A Proteome-Wide CDK/CRK-Specific Kinase Inhibitor Promotes Tumor Cell Death in the Absence of Cell Cycle Progression

Caligiuri, Maureen; Becker, Frank; Murthi, Krishna K.; Kaplan, Faith A.; Dédier, Séverine; Kaufmann, Christine; Machl, Andy; Zybarth, Gabriele; Judson, Richard; Bockovich, Nick; Kluge, Art; Kley, Nikolai

doi:10.1016/j.chembiol.2005.08.008

Cited by 41 publications

(38 citation statements)

References 52 publications

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“…CCRK was identified in a large-scale small interfering RNA (siRNA) screen for suppressors of apoptosis (36). A specific chemical inhibitor of CDK family members (RGB-286147) inhibits CCRK and promotes apoptosis in the absence of cell cycle progression (6). This is intriguing because it supports structural similarity of CCRK to other CDKs (35), but the target(s) for RGB-286147 that promotes apoptosis is unknown.…”

Section: Discussionmentioning

confidence: 91%

Identification of Yin-Yang Regulators and a Phosphorylation Consensus for Male Germ Cell-Associated Kinase (MAK)-Related Kinase

Fu¹,

Larson²,

Chitta³

et al. 2006

Molecular and Cellular Biology

127

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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.

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Section: Discussionmentioning

confidence: 91%

Identification of Yin-Yang Regulators and a Phosphorylation Consensus for Male Germ Cell-Associated Kinase (MAK)-Related Kinase

Fu¹,

Larson²,

Chitta³

et al. 2006

Molecular and Cellular Biology

127

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“…Genomic profile by DNA microarrays in that model revealed the down-regulation of the cell cycle-related kinase (CCRK) after development of HF. Although this protein has not been characterized in the heart before, it is known that CCRK plays a major role in promoting the growth of cancer cells (7). Therefore, the goal of the present study is to characterize the expression of CCRK in the normal and failing heart and to determine whether this kinase is involved in cardiac mechanisms of cell growth and survival.…”

mentioning

confidence: 99%

“…Cells lacking CCRK were incapable of growth, whereas cells with a reduced level of CCRK grew at a significantly slower rate than control cells (7,16). Most interestingly, it was shown that a specific inhibition of CCRK promotes tumor cell death (7).…”

mentioning

confidence: 99%

“…A well characterized cyclin-dependent protein kinase-activating kinase is made of a complex that includes the catalytic subunit p40 MO15 (also called Cdk7 (cyclin-dependent kinase-7)), the regulatory subunit cyclin H, and the assembly factor MAT1 and activates the transcription factor IIH, which phosphorylates RNA polymerase II, as well as multiple components of the transcriptional machinery, including p53, RAR␣, and Oct-1 (7)(8)(9)(10)(11)(12)(13)(14)(15). CCRK is distinct from MO15 and behaves differently in terms of size, substrate specificity, and immunological properties (16).…”

mentioning

confidence: 99%

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Characterization of a Novel Cardiac Isoform of the Cell Cycle-related Kinase That Is Regulated during Heart Failure

