MAT1 (MENAGE-A-TROIS) A NEW RING FINGER PROTEIN SUBUNIT STABILIZING CYCLIN H-CDK7 COMPLEXES IN STARFISH AND XENOPUS CAK

The kinase responsible for Thr161-Thr160 phosphorylation and activatio n of cdc2/cdk2 (CAK:cdk-activating kinase) has been shown previously t o comprise at least two subunits, cdk7 and cyclin H. An additional pro tein 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 f inger family of proteins with a characteristic C3HC4 motif located in the N-terminal domain. We demonstrate that the RING finger protein (MA T1: 'menage a trois') is a new subunit of CAK in both vertebrate and i nvertebrates. However, CAK may also exist in oocytes as heterodimeric complexes between cyclin H and cdk7 only. Stable heterotrimeric CAK co mplexes were generated in reticulocyte lysates programmed with mRNAs e ncoding Xenopus cdk7, cyclin H and MAT1. In contrast, no heterodimeric cyclin H-cdk7 complex could be immunoprecipitated from reticulocyte l ysates programmed with cdk7 and cyclin H mRNAs only. Stabilization of CAK complexes by MAT1 does not involve phosphorylation of Thr176, as t he Thr176-->Ala mutant of Xenopus cdk7 could engage as efficiently as wild-type cdk7 in ternary complexes. Even though starfish MAT1 is almo st identical to Xenopus MAT1 in the RING finger domain, the starfish s ubunit could not replace the Xenopus subunit and stabilize cyclin H-cd k7 in reticulocyte lysate, suggesting that the MAT1 subunit does not ( or not only) interact with cyclin H-cdk7 through the RING finger domai n.