[Show abstract][Hide abstract] ABSTRACT: Inhibition of protein kinases in the fight against disease remains a constant challenge for medicinal chemists, who have screened multitudes of predominantly planar organic scaffolds, natural and synthetic, to identify potent-albeit not always selective-kinase inhibitors. Herein, in an effort to investigate the potential biological utility of metal-based compounds as inhibitors against the cancer-relevant targets mitogen-activated protein kinase and cyclin-dependent kinase 2, we explore various parameters in planar platinum(II) complexes with substituted phenanthroline ligands and aliphatic diamine chelate co-ligands, to identify combinations that yield promising inhibitory activity. The individual ligands' steric requirements as well as their pattern of hydrogen bond donors/acceptors appear to alter inhibitory potency when modulated. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12154-011-0059-5) contains supplementary material, which is available to authorized users.
Journal of Chemical Biology 10/2011; 4(4):159-65. DOI:10.1007/s12154-011-0059-5
[Show abstract][Hide abstract] ABSTRACT: The elucidation of signalling pathways relies heavily upon the identification of protein kinase substrates. Recent investigations have demonstrated the efficacy of chemical genetics using ATP analogues and modified protein kinases for specific substrate labelling. Here we combine N(6) -(cyclohexyl)ATPγS with an analogue-sensitive cdk2 variant to thiophosphorylate its substrates and demonstrate a pH-dependent, chemoselective, one-step alkylation to facilitate the detection or isolation of thiophosphorylated peptides.
[Show abstract][Hide abstract] ABSTRACT: D-type cyclins predominantly regulate progression through the cell cycle by their interactions with cyclin-dependent kinases (cdks). Here, we show that stimulating mitogenesis of Swiss 3T3 cells with phorbol esters or forskolin can induce divergent responses in the expression levels, localization and activation state of cyclin D1 and cyclin D3. Phorbol ester-mediated protein kinase C stimulation induces S phase entry which is dependent on MAPK activation and increases the levels and activation of cyclin D1, whereas forskolin-mediated cAMP-dependent protein kinase A stimulation induces mitogenesis that is independent of MAPK, but dependent upon mTor and specifically increases the level and activation of cyclin D3. These findings uncover additional levels of complexity in the regulation of the cell cycle at the level of the D-type cyclins and thus may have important therapeutic implications in cancers where specific D-cyclins are overexpressed.
[Show abstract][Hide abstract] ABSTRACT: Several mammalian forkhead transcription factors have been shown to impact on cell cycle regulation and are themselves linked to cell cycle control systems. Here we have investigated the little studied mammalian forkhead transcription factor FOXK2 and demonstrate that it is subject to control by cell cycle-regulated protein kinases. FOXK2 exhibits a periodic rise in its phosphorylation levels during the cell cycle, with hyperphosphorylation occurring in mitotic cells. Hyperphosphorylation occurs in a cyclin-dependent kinase (CDK)·cyclin-dependent manner with CDK1·cyclin B as the major kinase complex, although CDK2 and cyclin A also appear to be important. We have mapped two CDK phosphorylation sites, serines 368 and 423, which play a role in defining FOXK2 function through regulating its stability and its activity as a transcriptional repressor protein. These two CDK sites appear vital for FOXK2 function because expression of a mutant lacking these sites cannot be tolerated and causes apoptosis.
[Show abstract][Hide abstract] ABSTRACT: Cyclin-dependent kinase 2 (cdk2) is a central regulator of the mammalian cell cycle. Here we describe the properties of a mutant form of cdk2 identified during large-scale sequencing of protein kinases from cancerous tissue. The mutation substituted a leucine for a proline in the PSTAIRE helix, the central motif in the interaction of the cdk with its regulatory cyclin subunit. We demonstrate that whilst the mutant cdk2 is considerably impaired in stable cyclin association, it is still able to generate an active kinase that can functionally complement defective cdks in vivo. Molecular dynamic simulations and biophysical measurements indicate that the observed biochemical properties likely stem from increased flexibility within the cyclin-binding helix.
