Cyclin-dependent Protein Kinase Family Research Articles

An Interpretable Multitask Framework BiLAT Enables Accurate Prediction of Cyclin-Dependent Protein Kinase Inhibitors.

The cyclin-dependent protein kinases (CDKs) are protein-serine/threonine kinases with crucial effects on the regulation of cell cycle and transcription. CDKs can be a hallmark of cancer since their excessive expression could lead to impaired cell proliferation. However, the selectivity profile of most developed CDK inhibitors is not enough, which have hindered the therapeutic use of CDK inhibitors. In this study, we propose a multitask deep learning framework called BiLAT based on SMILES representation for the prediction of the inhibitory activity of molecules on eight CDK subtypes (CDK1, 2, 4-9). The framework is mainly composed of an improved bidirectional long short-term memory module BiLSTM and the encode layer of the Transformer framework. Additionally, the data enhancement method of SMILES enumeration is applied to improve the performance of the model. Compared with baseline predictive models based on three conventional machine learning methods and two multitask deep learning algorithms, BiLAT achieves the best performance with the highest average AUC, ACC, F1-score, and MCC values of 0.938, 0.894, 0.911, and 0.715 for the test set. Moreover, we constructed a targeted external data set CDK-Dec for the CDK family, which mainly contains bait values screened by 3D similarity with active compounds. This dataset was utilized in the subsequent evaluation of our model. It is worth mentioning that the BiLAT model is interpretable and can be used by chemists to design and synthesize compounds with improved activity. To further verify the generalization ability of the multitask BiLAT model, we also conducted another evaluation on three public datasets (Tox21, ClinTox, and SIDER). Compared with several currently popular models, BiLAT shows the best performance on two datasets. These results indicate that BiLAT is an effective tool for accelerating drug discovery.

MEDB-80. CDK8 promotes stemness of MYC-driven medulloblastoma

Cyclin-dependent kinase 8 (CDK8) belongs to the transcription-related cyclin dependent protein kinase family. CDK8 and cyclin C associate with the mediator complex to regulate gene transcription. Although CDK8 has been shown to be implicated in the malignancy of several types of cancer, its functional role and mechanism in medulloblastoma remains largely unknown. Here, we demonstrate how CDK8 plays an essential role in maintaining stemness and tumorigenicity in medulloblastoma stem cell. CDK8 inhibition suppresses stem cell associated signaling in medulloblastoma cells and inhibits tumor cell self-renewal. Additionally, CDK8 is amplified in MYC-driven medulloblastoma, is positively correlated with c-MYC expression in human medulloblastoma specimens and associates with poor survival in patients. Using cut&run assay, we found CDK8 associates with MED1 to activate transcription of MYC target genes. CDK8 attributes to MYC-driven transcriptional programs mediating DNA repair. Pharmaceutic inhibitors and genetic depletion result in cessation of tumor growth in xenograft mouse models and increase in apoptosis and DNA damage. Collectively, our studies establish the selective inhibition of CDK8 inhibition as a viable therapeutic strategy in MYC-driven medulloblastoma.

Abstract 1781: Transcriptional activation of CBFβ by CDK11p110 is necessary to promote osteosarcoma cell proliferation

