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A transcription corepressor, MAT1-mediated transcriptional repressor (MMTR), was found in mouse embryonic stem cell lines. MMTR orthologs (DMAP1) are found in a wide variety of life forms from yeasts to humans. MMTR down-regulation in differentiating mouse embryonic stem cells in vitro resulted in activation of many unrelated genes, suggesting its role as a general transcriptional repressor. In luciferase reporter assays, the transcriptional repression activity resided at amino acids 221 to 468. Histone deacetylase 1 (HDAC1) interacts with MMTR both in vitro and in vivo and also interacts with MMTR in the nucleus. Interestingly, MMTR activity was only partially rescued by competition with dominant-negative HDAC1(H141A) or by treatment with an HDAC inhibitor, trichostatin A (TSA). To identify the protein responsible for HDAC1-independent MMTR activity, we performed a yeast two-hybrid screen with the full-length MMTR coding sequence as bait and found MAT1. MAT1 is an assembly/targeting factor for cyclin-dependent kinase-activating kinase which constitutes a subcomplex of TFIIH. The coiled-coil domain in the middle of MAT1 was confirmed to interact with the C-terminal half of MMTR, and the MMTR-mediated transcriptional repression activity was completely restored by MAT1 in the presence of TSA. Moreover, intact MMTR was required to inhibit phosphorylation of the C-terminal domain in the RNA polymerase II largest subunit by TFIIH kinase in vitro. Taken together, these data strongly suggest that MMTR is part of the basic cellular machinery for a wide range of transcriptional regulation via interaction with TFIIH and HDAC.

MAK (male germ cell-associated protein kinase) and MRK/ICK (MAK-related kinase/intestinal cell kinase) are human homologs of Ime2p in Saccharomyces cerevisiae and of Mde3 and Pit1 in Schizosaccharomyces pombe and are similar to human cyclin-dependent kinase 2 (CDK2) and extracellular signal-regulated kinase 2 (ERK2). MAK and MRK require dual phosphorylation in a TDY motif catalyzed by an unidentified human threonine kinase and tyrosine autophosphorylation. Herein, we establish that human CDK-related kinase CCRK (cell cycle-related kinase) is an activating T157 kinase for MRK, whereas active CDK7/cyclin H/MAT1 complexes phosphorylate CDK2 but not MRK. Protein phosphatase 5 (PP5) interacts with MRK in a complex and dephosphorylates MRK at T157 in vitro and in situ. Thus, CCRK and PP5 are yin-yang regulators of T157 phosphorylation. To determine a substrate consensus, we screened a combinatorial peptide library with active MRK. MRK preferentially phosphorylates R-P-X-S/T-P sites, with the preference for arginine at position -3 (P-3) being more stringent than for prolines at P-2 and P+1. Using the consensus, we identified a putative phosphorylation site (RPLT(1080)S) for MRK in human Scythe, an antiapoptotic protein that interacts with MRK. MRK phosphorylates Scythe at T1080 in vitro as determined by site-directed mutagenesis and mass spectrometry, supporting the consensus and suggesting Scythe as a physiological substrate for MRK.

Cyclin-dependent kinase 7 (CDK7) is the catalytic subunit of the metazoan CDK-activating kinase (CAK), which activates CDKs, such as CDC2 and CDK2, through phosphorylation of a conserved threonine residue in the T loop. Full activation of CDK7 requires association with a positive regulatory subunit, cyclin H, and phosphorylation of a conserved threonine residue at position 170 in its own T loop. We show that threonine-170 of CDK7 is phosphorylated in vitro by its targets, CDC2 and CDK2, which also phosphorylate serine-164 in the CDK7 T loop, a site that perfectly matches their consensus phosphorylation site. In contrast, neither CDK4 nor CDK7 itself can phosphorylate the CDK7 T loop in vitro. The ability of CDC2 or CDK2 and CDK7 to phosphorylate each other but not themselves implies that each kinase can discriminate among closely related sequences and can recognize a substrate site that diverges from its usual preferred site. To understand the basis for this paradoxical substrate specificity, we constructed a chimeric CDK with the T loop of CDK7 grafted onto the body of CDK2. Surprisingly, the hybrid enzyme, CDK2-7, was efficiently activated in cyclin A-dependent fashion by CDK7 but not at all by CDK2. CDK2-7, moreover, phosphorylated wild-type CDK7 but not CDK2. Our results suggest that the primary amino acid sequence of the T loop plays only a minor role, if any, in determining the specificity of cyclin-dependent CAKs for their CDK substrates and that protein-protein interactions involving sequences outside the T loop can influence substrate specificity both positively and negatively.

