk/59959