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.

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