The retinoblastoma family of proteins, also known as pocket proteins, includes the product of the retinoblastoma susceptibility gene and the functionally and structurally related proteins p107 and p130. Pocket proteins control growth processes in many cell types, and this has been linked to the ability of pocket proteins to interact with a multitude of cellular proteins that regulate gene expression at various levels. By regulating gene expression, pocket proteins control cell cycle progression, cell cycle entry and exit, cell dierentiation and apoptosis. This review will focus on the mechanisms of regulation of pocket proteins and how modulation of pocket protein levels and phosphorylation status regulate association with their cellular targets. The coordinated regulation of pocket proteins provides the cells with a competence mechanism for passage through certain cell growth and dierentiation transitions.

Members of the cell division cycle 2 (CDC2) family of kinases play a pivotal role in the regulation of the eukaryotic cell cyde. In this communication, we report the isolation of a cDNA that encodes a CDC2-related human protein kinase temporarily designated PITALRE for the characteristic Pro-Ile-Thr-Ala-Leu-Arg-Glu motif. Its deduced amino acid sequence is 47% identical to that of the human cholinesterase-related cell division controller (CHED) kinase, which is required during hematopoiesis, and 42% identical to the Saccharomyces cerewasle SGV1 gene product, a putative kinase involved in the response to pheromone via its guanine nucleotide-binding protein a subunit. PITALRE expression is ubiquitous, but its expression levels are different in various human tissues. PITALRE is an =43-kDa protein that associates with three cellular polypeptides of 80, 95, and 155 kDa. PITALRE is lad primarily to the nucleus. In addition, we have identified a retinoblastoma protein kinase activity associated with PITALRE Immunocomplexes that cannot phosphorylate histone Hi, suggesting that the target phosphorylation site of PITALRE differs from that of CDC2 kinase. Interestingly, the retinoblastoma kinase activity associated with PITALRE does not oscillate during the cell cycle.The cell cycle in eukaryotes is regulated by a sequence of restriction points. In yeast, the first restriction point occurs during the G1 phase prior to the DNA synthesis and the second occurs before the initiation of mitosis. In Saccharomyces cerevisiae, the cell division cycle 28 (CDC28) kinase controls both restriction points through association with the CLN cyclins in G1 and with CLB cyclins in G2/M (1). In vertebrate cells, the regulatory mechanisms involved in cell cycle progression are more complex. CDC2 kinase, in association with cyclin B, appears to be a universal regulator of the eukaryotic entry into mitosis. However, in G1, just before the onset of DNA synthesis, cyclin-dependent kinase 2 (CDK2), but not CDC2, is required (2, 3). Additional mammalian CDC2-related kinases have been isolated that share >40% identity at the amino acid level (4)(5)(6)(7)(8)(9)(10)(11) This indicates that CDK4-cyclin D complexes possess a different phosphorylation specificity than the CDC2 kinase. Nevertheless, no kinase activity has been detected in CDK4 immunocomplexes (12). The association of CDK5 with cyclins D1/D3 and with proliferating cell nuclear antigen (PCNA) suggests a role for this kinase in the cell cycle (13). However, the high levels of expression of cdk5 found in neurons, cells no longer dividing, indicate a role for CDK5 in terminally differentiated cells (11). The study of CDC2 and CDC2-related kinases over the past few years has revealed a key role for these kinases in the regulation of the cell cycle. Most recently, an involvement in differentiation processes has also been proposed (8,11).With the aim of isolating additional putative controllers of the mammalian cell cycle, we performed a combination of PCR amplification and low-stringency ...

SUMMARY: Cyclin-Dependent Kinase 9 (CDK9) promotes transcriptional elongation through RNAPII pause release. We now report that CDK9 is also essential for maintaining gene silencing at heterochromatic loci. Through a live cell drug screen with genetic confirmation, we discovered that CDK9 inhibition reactivates epigenetically silenced genes in cancer, leading to restored tumor suppressor gene expression, cell differentiation, and activation of endogenous retrovirus genes. CDK9 inhibition dephosphorylates the SWI/SNF protein BRG1, which contributes to gene reactivation. By optimization through gene expression, we developed a highly selective CDK9 inhibitor (MC180295, IC50=5nM) that has broad anti-cancer activity in-vitro and is effective in in-vivo cancer models. Additionally, CDK9 inhibition sensitizes to the immune checkpoint inhibitor α-PD-1 in vivo, making it an excellent target for epigenetic therapy of cancer.