Abstract Aberrant expression and activation of cyclin-dependent protein kinase (CDK) is a hallmark of cancer. CDK11 is a protein kinase in the CDK family and plays a crucial role in cancer cell growth and proliferation. However, the molecular mechanisms of CDK11 in osteosarcoma and CDK11 transcriptional regulated genes are largely unknown. In this study, we performed global transcriptional analysis using gene array technology to investigate the transcriptional role of CDK11 in osteosarcoma. The promoter luciferase assay, chromatin immunoprecipitation assay, and Gel Shift assay were used to identify direct transcriptional target of CDK11. Clinical significance relevance and function of CBFβ were further accessed in osteosarcoma tissue microarray and in osteosarcoma cell lines. We identified a transcriptional role of protein-DNA interaction for CDK11p110, but not CDK11p58, in the regulation of core-binding factor subunit beta (CBFβ) expression in osteosarcoma cells. The CBFβ promoter luciferase assay, chromatin immunoprecipitation assay, and Gel Shift assay confirmed that CBFβ is a direct transcriptional target of CDK11. High expression of CBFβ is associated with poor outcome in osteosarcoma patients. Expression of CBFβ contributes to the proliferation and metastatic behavior of osteosarcoma cells. These data establish CBFβas a mediator of CDK11p110dependent oncogenesis and suggest that targeting the CDK11-CBFβ pathway may be a promising therapeutic strategy for osteosarcoma treatment. Citation Format: Yong Feng, Francis Hornicek, Zhenfeng Duan. Transcriptional activation of CBFβ by CDK11p110 is necessary to promote osteosarcoma cell proliferation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1781.

Structure-based discovery of cyclin-dependent protein kinase inhibitors.

The cell fate-determining roles played by members of the cyclin-dependent protein kinase (CDK) family explain why their dysregulation can promote proliferative diseases, and identify them as potential targets for drug discovery in oncology and beyond. After many years of research, the first efficacious CDK inhibitors have now been registered for clinical use in a defined segment of breast cancer. Research is underway to identify inhibitors with appropriate CDK-inhibitory profiles to recapitulate this success in other disease settings. Here, we review the structural data that illustrate the interactions and properties that confer upon inhibitors affinity and/or selectivity toward different CDK family members. We conclude that where CDK inhibitors display selectivity, that selectivity derives from exploiting active site sequence peculiarities and/or from the capacity of the target CDK(s) to access conformations compatible with optimizing inhibitor-target interactions.

Chemically induced degradation of CDK9 by a proteolysis targeting chimera (PROTAC).

Cyclin-dependent kinase 9 (CDK9), a member of the cyclin-dependent protein kinase (CDK) family, is involved in transcriptional elongation of several target genes. CDK9 is ubiquitously expressed and has been shown to contribute to a variety of malignancies such as pancreatic, prostate and breast cancers. Here we report the development of a heterobifunctional small molecule proteolysis targeting chimera (PROTAC) capable of cereblon (CRBN) mediated proteasomal degradation of CDK9. In HCT116 cells, it selectively degrades CDK9 while sparing other CDK family members. This is the first example of a PROTAC that selectively degrades CDK9.

Identification and Characterization of the Cyclin-Dependent Kinases Gene Family in Silkworm, Bombyx mori.

Cyclin-dependent protein kinases (CDKs) play key roles at different checkpoint regulations of the eukaryotic cell cycle. However, only few studies of lepidoptera CDK family proteins have been reported so far. In this study, we performed the cDNA sequencing of 10 members of the CDK family in Bombyx mori. Gene structure analysis suggested that CDK12 and CDC2L1 owned two and three isoforms, respectively. Phylogenetic analysis showed that CDK genes in different species were highly conserved, implying that they evolved independently even before the split between vertebrates and invertebrates. We found that the expression levels of BmCDKs in 13 tissues of fifth-instar day 3 larvae were different: CDK1, CDK7, and CDK9 had a high level of expression, whereas CDK4 was low-level expressed and was detected only in the testes and fat body cells. Similar expression profiles of BmCDKs during embryo development were obtained. Among the variants of CDK12, CDK12 transcript variant A had the highest expression, and the expression of CDC2L1 transcript variant A was the highest among the variants of CDC2L1. It was shown from the RNAi experiments that the silencing of CDK1, CDK10, CDK12, and CDC2L1 could influence the cells from G0/G1 to S phase transition.

