Cdk-activating Kinase Complex Research Articles

CVB3 VP1 interacts with MAT1 to inhibit cell proliferation by interfering with Cdk-activating kinase complex activity in CVB3-induced acute pancreatitis.

Coxsackievirus B3 (CVB3) belongs to the genus Enterovirus of the family Picornaviridae and can cause acute acinar pancreatitis in adults. However, the molecular mechanisms of pathogenesis underlying CVB3-induced acute pancreatitis have remained unclear. In this study, we discovered that CVB3 capsid protein VP1 inhibited pancreatic cell proliferation and exerted strong cytopathic effects on HPAC cells. Through yeast two-hybrid, co-immunoprecipitation, and confocal microscopy, we show that Menage a trois 1 (MAT1), a subunit of the Cdk-Activating Kinase (CAK) complex involved in cell proliferation and transcription, is a novel interaction protein with CVB3 VP1. Moreover, CVB3 VP1 inhibited MAT1 accumulation and localization, thus interfering with its interaction with CDK7. Furthermore, CVB3 VP1 could suppress CAK complex enzymic phosphorylation activity towards RNA Pol II and CDK4/6, direct substrates of CAK. VP1 also suppresses phosphorylation of retinoblastoma protein (pRb), an indirect CAK substrate, especially at phospho-pRb Ser780 and phospho-pRb Ser807/811 residues, which are associated with cell proliferation. Finally, we present evidence using deletion mutants that the C-terminal domain (VP1-D8, 768-859aa) is the minimal VP1 region required for its interaction with MAT1, and furthermore, VP1-D8 alone was sufficient to arrest cells in G1/S phase as observed during CVB3 infection. Taken together, we demonstrate that CVB3 VP1 can inhibit CAK complex assembly and activity through direct interaction with MAT1, to block MAT1-mediated CAK-CDK4/6-Rb signaling, and ultimately suppress cell proliferation in pancreatic cells. These findings substantially extend our basic understanding of CVB3-mediated pancreatitis, providing strong candidates for strategic therapeutic targeting.

Structural basis for CDK7 activation by MAT1 and Cyclin H

Cyclin-dependent kinase 7 (CDK7), Cyclin H, and the RING-finger protein MAT1 form the heterotrimeric CDK-activating kinase (CAK) complex which is vital for transcription and cell-cycle control. When associated with the general transcription factor II H (TFIIH) it activates RNA polymerase II by hyperphosphorylation of its C-terminal domain (CTD). In the absence of TFIIH the trimeric complex phosphorylates the T-loop of CDKs that control cell-cycle progression. CAK holds a special position among the CDK branch due to this dual activity and the dependence on two proteins for activation. We solved the structure of the CAK complex from the model organism Chaetomium thermophilum at 2.6-Å resolution. Our structure reveals an intricate network of interactions between CDK7 and its two binding partners MAT1 and Cyclin H, providing a structural basis for the mechanism of CDK7 activation and CAK activity regulation. In vitro activity measurements and functional mutagenesis show that CDK7 activation can occur independent of T-loop phosphorylation and is thus exclusively MAT1-dependent by positioning the CDK7 T-loop in its active conformation.

In TFIIH the Arch domain of XPD is mechanistically essential for transcription and DNA repair

The XPD helicase is a central component of the general transcription factor TFIIH which plays major roles in transcription and nucleotide excision repair (NER). Here we present the high-resolution crystal structure of the Arch domain of XPD with its interaction partner MAT1, a central component of the CDK activating kinase complex. The analysis of the interface led to the identification of amino acid residues that are crucial for the MAT1-XPD interaction. More importantly, mutagenesis of the Arch domain revealed that these residues are essential for the regulation of (i) NER activity by either impairing XPD helicase activity or the interaction of XPD with XPG; (ii) the phosphorylation of the RNA polymerase II and RNA synthesis. Our results reveal how MAT1 shields these functionally important residues thereby providing insights into how XPD is regulated by MAT1 and defining the Arch domain as a major mechanistic player within the XPD scaffold.

Discovery and Characterization of SY-1365, a Selective, Covalent Inhibitor of CDK7.