[Show abstract][Hide abstract] ABSTRACT: Cells respond to DNA damage by either repairing the damage or committing to a death or senescence pathway, dependent on the level of damage sustained. In this study, we show that the protein levels of cyclin D1 and the CDK inhibitor, p21(CIP1), respond in a dose-dependent manner to the DNA damaging agent, 4-nitroquinoline 1-oxide (4NQO). Cyclin D1 responses were independent of p53 and resulted in a partial loss of Retinoblastoma protein phosphorylation. The differential responses of cyclin D1 and p21(CIP1) were associated with distinct cellular responses: in low dose treatments the cells recovered after a lag period whilst at medium and high doses, the cells died through seemingly distinct mechanisms. Our data suggest that the balance between cyclin D1 and p21(CIP1) following exposure to DNA damage may play a key role in determining the subsequent cellular responses.
Archives of Biochemistry and Biophysics 03/2010; 497(1-2):55-61. DOI:10.1016/j.abb.2010.03.003 · 3.02 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Enantioenriched tetrafluorinated aryl-C-nucleosides were synthesised in four steps from 1-benzyloxy-4-bromo-3,3,4,4-tetrafluorobutan-2-ol. The presence of the tetrafluorinated ethylene group is compatible with O-phosphorylation of the primary alcohol, as demonstrated by the successful preparation of the tetrafluorinated naphthyl-C-nucleotide.
[Show abstract][Hide abstract] ABSTRACT: The protein kinase superfamily is one of the most important families of enzymes in molecular biology. Protein kinases typically catalyze the transfer of the γ-phosphate from ATP to a protein substrate (a highly ubiquitous cellular reaction), thereby controlling key areas of cell regulation. Deregulation of protein kinases is known to contribute to many human diseases, and selective inhibitors of protein kinases are a major area of interest in medicinal chemistry. However, a detailed understanding of many kinase pathways is currently lacking. Before we can effectively design medicinally relevant selective kinase inhibitors, it is necessary to understand the role played by a given kinase in specific signal-transduction cascades and to decipher its protein targets. Here, we describe recent advances towards dissecting protein kinase function through the use of chemical genetics.
[Show abstract][Hide abstract] ABSTRACT: Mutant kinase kinetics: Protein kinases with enlarged ATP binding sites are increasingly being used as tools to probe the functioning signal transduction cascades. Using human cyclin-dependent kinase 2 as a model system, we demonstrate that enlargement of the ATP binding site does not substantially alter either the catalysis kinetics nor substrate or phosphorylation site selection.
Chemical genetic studies with enlarged ATP binding sites and unnatural ATP analogues have been applied to protein kinases for characterisation and substrate identification. Although this system is becoming widely used, there are limited data available about the kinetic profile of the modified system. Here we describe a detailed comparison of the wild-type cdk2 and the mutant gatekeeper kinase to assess the relative efficiencies of these kinases with ATP and unnatural ATP analogues. Our data demonstrate that mutation of the kinase alters neither the substrate specificity nor the phosphorylation site specificity. We find comparable KM/Vmax values for mutant cdk2 and wild-type kinase. Furthermore, F80G cdk2 is efficiently able to compensate for a defective cdk in a biological setting.
[Show abstract][Hide abstract] ABSTRACT: We hereby present a simple yet novel chemical synthesis of a family of gamma-modified ATPs bearing functional groups on the gamma-phosphate that are amenable to further derivatization by highly selective chemical manipulations (e.g., click chemistry, Staudinger ligations). A preliminary screen of these compounds as phosphate donors with a typical wild type protein kinase (cdk2) and one of its known substrates p27(kip1) is also presented.
[Show abstract][Hide abstract] ABSTRACT: Members of the gamma2-herpesvirus family encode cyclin-like proteins that have the ability to deregulate mammalian cell cycle control. Here we report the key features of the viral cyclin encoded by Murine Herpesvirus 68, M cyclin. M cyclin preferentially associated with and activated cdk2; the M cyclin/cdk2 holoenzyme displayed a strong reliance on phosphorylation of the cdk T loop for activity. cdk2 associated with M cyclin exhibited substantial resistance to the cdk inhibitor proteins p21(Cip) and p27(Kip). Furthermore, M cyclin directed cdk2 to phosphorylate p27(Kip1) on threonine 187 (T187) and cellular expression of M cyclin led to down-regulation of p27(Kip1) and the partial subversion of the associated G1 arrest. Mutation of T187 to a non-phosphorylatable alanine rendered the p27(Kip1)-imposed G1 arrest resistant to M cyclin expression. Unlike the related K cyclin, M cyclin was unable to circumvent the G1 arrest associated with p21(Cip1) and was unable to direct its associated catalytic subunit to phosphorylate this cdk inhibitor. These results imply that M cyclin has properties that are distinct from other viral cyclins and that M cyclin expression alone is insufficient for S phase entry.