Structural characterization of the cyclin-dependent protein kinase family

Structural studies of members of the CDK (cyclin-dependent protein kinase) family have made a significant contribution to our understanding of the regulation of protein kinases. The structure of monomeric unphosphorylated CDK2 was the first of an inactive protein kinase to be determined and, since then, structures of other members of the CDK family, alone, in complex with regulatory proteins and in differing phosphorylation states, have enhanced our understanding of the molecular mechanisms regulating protein kinase activity. Recently, our knowledge of the structural biology of the CDK family has been extended by determination of structures for members of the transcriptional CDK and CDK-like kinase branches of the extended family. We include these recent structures in the present review and consider them in the light of current models for CDK activation and regulation.

Zebrafish Cyclin-Dependent Protein Kinase–Like 1 (zcdkl1): Identification and Functional Characterization

The cyclin-dependent protein kinase family regulates a wide range of cellular functions such as cell cycle progression, differentiation, and apoptosis. In this study, we identified a zebrafish cyclin-dependent protein kinase-like 1 protein called zebrafish cdkl1 (zcdkl1), which shared a high degree of homology and conserved synteny with mammalian orthologs. zcdkl1 exhibited abilities for phosphorylation of myelin basic protein and histone H1. RT-PCR analysis revealed that zcdkl1 was expressed starting from fertilization and continuing thereafter. In adult tissues, zcdkl1 was predominantly detected in brain, ovary, and testis, and was expressed at low levels in other tissues. At 50% epiboly stage, zcdkl1 was widely expressed. At 12 to 48 h post-fertilization, zcdkl1 was predominantly expressed in the hypochord, the medial and lateral floor plate, and the pronephric duct. Interference of zcdkl1 expression resulted in abnormalities, such as brain and eye malformation, pericardial edema, and body axis curvature. Disruption of zcdkl1 reduced neurogenin-1 in the brain and sonic hedgehog expression in the floor plate region. These deformities were apparently rescued by co-injection of zcdkl1 mRNA. Findings of this study indicate that zcdkl1 plays an essential role in zebrafish development.

A Novel Pyrazolo[1,5-a]pyrimidine Is a Potent Inhibitor of Cyclin-Dependent Protein Kinases 1, 2, and 9, Which Demonstrates Antitumor Effects in Human Tumor Xenografts Following Oral Administration

Cyclin-dependent protein kinases (CDKs) are central to the appropriate regulation of cell proliferation, apoptosis, and gene expression. Abnormalities in CDK activity and regulation are common features of cancer, making CDK family members attractive targets for the development of anticancer drugs. Here, we report the identification of a pyrazolo[1,5-a]pyrimidine derived compound, 4k (BS-194), as a selective and potent CDK inhibitor, which inhibits CDK2, CDK1, CDK5, CDK7, and CDK9 (IC₅₀= 3, 30, 30, 250, and 90 nmol/L, respectively). Cell-based studies showed inhibition of the phosphorylation of CDK substrates, Rb and the RNA polymerase II C-terminal domain, down-regulation of cyclins A, E, and D1, and cell cycle block in the S and G₂/M phases. Consistent with these findings, 4k demonstrated potent antiproliferative activity in 60 cancer cell lines tested (mean GI₅₀= 280 nmol/L). Pharmacokinetic studies showed that 4k is orally bioavailable, with an elimination half-life of 178 min following oral dosing in mice. When administered at a concentration of 25 mg/kg orally, 4k inhibited human tumor xenografts and suppressed CDK substrate phosphorylation. These findings identify 4k as a novel, potent CDK selective inhibitor with potential for oral delivery in cancer patients.