Recent studies suggest that targeting transcriptional machinery can lead to potent and selective anticancer effects in cancers dependent on high and constant expression of certain transcription factors for growth and survival. Cyclin-dependent kinase 7 (CDK7) is the catalytic subunit of the CDK-activating kinase complex. Its function is required for both cell-cycle regulation and transcriptional control of gene expression. CDK7 has recently emerged as an attractive cancer target because its inhibition leads to decreased transcript levels of oncogenic transcription factors, especially those associated with super-enhancers. Here, we describe a selective CDK7 inhibitor SY-1365, which is currently in clinical trials in populations of patients with ovarian and breast cancer (NCT03134638). In vitro, SY-1365 inhibited cell growth of many different cancer types at nanomolar concentrations. SY-1365 treatment decreased MCL1 protein levels, and cancer cells with low BCL2L1 (BCL-XL) expression were found to be more sensitive to SY-1365. Transcriptional changes in acute myeloid leukemia (AML) cell lines were distinct from those following treatment with other transcriptional inhibitors. SY-1365 demonstrated substantial antitumor effects in multiple AML xenograft models as a single agent; SY-1365-induced growth inhibition was enhanced in combination with the BCL2 inhibitor venetoclax. Antitumor activity was also observed in xenograft models of ovarian cancer, suggesting the potential for exploring SY-1365 in the clinic in both hematologic and solid tumors. Our findings support targeting CDK7 as a new approach for treating transcriptionally addicted cancers. SIGNIFICANCE: These findings demonstrate the molecular mechanism of action and potent antitumor activity of SY-1365, the first selective CDK7 inhibitor to enter clinical investigation.

Mms19 is a mitotic gene that permits Cdk7 to be fully active as a Cdk-activating kinase

ABSTRACTMms19 encodes a cytosolic iron-sulphur assembly component. We found that Drosophila Mms19 is also essential for mitotic divisions and for the proliferation of diploid cells. Reduced Mms19 activity causes severe mitotic defects in spindle dynamics and chromosome segregation, and loss of zygotic Mms19 prevents the formation of imaginal discs. The lack of mitotic tissue in Mms19P/P larvae can be rescued by overexpression of the Cdk-activating kinase (CAK) complex, an activator of mitotic Cdk1, suggesting that Mms19 functions in mitosis to allow CAK (Cdk7/Cyclin H/Mat1) to become fully active as a Cdk1-activating kinase. When bound to Xpd and TFIIH, the CAK subunit Cdk7 phosphorylates transcriptional targets and not cell cycle Cdks. In contrast, free CAK phosphorylates and activates Cdk1. Physical and genetic interaction studies between Mms19 and Xpd suggest that their interaction prevents Xpd from binding to the CAK complex. Xpd bound to Mms19 therefore frees CAK complexes, allowing them to phosphorylate Cdk1 and facilitating progression to metaphase. The structural basis for the competitive interaction with Xpd seems to be the binding of Mms19, core TFIIH and CAK to neighbouring or overlapping regions of Xpd.

Expression of CDK7, Cyclin H, and MAT1 Is Elevated in Breast Cancer and Is Prognostic in Estrogen Receptor-Positive Breast Cancer.

CDK-activating kinase (CAK) is required for the regulation of the cell cycle and is a trimeric complex consisting of cyclin-dependent kinase 7 (CDK7), Cyclin H, and the accessory protein, MAT1. CDK7 also plays a critical role in regulating transcription, primarily by phosphorylating RNA polymerase II, as well as transcription factors such as estrogen receptor-α (ER). Deregulation of cell cycle and transcriptional control are general features of tumor cells, highlighting the potential for the use of CDK7 inhibitors as novel cancer therapeutics. mRNA and protein expression of CDK7 and its essential cofactors cyclin H and MAT1 were evaluated in breast cancer samples to determine if their levels are altered in cancer. Immunohistochemical staining of >900 breast cancers was used to determine the association with clinicopathologic features and patient outcome. We show that expressions of CDK7, cyclin H, and MAT1 are all closely linked at the mRNA and protein level, and their expression is elevated in breast cancer compared with the normal breast tissue. Intriguingly, CDK7 expression was inversely proportional to tumor grade and size, and outcome analysis showed an association between CAK levels and better outcome. Moreover, CDK7 expression was positively associated with ER expression and in particular with phosphorylation of ER at serine 118, a site important for ER transcriptional activity. Expressions of components of the CAK complex, CDK7, MAT1, and Cyclin H are elevated in breast cancer and correlate with ER. Like ER, CDK7 expression is inversely proportional to poor prognostic factors and survival. Clin Cancer Res; 22(23); 5929-38. ©2016 AACR.