Qiu¹,

Dai²,

Jain

et al. 2008

Journal of Biological Chemistry

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Myocardial infarction (MI) is often followed by heart failure (HF), but the mechanisms precipitating the transition to HF remain largely unknown. A genomic profile was performed in a monkey model of MI, from the myocardium adjacent to chronic (2-month) MI followed by 3 weeks of pacing to develop HF. The transcript of the gene encoding the cell cycle-related kinase (CCRK) was down-regulated by 50% in HF heart compared with control (p < 0.05), which was confirmed by quantitative PCR. The CCRK sequence cloned from a heart library showed a conservation of the N-terminal kinase domain when compared with the "generic" isoform cloned previously but a different C-terminal half due to alternative splicing with frameshift. The homology of the cardiac sequence was 100% between mice and humans. Expression of the corresponding protein, measured upon generation of a monoclonal antibody, was limited to heart, liver, and kidney. Upon overexpression in cardiac myocytes, both isoforms promote cell growth and reduce apoptosis by chelerythrine (p < 0.05 versus control). Using a yeast two-hybrid screening, we found an interaction of the generic but not the cardiac CCRK with cyclin H and casein kinase 2. In addition, only the generic CCRK phosphorylates the cyclin-dependent kinase 2, which was accompanied by a doubling of myocytes in the S and G 2 phases of the cell cycle (p < 0.05 versus control). Therefore, the heart expresses a splice variant of CCRK, which promotes cardiac cell growth and survival; differs from the generic isoform in terms of protein-protein interactions, substrate specificity and regulation of the cell cycle; and is downregulated significantly in HF.Because the fully differentiated cardiac myocyte has a limited capacity for regeneration, most forms of heart disease, including ischemic heart disease and heart failure (HF), 2 are characterized by a loss of cardiomyocytes (1-3). Therefore, deciphering the molecular mechanisms balancing cell growth and apoptosis may help develop strategies to improve the prognosis of both ischemic heart disease and HF, particularly with regard to stem cell therapy (4). Our previous studies have shown that genomic profiling in large mammalian models of heart disease allows the detection of novel potential candidates to promote cardiac cell growth and survival in a context of reversible ischemia or pressure overload (5, 6). In the present study, we extended this investigation to a model of postischemic cardiomyopathy in monkeys. We reasoned that a nonhuman primate model would be ideal, since it is phylogenetically and genetically closer to humans. Genomic profile by DNA microarrays in that model revealed the down-regulation of the cell cycle-related kinase (CCRK) after development of HF. Although this protein has not been characterized in the heart before, it is known that CCRK plays a major role in promoting the growth of cancer cells (7). Therefore, the goal of the present study is to characterize the expression of CCRK in the normal and failing heart and to determine whether this k...

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“…Roscovitine and CDK9 Inhibitor II (4-(3,5-diamino-1H-pyrazol-4-ylazo)-phenol) inhibited tracer binding weakly, with K i values >10 µM. CDK/CRK inhibitor (RGB-286147) inhibited with a K i near 120 nM, which is within the range of IC 50 values seen for a variety of CDKs with this inhibitor, 32 and CDK2/9 inhibitor ((4-(2-Amino-4-methylthiazol-5-yl)pyrimidin-2-yl)-(3-nitrophenyl)amine) demonstrated a K i of 72 nM, also within the range of potencies seen for this inhibitor with other CDKs. 33 The adenosine mimetic 5-iodotubercidin and the broad-spectrum kinase inhi bitor staurosporine bound tightest, with K i values of 23 nM and 2.8 nM, respectively.…”

Section: Characterization Of Compounds That Bind To Cdksmentioning

confidence: 74%

Development and Applications of a Broad-Coverage, TR-FRET-Based Kinase Binding Assay Platform

Lebakken¹,

Riddle²,

Singh³

et al. 2009

SLAS Discovery

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The expansion of kinase assay technologies over the past decade has mirrored the growing interest in kinases as drug targets. As a result, there is no shortage of convenient, fluorescence-based methods available to assay targets that span the kinome. The authors recently reported on the development of a non-activity-based assay to characterize kinase inhibitors that depended on displacement of an Alexa Fluor  647 conjugate of staurosporine (a "tracer") from a particular kinase. Kinase inhibitors were characterized by a change in fluorescence lifetime of the tracer when it was bound to a kinase relative to when it was displaced by an inhibitor. Here, the authors report on improvements to this strategy by reconfiguring the assay in a time-resolved fluorescence resonance energy transfer (TR-FRET) format that simplifies instrumentation requirements and allows for the use of a substantially lower concentration of kinase than was required in the fluorescence-lifetime-based format. The authors use this new assay to demonstrate several aspects of the binding assay format that are advantageous relative to traditional activity-based assays. The TR-FRET binding format facilitates the assay of compounds against lowactivity kinases, allows for the characterization of type II kinase inhibitors either using nonactivated kinases or by monitoring compound potency over time, and ensures that the signal being detected is specific to the kinase of interest and not a contaminating kinase. (Journal of Biomolecular Screening 2009:924-935)

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A Proteome-Wide CDK/CRK-Specific Kinase Inhibitor Promotes Tumor Cell Death in the Absence of Cell Cycle Progression

Cited by 41 publications

References 52 publications

Identification of Yin-Yang Regulators and a Phosphorylation Consensus for Male Germ Cell-Associated Kinase (MAK)-Related Kinase

Identification of Yin-Yang Regulators and a Phosphorylation Consensus for Male Germ Cell-Associated Kinase (MAK)-Related Kinase

Characterization of a Novel Cardiac Isoform of the Cell Cycle-related Kinase That Is Regulated during Heart Failure

Development and Applications of a Broad-Coverage, TR-FRET-Based Kinase Binding Assay Platform

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