A protein kinase activity that phosphorylates the C-terminal domain (CTD) of RNA polymerase II and is associated with the basal transcription-repair factor TFIIH (also called BTF2) resides with MO15, a cyclin-dependent protein kinase that was first found to be involved in cell cycle regulation. Using in vivo and in vitro repair assays, we show that MO15 is important for nucleotide excision repair, most likely through its association with TFIIH, thus providing an unexpected link among three important cellular mechanisms.

The kinase responsible for Thr161‐Thr160 phosphorylation and activation of cdc2/cdk2 (CAK:cdk‐activating kinase) has been shown previously to comprise at least two subunits, cdk7 and cyclin H. An additional protein co‐purified with CAK in starfish oocytes, but its sequencing did not reveal any similarity with any known protein. In the present work, a cDNA encoding this protein is cloned and sequenced in both starfish and Xenopus oocytes. It is shown to encode a new member of the RING finger family of proteins with a characteristic C3HC4 motif located in the N‐terminal domain. We demonstrate that the RING finger protein (MAT1: ‘menage à trois’) is a new subunit of CAK in both vertebrate and invertebrates. However, CAK may also exist in oocytes as heterodimeric complexes between cyclin H and cdk7 only. Stable heterotrimeric CAK complexes were generated in reticulocyte lysates programmed with mRNAs encoding Xenopus cdk7, cyclin H and MAT1. In contrast, no heterodimeric cyclin H‐cdk7 complex could be immunoprecipitated from reticulocyte lysates programmed with cdk7 and cyclin H mRNAs only. Stabilization of CAK complexes by MAT1 does not involve phosphorylation of Thr176, as the Thr176–>Ala mutant of Xenopus cdk7 could engage as efficiently as wild‐type cdk7 in ternary complexes. Even though starfish MAT1 is almost identical to Xenopus MAT1 in the RING finger domain, the starfish subunit could not replace the Xenopus subunit and stabilize cyclin H‐cdk7 in reticulocyte lysate, suggesting that the MAT1 subunit does not (or not only) interact with cyclin H‐cdk7 through the RING finger domain.

It is proposed that the CDK7‐cyclin H complex functions in cell cycle progression, basal transcription and DNA repair. Here we report that in vitro reconstitution of an active CDK7‐cyclin H complex requires stoichiometric amounts of a novel 36 kDa assembly factor termed MAT1 (ménage à trois 1). Sequencing of MAT1 reveals a putative zinc binding motif (a C3HC4 RING finger) in the N‐terminus; however, this domain is not required for ternary complex formation with CDK7‐cyclin H. MAT1 is associated with nuclear CDK7‐cyclin H at all stages of the cell cycle in vivo. Ternary complexes of CDK7, cyclin H and MAT1 display kinase activity towards substrates mimicking both the T‐loop in CDKs and the C‐terminal domain of RNA polymerase II, regardless of whether they are immunoprecipitated from HeLa cells or reconstituted in a reticulocyte lysate. MAT1 constitutes the first example of an assembly factor that appears to be essential for the formation of an active CDK‐cyclin complex.

Abstract. The activity of cyclin-dependent kinases (cdl~) depends on the phosphorylation of a residue corresponding to threonine 161 in human p34 ~c2. One enzyme responsible for phosphorylating this critical residue has recently been purified from Xenopus and starfish. It was termed CAK (for cdk-activating _ki-nase), and it was shown to contain p40 M°15 as its catalytic subunit. In view of the cardinal role of cdks in cell cycle control, it is important to learn if and how CAK activity is regulated during the somatic cell cycle. Here, we report a molecular characterization of a human p40 M°15 homologue and its associated CAK activity. We have cloned and sequenced a cDNA coding for human p40 M°tS, and raised specific polyclonal and monoclonal antibodies against the corresponding protein expressed in Escherichia coll. These tools were then used to demonstrate that p40 M°~5 protein expression and CAK activity are constant throughout the somatic cell cycle. Gel filtration suggests that active CAK is a multiprotein complex, and immunoprecipitation experiments identify two polypeptides of 34 and 32 kD as likely complex partners of p40 u°15. The association of the three proteins is near stoichiometric and invariant throughout the cell cycle. Immunocytochemistry and biochemical enucleation experiments both demonstrate that p40 u°15 is nuclear at all stages of the cell cycle (except for mitosis, when the protein redistributes throughout the cell), although the p34cdc2/cyclin B complex, one of the major purported substrates of CAK, occurs in the cytoplasm until shortly before mitosis. The absence of obvious changes in CAK activity in exponentially growing cells constitutes a surprise. It suggests that the phosphorylation state of threonine 161 in p34 ~c2 (and the corresponding residue in other cdks) may be regulated primarily by the availability of the cdk/cyclin substrates, and by phosphatase(s).