Nucleotide excision repair/transcription gene defects in the fetus and impaired TFIIH-mediated function in transcription in placenta leading to preeclampsia.

BackgroundPreeclampsia is a significant cause of maternal and fetal mortality and morbidity worldwide. We previously reported associations between trichothiodystrophy (TTD) nucleotide excision repair (NER) and transcription gene mutations in the fetus and the risk of gestational complications including preeclampsia. TTD NER/transcription genes, XPD, XPB and TTD-A, code for subunits of Transcription Factor (TF)IIH. Interpreting XPD mutations in the context of available biochemical data led us to propose adverse effects on CDK-activating kinase (CAK) subunit of TFIIH and TFIIH-mediated functions as a relevant mechanism in preeclampsia. In order to gain deeper insight into the underlying biologic mechanisms involving TFIIH-mediated functions in placenta, we analyzed NER/transcription and global gene expression profiles of normal and preeclamptic placentas and studied gene regulatory networks.ResultsWe found high expression of TTD NER/transcription genes in normal human placenta, above the mean of their expression in all organs. XPD and XPB were consistently expressed from 14 to 40 weeks gestation while expression of TTD-A was strongly negatively correlated (r = -0.7, P < 0.0001) with gestational age. Analysis of gene expression patterns of placentas from a case-control study of preeclampsia using Algorithm for Reconstruction of Accurate Cellular Networks (ARACNE) revealed GTF2E1, a component of TFIIE which modulates TFIIH, among major regulators of differentially-expressed genes in preeclampsia. The basal transcription pathway was among the largest dysregulated protein-protein interaction networks in this preeclampsia dataset. Within the basal transcription pathway, significantly down-regulated genes besides GTF2E1 included those coding for the CAK complex of TFIIH, namely CDK7, CCNH, and MNAT1. Analysis of other relevant gene expression and gene regulatory network data also underscored the involvement of transcription pathways and identified JUNB and JUND (components of transcription factor AP-1) as transcription regulators of the network involving the TTD genes, GTF2E1, and selected gene regulators implicated in preeclampsia.ConclusionsOur results indicate that TTD NER/transcription genes are expressed in placenta during gestational periods critical to preeclampsia development. Our overall findings suggest that impairment of TFIIH-mediated function in transcription in placenta is a likely mechanism leading to preeclampsia and provide etiologic clues which may be translated into therapeutic and preventive measures.

Phosphorylation of the Retinoic Acid Receptor Alpha Induces a Mechanical Allosteric Regulation and Changes in Internal Dynamics

Nuclear receptor proteins constitute a superfamily of proteins that function as ligand dependent transcription factors. They are implicated in the transcriptional cascades underlying many physiological phenomena, such as embryogenesis, cell growth and differentiation, and apoptosis, making them one of the major signal transduction paradigms in metazoans. Regulation of these receptors occurs through the binding of hormones, and in the case of the retinoic acid receptor (RAR), through the binding of retinoic acid (RA). In addition to this canonical scenario of RAR activity, recent discoveries have shown that RAR regulation also occurs as a result of phosphorylation. In fact, RA induces non-genomic effects, such as the activation of kinase signaling pathways, resulting in the phosphorylation of several targets including RARs themselves. In the case of RARα, phosphorylation of Ser369 located in loop L9–10 of the ligand-binding domain leads to an increase in the affinity for the protein cyclin H, which is part of the Cdk-activating kinase complex of the general transcription factor TFIIH. The cyclin H binding site in RARα is situated more than 40 Å from the phosphorylated serine. Using molecular dynamics simulations of the unphosphorylated and phosphorylated forms of the receptor RARα, we analyzed the structural implications of receptor phosphorylation, which led to the identification of a structural mechanism for the allosteric coupling between the two remote sites of interest. The results show that phosphorylation leads to a reorganization of a local salt bridge network, which induces changes in helix extension and orientation that affects the cyclin H binding site. This results in changes in conformation and flexibility of the latter. The high conservation of the residues implicated in this signal transduction suggests a mechanism that could be applied to other nuclear receptor proteins.

Abstract 694: Development of selective and potent CDK7 inhibitors for breast cancer therapy.

