T-loop phosphorylation stabilizes the CDK7-cyclin H-MAT1 complex in vivo and regulates its CTD kinase activity

Larochelle, Stéphane; Chen, Jian; Knights, Ronald; Pandur, Judit; Morcillo, Patrick; Erdjument‐Bromage, Hediye; Tempst, Paul; Suter, Beat; Fisher, Robert P

doi:10.1093/emboj/20.14.3749

Cited by 133 publications

(153 citation statements)

References 42 publications

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“…This suggests that the Pmh1 immunoprecipitation could contain other fission yeast TFIIH subunits [38]. These observations together with the demonstration that T-loop phosphorylation of Mcs6 is not essential [6] are consistent with a model where association of Pmh1 with the Mcs6 CDK and Mcs2 cyclin alleviates the requirement of T-loop phosphorylation as observed in other species [8,9,21,42], and that Pmh1 represents the Mat1 homolog in fission yeast.…”

Section: Pmh1 Is the Mat1 Homolog Of Fission Yeast And Activates The supporting

confidence: 85%

Mcs2 and a novel CAK subunit Pmh1 associate with Skp1 in fission yeast

Bamps

Westerling

Pihlak

et al. 2004

Biochemical and Biophysical Research Communications

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The Mcs6 CDK together with its cognate cyclin Mcs2 represents the CDK-activating kinase (CAK) of fission yeast Cdc2. We have attempted to determine complexes in which Mcs6 and Mcs2 mediate this and possible other functions. Here we characterize a novel interaction between Mcs2 and Skp1, a component of the SCF (Skp1-Cullin-F box protein) ubiquitin ligase. Furthermore, we identify a novel protein termed Pmh1 through its association with Skp1. Pmh1 associates with the Mcs6-Mcs2 complex, enhancing its kinase activity, and represents the apparent homolog of metazoan Mat1. Association of Mcs2 or Pmh1 with Skp1 does not appear to be involved in proteolytic degradation, as these complexes do not contain Pcu1, and levels of Mcs2 or Pmh1 are not sensitive to inhibition of SCF and the 26S proteasome. The identified interactions between Skp1 and two regulatory CAK subunits may reflect a novel mechanism to modulate activity and specificity of the Mcs6 kinase.

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Section: Pmh1 Is the Mat1 Homolog Of Fission Yeast And Activates The supporting

confidence: 85%

Mcs2 and a novel CAK subunit Pmh1 associate with Skp1 in fission yeast

Bamps

Westerling

Pihlak

et al. 2004

Biochemical and Biophysical Research Communications

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“…In the case of yeast Kin28(Cdk7), changing the T-loop threonine to either Ala, Asp, Val, Glu, or Gln had no impact on cell growth, indicating that T-loop phosphorylation was not essential for this Cdk (41). Mutating the Drosophila Cdk7 T-loop threonine to alanine did not affect viability of the organism at 18 or 25°C but did cause lethality at 29°C (46).…”

Section: Discussionmentioning

confidence: 99%

“…Although many of the side chains that contact the nucleotide or divalent cation are essential for function, there are exceptions (e.g. Thr 46 in SpCdk9). Our findings that Arg 221 is important for SpCdk9 function provides evidence in support of the structural data implicating Cdk2 Arg 169 in the formation of a proline-specific substrate binding pocket (33).…”

Section: Discussionmentioning

confidence: 99%

Characterization of the Schizosaccharomyces pombe Cdk9/Pch1 Protein Kinase

Pei

Shuman

2003

Journal of Biological Chemistry

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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 1 TPAWNSGSK 9 . 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 bur1⌬ 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.mRNA synthesis by RNA polymerase (pol) 1 II is regulated by phosphorylation of several of the protein components of the transcription apparatus (1). Phosphorylation of the carboxylterminal domain (CTD) of the largest subunit of pol II has been the focus of much attention because the CTD acts as an essential scaffold for the binding of macromolecular assemblies that regulate mRNA synthesis and cotranscriptional mRNA processing (2). The pol II CTD is composed of a heptapeptide motif of consensus sequence YSPTSPS repeated in tandem. The mammalian pol II CTD has 52 heptad repeats, the fission yeast Schizosaccharomyces pombe CTD has 29 repeats, and the budding yeast Saccharomyces cerevisiae CTD has 26 copies. The pol II CTD undergoes a cycle of extensive phosphorylation and dephosphorylation at positions Ser 5 and Ser 2 , which is coordinated with the transcription cycle (3). Multiple CTD kinases are present in eukaryotic cells, e.g. budding yeast has four CTD kinases, two of which (Kin28 and Bur1) are essential (3). CTD kinases are heteromeric enzymes consisting of a cyclin subunit plus a cyclin-dependent kinase (Cdk) subun...

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“…The kinase responsible for mitotic TFIIH inactivation remains to be identi®ed, but the CDC2-cyclin B complex (MPF) and/or a kinase acting downstream of MPF are likely candidates (Long et al, 1998;Garrett et al, 2001). Interestingly, and in contrast to TFIIH-associated CAK, both S164 phosphorylation and T170 phosphorylation by CDC2 stabilize the free trimeric CAK complex (Larochelle et al, 2001), and there is evidence for a positive feedback loop by reciprocal T-loop phosphorylation of CDC2 and CDK7 Garrett et al, 2001). Thus, CDK7 phosphorylation by CDKs appears to differentially affect free CAK complexes and CAK associated with higher-order protein complexes such as TFIIH.…”

Section: Cdks Associated With the Rnap II Transcription Machinerymentioning

confidence: 99%

Regulation of RNA polymerase II activity by CTD phosphorylation and cell cycle control

Oelgeschläger

2002

Journal Cellular Physiology

128

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The carboxyl-terminal domain (CTD) of the largest subunit of mammalian RNA polymerase II (RNAP II) consists of 52 repeats of a consensus heptapeptide and is subject to phosphorylation and dephosphorylation events during each round of transcription. RNAP II activity is regulated during the cell cycle and cell cycledependend changes in RNAP II activity correlate well with CTD phosphorylation. In addition, global changes in the CTD phosphorylation status are observed in response to mitogenic or cytostatic signals such as growth factors, mitogens and DNA-damaging agents. Several CTD kinases are members of the cyclindependent kinase (CDK) superfamily and associate with transcription initiation complexes. Other CTD kinases implicated in cell cycle regulation include the mitogen-activated protein kinases ERK-1/2 and the c-Abl tyrosine kinase. These observations suggest that reversible RNAP II CTD phosphorylation may play a key role in linking cell cycle regulatory events to coordinated changes in transcription.

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T-loop phosphorylation stabilizes the CDK7-cyclin H-MAT1 complex in vivo and regulates its CTD kinase activity

Cited by 133 publications

References 42 publications

Mcs2 and a novel CAK subunit Pmh1 associate with Skp1 in fission yeast

Mcs2 and a novel CAK subunit Pmh1 associate with Skp1 in fission yeast

Characterization of the Schizosaccharomyces pombe Cdk9/Pch1 Protein Kinase

Regulation of RNA polymerase II activity by CTD phosphorylation and cell cycle control

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