Cyclin T1 has been identi®ed recently as a regulatory subunit of CDK9 and as a component of the transcription elongation factor P-TEFb. Cyclin T1/CDK9 complexes phosphorylate the carboxy terminal domain (CTD) of RNA polymerase II (RNAP II) in vitro. Here we report that the levels of cyclin T1 are dramatically upregulated by two independent signaling pathways triggered respectively by PMA and PHA in primary human peripheral blood lymphocytes (PBLs). Activation of these two pathways in tandem is su cient for PBLs to enter and progress through the cell cycle. However, the expression of cyclin T1 is not growth and/or cell cycle regulated in other cell types, indicating that regulation of cyclin T1 expression is dependent on tissue-speci®c signaling pathways. Upregulation of cyclin T1 in stimulated PBLs results in induction of the CTD kinase activity of the cyclin T1/CDK9 complex, which in turn correlates directly with phosphorylation of RNAP II in vivo, linking for the ®rst time activation of the cyclin T1/ CDK9 pair with phosphorylation of RNAP II in vivo. In addition, we report here that endogenous CDK9 and cyclin T1 complexes associate with HIV-1 generated Tat in relevant cells and under physiological conditions (HIV-1 infected T cells). This, together with our results showing that HIV-1 replication in stimulated PBLs correlates with the levels of cyclin T1 protein and associated CTD kinase activity, suggests that the cyclin T1/CDK9 pair is one of the HIV-1 required host cellular cofactors generated during T cell activation.

No abstract

The positive transcription elongation factor b (P-TEFb) is involved in physiological and pathological events including inflammation, cancer, AIDS, and cardiac hypertrophy. The balance between its active and inactive form is tightly controlled to ensure cellular integrity. We report that the transcriptional repressor CTIP2 is a major modulator of P-TEFb activity. CTIP2 copurifies and interacts with an inactive P-TEFb complex containing the 7SK snRNA and HEXIM1. CTIP2 associates directly with HEXIM1 and, via the loop 2 of the 7SK snRNA, with P-TEFb. In this nucleoprotein complex, CTIP2 significantly represses the Cdk9 kinase activity of P-TEFb. Accordingly, we show that CTIP2 inhibits large sets of P-TEFb-and 7SK snRNA-sensitive genes. In hearts of hypertrophic cardiomyopathic mice, CTIP2 controls P-TEFb-sensitive pathways involved in the establishment of this pathology. Overexpression of the β-myosin heavy chain protein contributes to the pathological cardiac wall thickening. The inactive P-TEFb complex associates with CTIP2 at the MYH7 gene promoter to repress its activity. Taken together, our results strongly suggest that CTIP2 controls P-TEFb function in physiological and pathological conditions. D iscovered in 1995 (1), P-TEFb (CyclinT1/Cdk9) is involved in physiological and pathological transcriptionally regulated events such as cell growth, differentiation, cancer, cardiac hypertrophy, and AIDS (for review, see refs. 2 and 3). It has been suggested to be required for transcription of most RNA polymerase II-dependent genes. However, a recent study suggests that a subset of cellular genes are distinctively sensitive to Cdk9 inhibition (4). P-TEFb is dynamically regulated by both positive and negative regulators. In contrast to Brd4, which is associated with the active form of P-TEFb (5, 6), the 7SK small nuclear RNA (7SK snRNA) and HEXIM1 inhibit Cdk9 activity in the inactive P-TEFb complex (7-10). P-TEFb elongation complexes are crucial for HIV-1 gene transactivation and viral replication. Recently, new P-TEFb complexes containing the HIV-1 Tat protein have been characterized (11, 12), providing evidence for the recruitment of an inactive Tat/P-TEFb complex to the HIV-1 promoter (13). However, defining the diverse nature and functions of the different P-TEFb complexes will require further investigations. The cellular protein CTIP2 (Bcl11b) has been highlighted as a key transcription factor for thymocyte (14,15) and neuron development (16), odontogenesis (17), cancer evolution (18), and HIV-1 gene silencing (19). Besides AIDS, hypertrophic cardiomyopathy is a well-described P-TEFb-dependent pathology (for review, see refs. 20 and 21).Here, we report that CTIP2 represses P-TEFb function as part of an inactive P-TEFb complex. In hearts of hypertrophic cardiomyopathic mice, CTIP2 controls P-TEFb-sensitive pathways involved in the establishment of this pathology. Together with the inactive P-TEFb complex, CTIP2 associates with the β-myosin heavy chain promoter to repress its activity. Thereby, CTIP2 might contrib...