Abstract Cyclin dependent kinases (CDK) function as either regulators of cell cycle progression or transcription. CDK7 is unique from other CDKs because it has a dual role in the regulation of cell cycle and transcription making it a desirable target for drug development. It forms a complex with Cyclin H and MAT1 to form the CDK-activating kinase (CAK) that regulates CDK1, CDK2, CDK4 and CDK6, which are important in cell cycle progression. The CAK complex can also act as a transcriptional regulator as part of the TFIIH transcription factor by phosphorylating the COOH-terminal domain of RNA polymerase II (Pol II CTD). In addition, CDK7 as part of the CAK complex can inhibit the phosphorylation of serine 118 on the estrogen receptor (ER), a key player in breast cancer pathogenesis. Our target validation studies using siRNA have shown that CDK7 reduces growth of the human colorectal cancer cell line, HCT116, induces apoptosis and reduces phosphorylation of key biomarkers (RNA Poll II, CDK1 and CDK2). Using computer modelling of the CDK7 structure, we have designed CDK7 inhibitors, which were selected from a library of pyrazolo [1,5-a]pyrimidine-derived compounds. BS-181, is our first CDK7 inhibitor which inhibits CAK activity with an IC50 of 21 nmol/L. BS-181 has poor pharmacokinetic properties and is not orally bioavailable therefore after extensive studies and chemical modifications we have developed a more potent orally bioavailable CDK7 selective inhibitor, BS-181-L. Extensive in vitro ADME studies have highlighted that BS-181-L has good aqueous solubility, plasma proteins binding and no hERG liability. BS-181-L inhibits in vivo tumour cell growth in two cancer cell line models, MCF7 and HCT116 cells. In these studies BS-181-L shows no organ and haematological toxicity. We show BS-181-L down regulates key biomarkers in both the peripheral blood mononuclear cells (PBMCs) by flow cytometry and tumour tissue by immunohistochemistry (IHC) and western blotting. In addition, we show that BS-181-L reduces ER activity in reporter assays and ER (serine 118) phosphorylation. Furthermore, combination studies of BS-181-L and anti-estrogens show additive growth inhibitory effects on breast cancer cells. In summary, here we present data for a potent CDK7 inhibitor, BS-181-L, which has improved CDK7 selectivity, potency and bioavailability from our previously identified CDK7 inhibitor, BS-181. Furthermore, dual inhibition of CDK7 and ER has an additive effect in breast cancer cell growth. Taken together, this data highlights a potential new therapeutic approach for breast cancer. Citation Format: Hetal Patel, Silvia Ottaviani, Manikandan Periyasamy, Alexander Bondke, Brian Slafer, Richard Starkey, Sebastian H.B Kroll, Sean Delaney, Zahida Zahoor, Matthew J. Fuchter, Anthony G.M Barrett, R. Charles Coombes, Simak Ali. Development of selective and potent CDK7 inhibitors for breast cancer therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 694. doi:10.1158/1538-7445.AM2013-694

Abnormal XPD-induced nuclear receptor transactivation in DNA repair disorders: trichothiodystrophy and xeroderma pigmentosum

XPD (ERCC2) is a DNA helicase involved in nucleotide excision repair and in transcription as a structural bridge tying the transcription factor IIH (TFIIH) core with the cdk-activating kinase complex, which phosphorylates nuclear receptors. Mutations in XPD are associated with several different phenotypes, including trichothiodystrophy (TTD), with sulfur-deficient brittle hair, bone defects, and developmental abnormalities without skin cancer, xeroderma pigmentosum (XP), with pigmentary abnormalities and increased skin cancer, or XP/TTD with combined features, including skin cancer. We describe the varied clinical features and mutations in nine patients examined at the National Institutes of Health who were compound heterozygotes for XPD mutations but had different clinical phenotypes: four TTD, three XP, and two combined XP/TTD. We studied TFIIH-dependent transactivation by nuclear receptor for vitamin D (VDR) and thyroid in cells from these patients. The vitamin D stimulation ratio of CYP24 and osteopontin was associated with specific pairs of mutations (reduced in 5, elevated in 1) but not correlated with distinct clinical phenotypes. Thyroid receptor stimulation ratio for KLF9 was not significantly different from normal. XPD mutations frequently were associated with abnormal VDR stimulation in compound heterozygote patients with TTD, XP, or XP/TTD.

