Schizosaccharomyces pombe Mop1-Mcs2 is related to mammalian CAK.

Abstract

The cyclin‐dependent kinase (CDK)‐activating kinase, CAK, from mammals and amphibians consists of MO15/CDK7 and cyclin H, a complex which has been identified also as a RNA polymerase II C‐terminal domain (CTD) kinase. While the Schizosaccharomyces pombe cdc2 gene product also requires an activating phosphorylation, the enzyme responsible has not been identified. We have isolated an essential S.pombe gene, mop1, whose product is closely related to MO15 and to Saccharomyces cerevisiae Kin28. The functional similarity of Mop1 and MO15 is reflected in the ability of MO15 to rescue a mop1 null allele. This suggests that Mop1 would be a CDK, and indeed Mop1 associates with a previously characterized cyclin H‐related cyclin Mcs2 of S.pombe. Also, Mop1 and Mcs2 can associate with the heterologous partners human cyclin H and MO15, respectively. Moreover, the rescue of a temperature‐sensitive mcs2 strain by expression of mop1+ demonstrates a genetic interaction between mop1 and mcs2. In a functional assay, immunoprecipitated Mop1‐Mcs2 acts both as an RNA polymerase II CTD kinase and as a CAK. The CAK activity of Mop1‐Mcs2 distinguishes it from the related CDK‐cyclin pair Kin28‐Ccl1 from S.cerevisiae, and supports the notion that Mop1‐Mcs2 may represent a homolog of MO15‐cyclin H in S.pombe with apparent dual roles as a RNA polymerase CTD kinase and as a CAK.

pch1 +, a Second Essential C-type Cyclin Gene in Schizosaccharomyces pombe

“…The third cyclin gene, mcs2 ϩ , encodes an essential C-type cyclin that shares some of the characteristics of pch1 ϩ , the cyclin gene described in this study (18). Recent studies have shown that Mcs2 is the cyclin partner of Mcs6 kinase, which is also known as Crk1 or Mop1 (19,20). The Mcs2-Mcs6 kinase can carry out the activating threonine 167 phosphorylation of Cdc2 in vitro, although it is unknown whether it performs this function in vivo.…”

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

“…LexA-Mcs6, LexA-Mcs2, and B42-Mcs2 were expressed in budding yeast from the pEG202 and pJG4-5 vectors, respectively [3]. To generate skp1-pJG4-5, the skp1 ORF was amplified by PCR with the following primers: ACG-CGT-CGA-CGG-ATC-CGA-ATT-CAC-CAT-GTC-CAA-AAT-CAA-ACT-GAT-TTC-ATC-T and TCC-CCC-GGG-CTC-GAG-CTA-TC T-GTC-TTC-GGC-CCA-TTC-AAT.…”

“…Protein amounts were estimated by silver staining and 100 ng of purified proteins was used for kinase assays. Bacterially expressed GST-CTD (a kind gift from David Chao and Richard Young) proteins were purified as described [3,35].…”

“…In fission yeast Schizosaccharomyces pombe Mcs2 represents the cognate cyclin of the Mcs6 CDK [1][2][3]. Both mcs6 and mcs2 were first identified as mutant alleles (mcs6-13 and mcs2-75) capable of suppressing a mitotic catastrophe resulting from a hyperactive Cdc2 in a cdc2-3w wee1-50 double mutant strain [4].…”

“…As part of TFIIH, Cdk7 can phosphorylate the carboxyl terminal domain (CTD) of the large subunit of RNA polymerase II (reviewed in [14]) as well as other transcription factors (reviewed in [15]), and is thought to be involved in regulation of transcription in vivo [12,[16][17][18]. The ability of Mcs6 immunocomplexes to also phosphorylate RNA Pol II CTD in vitro [2,3] suggests that this function could also be operational in fission yeast.…”