Mitogenic stimulation leads to activation of G 1 cyclindependent kinases (CDKs), which phosphorylate pocket proteins and trigger progression through the G 0 /G 1 and G 1 /S transitions of the cell cycle. However, the individual role of G 1 cyclin-CDK complexes in the coordinated regulation of pocket proteins and their interaction with E2F family members is not fully understood. Here we report that individually or in concert cyclin D1-CDK and cyclin E-CDK complexes induce distinct and coordinated phosphorylation of endogenous pocket proteins, which also has distinct consequences in the regulation of pocket protein interactions with E2F4 and the expression of p107 and E2F1, both E2F-regulated genes. The up-regulation of these two proteins and the release of p130 and pRB from E2F4 complexes allows formation of E2F1 complexes not only with pRB but also with p130 and p107 as well as the formation of p107-E2F4 complexes. The formation of these complexes occurs in the presence of active cyclin D1-CDK and cyclin E-CDK complexes, indicating that whereas phosphorylation plays a role in the abrogation of certain pocket protein/E2F interactions, these same activities induce the formation of other complexes in the context of a cell expressing endogenous levels of pocket and E2F proteins. Of note, phosphorylated p130 "form 3," which does not interact with E2F4, readily interacts with E2F1. Our data also demonstrate that ectopic overexpression of either cyclin is sufficient to induce mitogen-independent growth in human T98G and Rat-1 cells, although the effects of cyclin D1 require downstream activation of cyclin E-CDK2 activity. Interestingly, in T98G cells, cyclin D1 induces cell cycle progression more potently than cyclin E. This suggests that cyclin D1 activates pathways independently of cyclin E that ensure timely progression through the cell cycle.G 1 cyclin-dependent kinases (CDKs) 1 regulate progression through the G 0 /G 1 transition and entry into the S-phase of the cell cycle following activation by mitogenic signaling pathways (1-5). G 1 CDKs phosphorylate the three members of the retinoblastoma family of pocket proteins, pRB, p107, and p130, resulting in cell cycle-dependent inactivation of their growth suppressor activities (6 -13) (reviewed in Ref. 14).Ectopic expression of cyclin D1 and cyclin E in primary or immortal, nontransformed mammalian fibroblasts shortens the G 1 phase of the cell cycle (15-17). The relatively modest effects of ectopic expression of G 1 cyclins in primary or immortal, nontransformed mammalian fibroblasts are probably due to a requirement for additional events to ensure full activation of these complexes. Whereas cyclins are limiting subunits for activation of their corresponding CDKs, full activation of cyclin-CDK complexes requires other events also dependent upon mitogenic stimulation (reviewed in . In agreement with this idea, microinjection of purified recombinant active cyclin D1-CDK4 or cyclin E-CDK2 complexes in human primary lung fibroblasts bypasses the requirement for mitogen...

Background and Aims-Hepatocellular carcinoma is the third leading cause of cancer mortality worldwide and current chemotherapeutic interventions for this disease are largely ineffective. The retinoblastoma tumor suppressor (RB) is functionally inactivated at relatively high frequency in hepatocellular carcinoma and hepatoma cell lines. Here we interrogated the ability of CDK4/6-inhibition to inhibit hepatocyte proliferation and the impact of RB-status on this process.