Lack of CAK complex accumulation at DNA damage sites in XP-B and XP-B/CS fibroblasts reveals differential regulation of CAK anchoring to core TFIIH by XPB and XPD helicases during nucleotide excision repair

Transcription factor II H (TFIIH) is composed of core TFIIH and Cdk-activating kinase (CAK) complexes. Besides transcription, TFIIH also participates in nucleotide excision repair (NER), verifying DNA lesions through its helicase components XPB and XPD. The assembly state of TFIIH is known to be affected by truncation mutations in xeroderma pigmentosum group G/Cockayne syndrome (XP-G/CS). Here, we showed that CAK component MAT1 was rapidly recruited to UV-induced DNA damage sites, co-localizing with core TFIIH component p62, and dispersed from the damage sites upon completion of DNA repair. While the core TFIIH–CAK association remained intact, MAT1 failed to accumulate at DNA damage sites in fibroblasts harboring XP-B or XP-B/CS mutations. Nevertheless, MAT1, XPD and XPC as well as XPG were able to accumulate at damage sites in XP-D fibroblasts, in which the core TFIIH-CAK association also remained intact. Interestingly, XPG recruitment was impaired in XP-B/CS fibroblasts derived from patients with mild phenotype, but persisted in XP-B/CS fibroblasts from severely affected patients resulting in a nonfunctional preincision complex. An examination of steady-state levels of RNA polymerase II (RNAPII) indicated that UV-induced RNAPII phosphorylation was dramatically reduced in XP-B/CS fibroblasts. These results demonstrated that the CAK rapidly disassociates from the core TFIIH upon assembly of nonfunctional preincision complex in XP-B and XP-B/CS cells. The persistency of nonfunctional preincision complex correlates with the severity exhibited by XP-B patients. The results suggest that XPB and XPD helicases differentially regulate the anchoring of CAK to core TFIIH during damage verification step of NER.

Tumor Suppressor Protein p53 Regulates Megakaryocytic Polyploidization and Apoptosis

The molecular mechanisms underlying differentiation of hematopoietic stem cells into megakaryocytes are poorly understood. Tumor suppressor protein p53 can act as a transcription factor affecting both cell cycle control and apoptosis, and we have previously shown that p53 is activated during terminal megakaryocytic (Mk) differentiation of the CHRF-288-11 (CHRF) cell line. Here, we use RNA interference to reduce p53 expression in CHRF cells and show that reduced p53 activity leads to a greater fraction of polyploid cells, higher mean and maximum ploidy, accelerated DNA synthesis, and delayed apoptosis and cell death upon phorbol 12-myristate 13-acetate-induced Mk differentiation. In contrast, reduced p53 expression did not affect the ploidy or DNA synthesis of CHRF cells in the absence of phorbol 12-myristate 13-acetate stimulation. Furthermore, primary Mk cells from cultures initiated with p53-null mouse bone marrow mononuclear cells displayed higher ploidy compared with wild-type controls. Quantitative reverse transcription-PCR analysis of p53-knockdown CHRF cells, compared with the "scrambled" control CHRF cells, revealed that six known transcriptional targets of p53 (BBC3, BAX, TP53I3, TP53INP1, MDM2, and P21) were down-regulated, whereas BCL2 expression, which is known to be negatively affected by p53, was up-regulated. These studies show that the functional role of the intrinsic activation of p53 during Mk differentiation is to control polyploidization and the transition to endomitosis by impeding cell cycling and promoting apoptosis.

Phosphorylation of Steroidogenic Factor 1 Is Mediated by Cyclin-Dependent Kinase 7

The nuclear receptor steroidogenic factor-1 (SF1) is critical for development and function of steroidogenic tissues. Posttranslational modifications are known to influence the transcriptional capacity of SF1, and it was previously demonstrated that serine 203 is phosphorylated. In this paper we report that serine 203 is phosphorylated by a cyclin-dependent kinase 7 (CDK7)-mediated process. As part of the CDK-activating kinase complex, CDK7 is a component of the basal transcription factor TFIIH, and phosphorylation of SF1 as well as SF1-dependent transcription was clearly reduced in cells carrying a mutation that renders the CDK-activating kinase complex unable to interact with the TFIIH core. Coimmunoprecipitation analyses revealed that SF1 and CDK7 reside in the same complex, and kinase assays demonstrated that immunoprecipitated CDK7 and purified TFIIH phosphorylate SF1 in vitro. The CDK inhibitor roscovitine blocked phosphorylation of SF1, and an inactive form of CDK7 repressed the phosphorylation level and the transactivation capacity of SF1. Structural studies have identified phosphoinositides as potential ligands for SF1. Interestingly, we found that mutations designed to block phospholipid binding dramatically decreased the level of SF1 phosphorylation. Together our results suggest a connection between ligand occupation and phosphorylation and association with the basic transcriptional machinery, indicating an intricate regulation of SF1 transactivation.