Experimental Cell Research 02/2008; 314(1):204-12. DOI:10.1016/j.yexcr.2007.09.016 · 3.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To investigate the potential functional cooperation between p27Kip1 and p130 in vivo, we generated mice deficient for both p27Kip1 and p130. In p27Kip1-/-; p130-/- mice, the cellularity of the spleens but not the thymi is significantly increased compared with that of their p27Kip1-/- counterparts, affecting the lymphoid, erythroid, and myeloid compartments. In vivo cell proliferation is significantly augmented in the B and T cells, monocytes, macrophages, and erythroid progenitors in the spleens of p27Kip1-/-; p130-/- animals. Immunoprecipitation and immunodepletion studies indicate that p130 can compensate for the absence of p27Kip1 in binding to and repressing CDK2 and is the predominant CDK-inhibitor associated with the inactive CDK2 in the p27Kip1-/- splenocytes. The finding that the p27Kip1-/-; p130-/- splenic B cells are hypersensitive to mitogenic stimulations in vitro lends support to the concept that the hyperproliferation of splenocytes is not a result of the influence of their microenvironment. In summary, our findings provide genetic and molecular evidence to show that p130 is a bona fide cyclin-dependent kinase inhibitor and cooperates with p27Kip1 to regulate hematopoietic cell proliferation in vivo.
[Show abstract][Hide abstract] ABSTRACT: p21 was originally described as functioning as a cell cycle regulator via inhibition of both cyclin-dependent kinases and processive DNA replication. Nowadays it is recognized to play other fundamental roles including transcriptional regulation and the modulation of apoptosis. Each of these functions of p21 is achieved through direct p21/protein interactions and the subcellular localization of p21 plays an important part in dictating the binding partners to which p21 is exposed. Over recent years, a number of phosphorylation sites in p21 have been identified, these being targeted by several important intracellular signalling protein kinases. Here we review the state of our knowledge of p21 phosphorylation with respect to the kinases involved and the molecular biological effects of each phosphorylation event.
[Show abstract][Hide abstract] ABSTRACT: K cyclin encoded by Kaposi's sarcoma-associated herpesvirus confers resistance to the cyclin-dependent kinase (cdk) inhibitors p16Ink4A, p21Cip1, and p27Kip1 on the associated cdk6. We have previously shown that K cyclin expression enforces S-phase entry on cells overexpressing p27Kip1 by promoting phosphorylation of p27Kip1 on threonine 187, triggering p27Kip1 down-regulation. Since p21Cip1 acts in a manner similar to that of p27Kip1, we have investigated the subversion of a p21Cip1-induced G1 arrest by K cyclin. Here, we show that p21Cip1 is associated with K cyclin both in overexpression models and in primary effusion lymphoma cells and is a substrate of the K cyclin/cdk6 complex, resulting in phosphorylation of p21Cip1 on serine 130. This phosphoform of p21Cip1 appeared unable to associate with cdk2 in vivo. We further demonstrate that phosphorylation on serine 130 is essential for K cyclin-mediated release of a p21Cip1-imposed G1 arrest. Moreover, we show that under physiological conditions of cell cycle arrest due to elevated levels of p21Cip1 resulting from oxidative stress, K cyclin expression enabled S-phase entry and was associated with p21Cip1 phosphorylation and partial restoration of cdk2 kinase activity. Thus, expression of the viral cyclin enables cells to subvert the cell cycle inhibitory function of p21Cip1 by promoting cdk6-dependent phosphorylation of this antiproliferative protein.