Recognition of Cdk2 by Cdk7

Cdk7, a member of the cyclin dependent protein kinase family, regulates the activities of other Cdks through phosphorylation on their activation segment, and hence contributes to control of the eukaryotic cell cycle. Cdk7 is itself phosphorylated on the activation segment. Cdk7 phosphorylates Cdk1, Cdk2, Cdk4, and Cdk6, but only Cdk1 and Cdk2 can phosphorylate Cdk7 and none of them is able to auto-phosphorylate. The activation segments of the Cdks are very similar in sequence. Their specificity does not appear to be dictated by the sequences surrounding the phosphorylation sites but by structural determinants at remote sites. Through mutagenesis studies, we have identified regions in Cdk2 responsible for its interaction with Cdk7. A model has been built that explains the molecular basis for the specificity observed in Cdk recognition. The two kinases are arranged in a quasi-symmetric head-to-tail arrangement in which the N-terminal lobe from one kinase docks against the C-terminal lobe from the other kinase, and the activation segments are within reach of the opposite catalytic sites. Further experiments demonstrate that cyclin A hydrophobic pocket is not a recruitment site for Cdk7.

The crystal structure of human CDK7 and its protein recognition properties

CDK7, a member of the cyclin-dependent protein kinase family, regulates the activities of other CDKs through phosphorylation on their activation segment and hence contributes to control of the eukaryotic cell cycle. CDK7 also assists in the regulation of transcription as part of the transcription factor TFIIH complex. For maximum activity and stability, CDK7 requires phosphorylation, association with cyclin H, and association with a third protein, MAT1. We have determined the crystal structure of human CDK7 in complex with ATP at 3 A resolution. The kinase is in the inactive conformation, similar to that observed for inactive CDK2. The activation segment is phosphorylated at Thr170 and is in a defined conformation that differs from that in phospho-CDK2 and phospho-CDK2/cyclin A. The functional properties of the enzyme against CDK2 and CTD as substrates are characterized through kinase assays. Experiments confirm that CDK7 is not a substrate for kinase-associated phosphatase.

The Crystal Structure of Human CDK7 and Its Protein Recognition Properties

CDK7, a member of the cyclin-dependent protein kinase family, regulates the activities of other CDKs through phosphorylation on their activation segment and hence contributes to control of the eukaryotic cell cycle. CDK7 also assists in the regulation of transcription as part of the transcription factor TFIIH complex. For maximum activity and stability, CDK7 requires phosphorylation, association with cyclin H, and association with a third protein, MAT1. We have determined the crystal structure of human CDK7 in complex with ATP at 3 A resolution. The kinase is in the inactive conformation, similar to that observed for inactive CDK2. The activation segment is phosphorylated at Thr170 and is in a defined conformation that differs from that in phospho-CDK2 and phospho-CDK2/cyclin A. The functional properties of the enzyme against CDK2 and CTD as substrates are characterized through kinase assays. Experiments confirm that CDK7 is not a substrate for kinase-associated phosphatase.

Phosphorylation of Phosphatase Inhibitor-2 at Centrosomes during Mitosis

Inhibitor-2 (I-2) is a regulator of protein phosphatase type-1 (PP1), known to be phosphorylated in vitro by multiple kinases. In particular Thr72 is a Thr-Pro phosphorylation site conserved from yeast to human, but there is no evidence that this phosphorylation responds to any physiological signals. Here, we used electrophoretic mobility shift and immunoblotting with a site-specific phospho-Thr72 antibody to establish Thr72 phosphorylation in HeLa cells and show a 25-fold increase in phosphorylation during mitosis. Mass spectrometry demonstrated I-2 in actively growing HeLa cells was also phosphorylated at three other sites, Ser120, Ser121, and an additional Ser located between residues 70 and 90. In vitro kinase assays using recombinant I-2 as a substrate showed that the Thr72 kinase(s) was activated during mitosis, and sensitivity to kinase inhibitors indicated that the principal I-2 Thr72 kinase was not GSK3 but instead a member of the cyclin-dependent protein kinase family. Immunocytochemistry confirmed Thr72 phosphorylation of I-2 during mitosis, with peak intensity at prophase, and revealed subcellular concentration of the phospho-Thr72 I-2 at centrosomes. Together, the data show dynamic changes in I-2 phosphorylation during mitosis and localization of phosphorylated I-2 at centrosomes, suggesting involvement in mammalian cell division.