Cyclin H is targeted to the nucleus by C-terminal nuclear localization sequences

Cdk-activating kinase (CAK) is a trimeric complex consisting of cdk7, cyclin H, and MAT1, which activates the cell-cycle-regulating cdks through T loop phosphorylation. In addition, other substrates of the CAK complex have been identified when CAK is assembled with the TFIIH core proteins, thereby regulating transcription and nucleotide excision repair. Little is known about the regulation of the CAK complex through cyclin H. In this study we further analyzed cyclin H regulation and identified two basic clusters in the C terminus of the protein as putative nuclear localization sequences (NLSs). Fusion constructs of full-length and truncated cyclin H sequences demonstrated the functionality of the NLSs. A peptide-binding assay revealed that at least one NLS interacts with the nuclear import receptors importin alpha/beta. Phosphorylation in the vicinity of the NLSs by cyclin C/cdk8 or protein kinase CK2, however, does not influence the nuclear translocation of cyclin H.

Solution Structure of the C-terminal Domain of TFIIH P44 Subunit Reveals a Novel Type of C4C4 Ring Domain Involved in Protein-Protein Interactions

The human general transcription factor TFIIH is involved in both transcription and DNA nucleotide excision repair. Among the 10 subunits of the complex, p44 subunit plays a crucial role in both mechanisms. Its N-terminal domain interacts with the XPD helicase, whereas its C-terminal domain is involved specifically in the promoter escape activity. By mutating an exposed and non-conserved cysteine residue into a serine, we produced a soluble mutant of p44-(321-395) suitable for solution structure determination. The domain adopts a C4C4 RING domain structure with sequential organization of beta-strands that is related to canonical RING domains by a circular permutation of the beta-sheet elements. Analysis of the molecular surface and mutagenesis experiments suggests that the binding of p44-(321-395) to TFIIH p34 subunit is not mediated by electrostatic interactions and, thus, differs from previously reported interaction mechanisms involving RING domains.

Hepatitis C Virus Core Functions as a Suppressor of Cyclin-dependent Kinase-activating Kinase and Impairs Cell Cycle Progression

We investigated how the hepatitis C virus (HCV) core protein affects the cell cycle profile and cell cycle-related molecules by using the HCV core-expressing stable transfectant. Analysis of the cell cycle profile showed that HCV core impaired G(1) to S transition. The E2F-mediated transcription, phosphorylation of the retinoblastoma protein, and cyclin-dependent kinase (CDK) 4 and CDK2 activities were suppressed in HCV core-expressing cells. The expression levels of G(1) phase-related CDKs/cyclins and various CDK inhibitors were not substantially affected by expression of HCV core. When influences of HCV core on CDK-activating kinase (CAK) were examined, the expression levels of the CAK components, CDK7, cyclin H, and MAT1, were not affected. However, formation of the ternary CAK complex, CAK activity, and the CDK2 level with activating phosphorylation were inhibited by expression of the HCV core. The direct effect of HCV core on CAK was further assessed in the cell-free system by adding the in vitro translated HCV core protein to the anti-CDK7 immunoprecipitate from the cell. The results showed that HCV core led to dissociation of MAT1 from the CAK complex and suppressed the CAK activity. Furthermore, the binding assay revealed that the HCV core was directed against CDK7. Their interaction occurred mainly in the nucleus by the immunostaining. In conclusion, the HCV core protein interacts with CAK and functions as an extrinsic suppressor of CAK. This may be the molecular basis of HCV core-mediated suppression of cell cycle progression. Our findings suggest a novel mechanism concerning HCV core-mediated alteration in the cell cycle machinery.

Modulation of MDM2/p53 and cyclin-activating kinase during the megakaryocyte differentiation of human erythroleukemia cells.

