Substrate specificity of the cdk-activating kinase (CAK) is altered upon association with TFIIH.
The transcription/DNA repair factor TFIIH consists of nine subunits, several exhibiting known functions: helicase/ATPase, kinase activity and DNA binding. Three subunits of TFIIH, cdk7, cyclin H and MAT1, form a ternary complex, cdk-activating kinase (CAK), found either on its own or as part of TFIIH. In the present work, we demonstrate that purified human CAK complex (free CAK) and recombinant CAK (rCAK) produced in insect cells exhibit a strong preference for the cyclin-dependent kinase 2 (cdk2) over a ctd oligopeptide substrate (which mimics the carboxy-terminal domain of the RNA polymerase II). In contrast, TFIIH preferentially phosphorylates the ctd as well as TFIIE alpha, but not cdk2. TFIIH was resolved into four subcomplexes: the kinase complex composed of cdk7, cyclin H and MAT1; the core TFIIH which contains XPB, p62, p52, p44 and p34; and two other subcomplexes in which XPD is found associated with either the kinase complex or with the core TFIIH. Using these fractions, we demonstrate that TFIIH lacking the CAK subcomplex completely recovers its transcriptional activity in the presence of free CAK. Furthermore, studies examining the interactions between TFIIH subunits provide evidence that CAK is integrated within TFIIH via XPB and XPD.
Rossignol
M
M
Institut de Génétique et de Biologie Moléculaire et Cellulaire, UPR 6520 (CNRS), Unité 184 (INSERM), Illkirch, CU de Strasbourg, France.
Kolb-Cheynel
I
I
Egly
J M
JM
D000818:Animals; D016367:Baculoviridae; D018844:Cyclin-Dependent Kinases; D004265:DNA Helicases / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera; D013379:Substrate Specificity; D051758:Transcription Factor TFIIH; D014157:Transcription Factors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D019473:Transcription Factors, TFII; D014162:Transfection; D000095043:Cyclin-Dependent Kinase-Activating Kinase
D011994:Recombinant Proteins; D014157:Transcription Factors; D019473:Transcription Factors, TFII; D051758:Transcription Factor TFIIH; D017346:Protein Serine-Threonine Kinases; D018844:Cyclin-Dependent Kinases; D004265:DNA Helicases; D000095043:Cyclin-Dependent Kinase-Activating Kinase