[Show abstract][Hide abstract] ABSTRACT: To defend against the potential damages induced by reactive oxygen species, proliferating cells enter a transient cell cycle
arrest. We treated mouse fibroblasts with H2O2 and found that sublethal doses of H2O2 induced a transient multi-phase cell cycle arrest at the G1, S, and G2 phases but not the M phase. Western blot analysis demonstrated that this transient cell cycle arrest is associated with the
down-regulation of cyclins D1 and D3 and up-regulation of the CKI p21Cip1 expression. We also demonstrate that the induction in p21Cip1 expression by H2O2 is at least partially mediated at the transcriptional level and can occur in the absence of p53 function. Further immunoprecipitation
kinase and immunodepletion assays indicated that in response to H2O2 treatment, the down-regulation of cyclin Ds expression are associated with repression of cyclin D-CDK4, whereas the accumulation
of p21Cip1 is responsible for the inhibition of cyclin E and A-CDK2 activity and associated with the down-regulation of cyclin B-CDC2
activity. These data could account for the cell cycle arrest at the G1, S, and G2 phases following H2O2stimulation. Deletion of p21Cip1, restoration of cyclin D expression, or overexpression of cyclin E alone is insufficient to effectively overcome the cell
cycle arrest caused by sublethal doses of H2O2. By contrast, overexpression of the humanHerpesvirus 8 K cyclin, which can mimic the function of cyclin D and E, is enough to override this transient cell cycle arrest. On the
basis of our findings, we propose a model in which moderate levels of H2O2 induce a transient multi-phase cell cycle arrest at least partially through up-regulation of p21Cip1 and down-regulation of cyclin D expression.
[Show abstract][Hide abstract] ABSTRACT: Viral DNA replication is generally dependent upon circumventing host cell cycle control to force S phase entry in an otherwise quiescent cell. Here we describe novel attributes of the cyclin encoded by Human Herpesvirus 8 (K cyclin) that enable it to subvert the quiescent state. K cyclin is most similar to the mammalian D-type cyclins in primary sequence but displays properties more akin to those of cyclin E. K cyclin (like cyclin E) can autonomously couple with its cognate cdk subunit and localize to the nucleus. D-type cyclins require mitogen stimulated accessory factors (such as p21(Cip1) and p27(Kip1)) to facilitate both of these processes. A striking difference between K cyclin and mammalian cyclins is that K cyclin binding to cdk6 can substantially activate the catalytic activity of the complex without the requirement for cyclin H/cdk7 phosphorylation of the cdk T-loop; this phosphorylation is obligatory for endogenous cyclin/cdk activity. However, K cyclin/cdk6 complexes are not totally immune from cell cycle control since CAK phosphorylation is necessary for complete activation. Thus, CAK phosphorylated K cyclin/cdk6 targets multiple sites in the retinoblastoma protein (pRb) whereas the unphosphorylated complex targets a single site. The restricted substrate specificity of the non-CAK phosphorylated K cyclin/cdk6 complex is insufficient to enable K cyclin-mediated S phase entry. Thus, the viral K cyclin is reliant upon endogenous CAK activity to subvert the quiescent state.
[Show abstract][Hide abstract] ABSTRACT: DNA tumour viruses have evolved a number of mechanisms by which they deregulate normal cellular growth control. We have recently described the properties of a cyclin encoded by human herpesvirus 8 (also known as Kaposi's sarcoma-associated herpesvirus) which is able to resist the actions of p16(Ink4a), p21(Cip1) and p27(Kip1) cdk inhibitors. Here we investigate the mechanism involved in the subversion of a G1 blockade imposed by overexpression of p27(Kip1). We demonstrate that binding of K cyclin to cdk6 expands the substrate repertoire of this cdk to include a number of substrates phosphorylated by cyclin-cdk2 complexes but not cyclin D1-cdk6. Included amongst these substrates is p27(Kip1) which is phosphorylated on Thr187. Expression of K cyclin in mammalian cells leads to p27(Kip1) downregulation, this being consistent with previous studies indicating that phosphorylation of p27(Kip1) on Thr187 triggers its downregulation. K cyclin expression is not able to prevent a G1 arrest imposed by p27(Kip1) in which Thr187 is mutated to non-phosphorylatable Ala. These results imply that K cyclin is able to bypass a p27(Kip1)-imposed G1 arrest by facilitating phosphorylation and downregulation of p27(Kip1) to enable activation of endogenous cyclin-cdk2 complexes. The extension of the substrate repertoire of cdk6 by K cyclin is likely to contribute to the deregulation of cellular growth by this herpesvirus-encoded cyclin.
The EMBO Journal 03/1999; 18(3):654-63. DOI:10.1093/emboj/18.3.654 · 10.43 Impact Factor