The eukaryotic cell cycle is regulated by cyclin-dependent kinases (CDKs). CDK4 and CDK6, which are activated by D-type cyclins during the G(1) phase of the cell cycle, are thought to be responsible for phosphorylation of the retinoblastoma gene product (pRb). The tumor suppressor p16(INK4A) inhibits phosphorylation of pRb by CDK4 and CDK6 and can thereby block cell cycle progression at the G(1)/S boundary. Phosphorylation of the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase II by general transcription factor TFIIH is believed to be an important regulatory event in transcription. TFIIH contains a CDK7 kinase subunit and phosphorylates the CTD. We have previously shown that p16(INK4A) inhibits phosphorylation of the CTD by TFIIH. Here we report that the ability of p16(INK4A) to inhibit CDK7-CTD kinase contributes to the capacity to induce cell cycle arrest. These results suggest that p16(INK4A) may regulate cell cycle progression by inhibiting not only CDK4-pRb kinase activity but also by modulating CDK7-CTD kinase activity. Regulation of CDK7-CTD kinase activity by p16(INK4A) thus may represent an alternative pathway for controlling cell cycle progression.

The assembly of functional holoenzymes composed of regulatory D-type cyclins and cyclin-dependent kinases (cdks) is rate limiting for progression through the G1 phase of the mammalian somatic cell cycle. Complexes between D-type cyclins and their major catalytic subunit, cdk4, are catalytically inactive until cyclin-bound cdk4 undergoes phosphorylation on a single threonyl residue (Thr-172). This step is catalyzed by a cdk-activating kinase (CAK) functionally analogous to the enzyme which phosphorylates cdc2 and cdk2 at Thr-161/160. Here, we demonstrate that the catalytic subunit of mouse cdc2/cdk2 CAK (a 39-kDa protein designated p39MO15) can assemble with a regulatory protein present in either insect or mammalian cells to generate a CAK activity capable of phosphorylating and enzymatically activating both cdk2 and cdk4 in complexes with their respective cyclin partners. A newly identified 37-kDa cyclin-like protein (cyclin H [R. P. Fisher and D. O. Morgan, Cell 78:713-724, 1994]) can assemble with p39MO15 to activate both cyclin A-cdk2 and cyclin D-cdk4 in vitro, implying that CAK is structurally reminiscent of cyclin-cdk complexes themselves. Antisera produced to the p39MO15 subunit can completely deplete mammalian cell lysates of CAK activity for both cyclin A-cdk2 and cyclin D-cdk4, with recovery of activity in the resulting immune complexes. By using an immune complex CAK assay, CAK activity for cyclin A-cdk2 and cyclin D-cdk4 was detected both in quiescent cells and invariantly throughout the cell cycle. Therefore, although it is essential for the enzymatic activation of cyclin-cdk complexes, CAK appears to be neither rate limiting for the emergence of cells from quiescence nor subject to upstream regulatory control by stimulatory mitogens.

Cell cycle progression is regulated by cyclin-dependent kinases (cdk's), which in turn are regulated by their interactions with stoichiometric inhibitors, such as p27(Kip1). Although p27 associates with cyclin D-cyclin-dependent kinase 4 (cdk4) constitutively, whether or not it inhibits this complex is dependent on the absence or presence of a specific tyrosine phosphorylation that converts p27 from a bound inhibitor to a bound noninhibitor under different growth conditions. This phosphorylation occurs within the 3-10 helix of p27 and may dislodge the helix from cdk4's active site to allow ATP binding. Here we show that the interaction of nonphosphorylated p27 with cdk4 also prevents the activating phosphorylation of the T-loop by cyclin H-cdk7, the cdk-activating kinase (CAK). Even though the cyclin H-cdk7 complex is present and active in contact-arrested cells, p27's association with cyclin D-cdk4 prevents T-loop phosphorylation. When p27 is tyrosine phosphorylated in proliferating cells or in vitro with the tyrosine Y kinase Abl, phosphorylation of cdk4 by cyclin H-cdk7 is permitted, even without dissociation of p27. This suggests that upon release from the contact-arrested state, a temporal order for the reactivation of inactive p27-cyclin D-cdk4 complexes must exist: p27 must be Y phosphorylated first, directly permitting cyclin H-cdk7 phosphorylation of residue T172 and the consequent restoration of kinase activity. The non-Y-phosphorylated p27-cyclin D-cdk4 complex could be phosphorylated by purified Csk1, a single-subunit CAK from fission yeast, but was still inactive due to p27's occlusion of the active site. Thus, the two modes by which p27 inhibits cyclin D-cdk4 are independent and may reinforce one another to inhibit kinase activity in contact-arrested cells, while maintaining a reservoir of preformed complex that can be activated rapidly upon cell cycle reentry.