Inhibiting cyclin-dependent kinase/cyclin activity for the treatment of cancer and cardiovascular disease.

Excessive cell proliferation contributes to the pathobiology of human diseases with a high health and socio-economic impact, including cancer and vascular occlusive diseases (e. g., atherosclerosis, in-stent restenosis, transplant vasculopathy, and vessel bypass graft failure). Recent advances in the understanding of the molecular networks governing the hyperplastic growth of tumors and vascular obstructive neointimal lesions have provided new perspectives for preventive and therapeutic strategies against these disorders. Mammalian cell proliferation requires the activation of several cyclin-dependent protein kinases (CDKs). Postranslational activation of CDKs is a complex process that involves their association with regulatory subunits called cyclins. The activity of CDK/cyclin holoenzymes is negatively regulated through their interaction with members of the CDK family of inhibitory proteins (CKIs). Moreover, over fifty low molecular weight pharmacological CDK inhibitors that target the ATP-binding pocket of the catalytic site of CDKs have been identified. In this review, we will discuss the use of pharmacological and gene therapy strategies against CDK/cyclins in animal models and clinical trials of cancer and cardiovascular disease.

Amphiphysin 1 Binds the Cyclin-dependent Kinase (cdk) 5 Regulatory Subunit p35 and Is Phosphorylated by cdk5 and cdc2

Amphiphysin 1 is a phosphoprotein expressed at high levels in neurons, where it participates in synaptic vesicle endocytosis and neurite outgrowth. It is a substrate for cyclin-dependent kinase (cdk) 5, a member of the cyclin-dependent protein kinase family, which has been functionally linked to neuronal migration and neurite outgrowth via its action on the actin cytoskeleton. The yeast homologue of amphiphysin, Rvs167, functions in endocytosis and actin dynamics, is phosphorylated by the cdk5 homologue Pho85, and binds the Pho85 regulatory subunit Pcl2. We show here that amphiphysin 1 interacts with the cdk5-activating subunit p35 and that this interaction is mediated by the conserved NH2-terminal region of amphiphysin. Amphiphysin 1 colocalizes with p35 in the growth cones of neurons and at actin-rich peripheral lamellipodia in transfected fibroblasts. Amphiphysin is phosphorylated by cdk5 in a region including serines 272, 276, and 285. Amphiphysin 1 is also phosphorylated by the cdc2/cyclin B kinase complex in the same region and undergoes mitotic phosphorylation in dividing cells. These data indicate that phosphorylation by members of the cyclin-dependent kinase family is a conserved property of amphiphysin and suggest that this phosphorylation may play an important physiological role both in mitosis and in differentiated cells.

Inhibition of CDK2 activity in vivo by an associated 20K regulatory subunit.

The major events of the cell division cycle are triggered by periodic changes in the activity of cyclin-dependent protein kinases (CDKs). In mammals, the members of the CDK family include CDK2 and CDC2, which are thought to be involved in the control of DNA replication and mitosis, respectively. The protein kinase activity of these enzymes is controlled by a complex array of mechanisms. Activation of the CDK catalytic subunit requires association with a positive regulatory subunit (cyclin) and phosphorylation (at Thr 160 in CDK2). This activated complex can be inhibited by additional phosphorylation at Thr 14 and Tyr 15. Here we report the identification of a new mechanism for the regulation of CDK2 activity. We find that CDK2/cyclin complexes in mouse fibroblasts associate tightly with a 20K protein (CAP20). Complexes containing CAP20 were isolated from cell lysates and found to have negligible kinase activity, indicating that CAP20 association in vivo may inhibit CDK2 activity. We purified CAP20 from 3T3 cells and found that low concentrations of the protein completely inhibit the kinase activity of CDK2 in vitro. Thus CAP20 represents a new negative regulatory subunit that inhibits the activity of CDK2/cyclin complexes in mammalian cells.