MAT1 ('menage à 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 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.
Devault
A
A
Centre de Recherches de Biochimie Macromoléculaire, CNRS UPR 9008, Montpellier, France.
Martinez
A M
AM
Fesquet
D
D
Labbé
J C
JC
Morin
N
N
Tassan
J P
JP
Nigg
E A
EA
Cavadore
J C
JC
Dorée
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D056748:Cyclin H; D018844:Cyclin-Dependent Kinases; D016213:Cyclins / Q000378:metabolism*; D000070819:Discoidin Domain Receptor 1; D008969:Molecular Sequence Data; D005810:Multigene Family; D016133:Polymerase Chain Reaction; D011233:Precipitin Tests; D011485:Protein Binding; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism*; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis; D012156:Reticulocytes / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D013215:Starfish / Q000235:genetics; D014981:Xenopus / Q000235:genetics; D000095043:Cyclin-Dependent Kinase-Activating Kinase
C534751:CCNH protein, human; D056748:Cyclin H; D016213:Cyclins; D011994:Recombinant Proteins; C092030:DDR1 protein, human; D000070819:Discoidin Domain Receptor 1; D020794:Receptor Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D018844:Cyclin-Dependent Kinases; D000095043:Cyclin-Dependent Kinase-Activating Kinase