The tumor suppressor gene BRCA1 encodes a protein of 1,863 amino acids that can interact with a plethora of factors involved in transcription, DNA repair, cell cycle regulation, apoptosis, genome integrity, and ubiquitination (16). Theses interactions underlie the extensive implication of BRCA1 in crucial processes related to tumorigenesis. However, how it can precisely ensure those functions still remains unclear. It has been suggested that BRCA1 could exert its tumor suppressor functions, particularly in DNA repair and cell cycle regulation, via its transcriptional activity (39, 53). Interestingly, various BRCA1 target genes carry out functions that could largely account for its involvement in DNA repair and cell cycle control. BRCA1 induces the expression of the DNA-damage-responsive genes p21WAF1/CIP1 (54) and GADD45 (24), the nucleotide excision repair (NER) genes DDB2 and XPC (26), and the cell cycle arrest genes p27 (62) and 14-3-3σ (5), while it represses the expression of the cell cycle-promoting gene cyclin B1 (35). Transcriptional activators generally possess a DNA binding domain that binds specific sequences on promoters and an activating region known to interact with general transcription factors, the RNA polII holoenzyme, and chromatin remodeling machines to recruit the transcriptional machinery to a target promoter (34, 47). In contrast to those activators, BRCA1 has been shown to bind DNA only in a nonspecific fashion (46, 64) and has been shown to be a component of RNA polII holoenzyme (4, 51). Moreover, we have previously shown that at high concentration, the BRCA1 C-terminal region (amino acids 1528 to 1863) can stimulate transcription in vivo and in vitro without the requirement for a DNA-tethering function (41). That evidence suggests that BRCA1 can stimulate transcription by a mechanism alternative to recruitment, for example, by modulating an enzymatic activity. In vitro transcription assays using a highly purified system have demonstrated that the activation by Gal4-BRCA1, in contrast to Gal4-VP16, is highly influenced by TFIIH concentrations (23). Furthermore, immunopurification of BRCA1 complexes copurifies with transcriptionally active RNA polymerase II and TFIIH (51), suggesting functional and physical links between BRCA1 and TFIIH. Interestingly, TFIIH plays important roles in DNA repair and cell cycle regulation in addition to transcription (14), much like BRCA1. TFIIH bears helicase and kinase activities required for open complex formation and promoter escape, respectively, during transcription initiation (17). The Cdk-activating kinase (CAK) subcomplex of TFIIH, formed by Cdk7, cyclin H, and MAT1 subunits, is responsible for the kinase activity (18). CAK can be found either in a free form or associated with TFIIH, and these states are believed to influence its substrate preference (65). Free CAK preferentially phosphorylates Cdk2, whereas TFIIH-associated CAK mainly phosphorylates the RNA polII carboxy-terminal domain (CTD) (50). The mammalian RNA polII CTD is composed of 52 repeats of the heptapeptide YSPTSPS that can be highly phosphorylated. Moreover, the CTD phosphorylation state varies along with the transcriptional cycle (15). The hypophosphorylated form of RNA polII (IIa) is preferentially recruited to a target promoter to form a stable preinitiation complex (PIC), while the hyperphosphorylated form (IIo) is usually associated with the coding region of a gene (31). The Cdk7 subunit of TFIIH phosphorylates the CTD on serine 5 at the early stages of the transcriptional cycle, just after PIC formation (2). Cdk9, a member of the elongation factor P-TEFb, phosphorylates the CTD to render RNA polII more processive during elongation (38). Cdk8, a component of the RNA polII holoenzyme, is thought to phosphorylate the CTD prior to the binding of RNA polII to DNA and, by doing so, reduce the number of RNA polII molecules competent for initiation (55). It is therefore evident that the regulation of RNA polII phosphorylation constitutes an important step to modulate gene expression. The RNA polII CTD phosphorylation status and activity are also modulated during the cell cycle (43). For example, the CTD becomes hyperphosphorylated during mitosis (3), and it was found that CTD phosphorylation by the mitogen-promoting factor in vitro results in the dissociation of transcription complexes (68). Furthermore, transcription and cell cycle regulation share common modulators of CTD phosphorylation, such as the CAK subcomplex of TFIIH. CAK has also been shown to function as a CAK in metazoans by phosphorylating cyclin-dependent kinases (see above) and therefore promotes cell cycle progression (57). In addition to being involved in transcription and cell cycle progression, CTD phosphorylation is believed to play an important role in transcription-coupled DNA repair and RNA polII ubiquitination (8, 48). In an attempt to elucidate the mechanism by which BRCA1 could modulate gene expression as well as being involved in other processes such as DNA repair and cell cycle control, we investigated whether BRCA1 could directly modulate the RNA polII CTD phosphorylation levels. We found that the BRCA1 C-terminal region (herein BRCA1-C) can strongly inhibit CTD phosphorylation elicited by a HeLa nuclear extract. We have shown that BRCA1-C can inhibit free and TFIIH-associated CAK activity with respect to the RNA polII CTD as well as other substrates such as Cdk2 and TFIIE. We also found that BRCA1-C can directly interact with Cdk7 and compete with ATP. Finally, we have shown that full-length BRCA1 is able to inhibit CTD phosphorylation in a transient transfection assay.