This study was undertaken to address the involvement of CDK activating kinase (CAK), p53, and MDM2 proteins in the mitotic arrest associated with the acquisition of a polyploid DNA content during megakaryocyte differentiation of human erythroleukemia (HEL) cells. To evaluate this mechanism we investigated HEL cells as a model system in which there is a marked increase in DNA content during megakaryocyte differentiation induced by phorbol-diesters. Specific cell-cycle phases were separated by centrifugal elutriation and SDS PAGE and Western analysis were performed to determine the relative abundance of these proteins. Kinase assays were carried out following immunoprecipitation of cellular lysates with the antibodies to the proteins. Polyploid HEL cells show an increase in the abundance of the CAK complex proteins, CDK7 and cyclin H, and a sixfold increase in CAK-specific activity. Increased CAK activity in polyploid HEL cells follows both the downregulation of p53 protein and its decreased association with CAK complex. Consistent with the reduction of p53, polyploid HEL cells undergo a dramatic increase in MDM2 protein abundance that in turn facilitates increased interaction of this protein with p53. These observations demonstrate that deregulated expression of MDM2 and p53 during megakaryocyte differentiation allow a relaxation of the control over genomic stability, allowing further replicative rounds of DNA synthesis.

A Yeast Four-hybrid System Identifies Cdk-activating Kinase as a Regulator of the XPD Helicase, a Subunit of Transcription Factor IIH

To understand the role of the various components of TFIIH, a DNA repair/transcription factor, a yeast four-hybrid system was designed. When the ternary Cdk-activating kinase (CAK) complex composed of Cdk7, cyclin H, and MAT1 was used as bait, the xeroderma pigmentosum (XP) D helicase of transcription factor IIH (TFIIH), among other proteins, was identified as an interacting partner. Deletion mutant analyses demonstrated that the coiled-coil and the hydrophobic domains of MAT1 interlink the CAK complex directly with the N-terminal domain of XPD. Using immunoprecipitates from cells coinfected with baculoviruses, we further validated the bridging function of XPD, which anchors CAK to the core TFIIH. In addition we show that upon interaction with MAT1, CAK inhibits the helicase activity of XPD. This inhibition is overcome upon binding to p44, a subunit of the core TFIIH. It is not surprising that under these conditions some XPD mutations affect interactions not only with p44, but also with MAT1, thus preventing either the CAK inhibitory function within CAK.XPD and/or the role of CAK within TFIIH and, consequently, explaining the variety of the XP phenotypes.

T-loop phosphorylation stabilizes the CDK7-cyclin H-MAT1 complex in vivo and regulates its CTD kinase activity.

Cyclin-dependent kinase (CDK)7-cyclin H, the CDK-activating kinase (CAK) and TFIIH-associated kinase in metazoans can be activated in vitro through T-loop phosphorylation or binding to the RING finger protein MAT1. Although the two mechanisms can operate independently, we show that in a physiological setting, MAT1 binding and T-loop phosphorylation cooperate to stabilize the CAK complex of Drosophila. CDK7 forms a stable complex with cyclin H and MAT1 in vivo only when phosphorylated on either one of two residues (Ser164 or Thr170) in its T-loop. Mutation of both phosphorylation sites causes temperature-dependent dissociation of CDK7 complexes and lethality. Furthermore, phosphorylation of Thr170 greatly stimulates the activity of the CDK7- cyclin H-MAT1 complex towards the C-terminal domain of RNA polymerase II without significantly affecting activity towards CDK2. Remarkably, the substrate-specific increase in activity caused by T-loop phosphorylation is due entirely to accelerated enzyme turnover. Thus phosphorylation on Thr170 could provide a mechanism to augment CTD phosphorylation by TFIIH-associated CDK7, and thereby regulate transcription.

Structural Characterization of the Cysteine-rich Domain of TFIIH p44 Subunit

In an effort to understand the structure function relationship of TFIIH, a transcription/repair factor, we focused our attention on the p44 subunit, which plays a central role in both mechanisms. The amino-terminal portion of p44 has been shown to be involved in the regulation of the XPD helicase activity; here we show that its carboxyl-terminal domain is essential for TFIIH transcription activity and that it binds three zinc atoms through two independent modules. The first contains a C4 zinc finger motif, whereas the second is characterized by a CX(2)CX(2-4)FCADCD motif, corresponding to interleaved zinc binding sites. The solution structure of this second module reveals an unexpected homology with the regulatory domain of protein kinase C and provides a framework to study its role at the molecular level.