Human immunodeficiency virus type 1 (HIV-1) Tat interacts with cyclin T1 (CycT1), a regulatory partner of CDK9 in the positive transcription elongation factor (P-TEFb) complex, and binds cooperatively with CycT1 to TAR RNA to recruit P-TEFb and promote transcription elongation. We show here that Tat also stimulates phosphorylation of affinity-purified core RNA polymerase II and glutathione S-transferase‐C-terminal-domain substrates by CycT1-CDK9, but not CycH-CDK7, in vitro. Interestingly, incubation of recombinant Tat‐PTEFb complexes with ATP enhanced binding to TAR RNA dramatically, and the C-terminal half of CycT1 masked binding of Tat to TAR RNA in the absence of ATP. ATP incubation lead to autophosphorylation of CDK9 at multiple C-terminal Ser and Thr residues, and full-length CycT1 (amino acids 728) [CycT1(1‐728)], but not truncated CycT1(1‐303), was also phosphorylated by CDK9. P-TEFb complexes containing a catalytically inactive CDK9 mutant (D167N) bound TAR RNA weakly and independently of ATP, as did a C-terminal truncated CDK9 mutant that was catalytically active but unable to undergo autophosphorylation. Analysis of different Tat proteins revealed that the 101-amino-acid SF2 HIV-1 Tat was unable to bind TAR with CycT1(1‐ 303) in the absence of phosphorylated CDK9, whereas unphosphorylated CDK9 strongly blocked binding of HIV-2 Tat to TAR RNA in a manner that was reversed upon autophosphorylation. Replacement of CDK9 phosphorylation sites with negatively charged residues restored binding of CycT1(1‐303)-D167N-Tat, and rendered D167N a more potent inhibitor of transcription in vitro. Taken together, these results demonstrate that CDK9 phosphorylation is required for high-affinity binding of Tat‐P-TEFb to TAR RNA and that the state of P-TEFb phosphorylation may regulate Tat transactivation in vivo.

The tumor suppressor protein p53 acts as a transcriptional activator that can mediate cellular responses to DNA damage by inducing apoptosis and cell cycle arrest. p53 is a nuclear phosphoprotein, and phosphorylation has been proposed to be a means by which the activity of p53 is regulated. The cyclin-dependent kinase (CDK)-activating kinase (CAK) was originally identified as a cellular kinase required for the activation of a CDK-cyclin complex, and CAK is comprised of three subunits: CDK7, cyclin H, and p36MAT1. CAK is part of the transcription factor IIH multiprotein complex, which is required for RNA polymerase II transcription and nucleotide excision repair. Because of the similarities between p53 and CAK in their involvement in the cell cycle, transcription, and repair, we investigated whether p53 could act as a substrate for phosphorylation by CAK. While CDK7-cyclin H is sufficient for phosphorylation of CDK2, we show that p36MAT1 is required for efficient phosphorylation of p53 by CDK7-cyclin H, suggesting that p36MAT1 can act as a substrate specificity-determining factor for CDK7-cyclin H. We have mapped a major site of phosphorylation by CAK to Ser-33 of p53 and have demonstrated as well that p53 is phosphorylated at this site in vivo. Both wild-type and tumor-derived mutant p53 proteins are efficiently phosphorylated by CAK. Furthermore, we show that p36 and p53 can interact both in vitro and in vivo. These studies reveal a potential mechanism for coupling the regulation of p53 with DNA repair and the basal transcriptional machinery.