No abstract

Chemical compounds that interfere with an enzymatic function of kinases are useful for gaining insight into the complicated biochemical processes in mammalian cells. Cyclin-dependent kinases (CDK) play an essential role in the control of the cell cycle and/or proliferation. These kinases as well as their regulators are frequently deregulated in diVerent human tumors. Aberrations in CDK activity have also been observed in viral infections, Alzheimer's, Parkinson's diseases, ischemia and some proliferative disorders. This led to an intensive search for small-molecule CDK inhibitors not only for research purposes, but also for therapeutic applications. Here, we discuss seventeen CDK inhibitors and their use in cancer research or therapy. This review should help researchers to decide which inhibitor is best suited for the speciWc purpose of their research. For this purpose, the targets, commercial availability and IC 50 values are provided for each inhibitor. The review will also provide an overview of the clinical studies performed with some of these inhibitors.

In response to binding of platelet-derived growth factor (PDGF), the PDGF receptor (PDGFR) I1 subunit is phosphorylated on tyrosine residues and associates with numerous signal transduction enzymes, including the GTPase-activating protein of ras (GAP) and phosphatidylinositol 3-kinase (P13K). Previous studies have shown that association of P13K requires phosphorylation of tyrosine 751 (Y751) in the kinase insert and that this region of receptor forms at least a portion of the binding site for P13K. In this study, the in vitro binding of GAP to the PDGFR was investigated. Like PI3K, GAP associates only with receptors that have been permitted to autophosphorylate, and GAP itself does not require tyrosine phosphate in order to stably associate with the phosphorylated PDGFR. To define which tyrosine residues are required for GAP binding, a panel of PDGFR phosphorylation site mutants was tested. Mutation of Y771 reduced the amount of GAP that associates to an undetectable level. In contrast, the F771 (phenylalanine at 771) mutant bound wild-type levels of PI3K, whereas the F740 and F751 mutants bound 3 and 23%, respectively, of the wild-type levels of PI3K but wild-type levels of GAP. The F740/F751 double mutant associated with wild-type levels of GAP, but no detectable P13K activity, while the F740/F751/F771 triple mutant could not bind either GAP or P13K. The in vitro and in vivo associations of GAP and PI3K activity to these PDGFR mutants were indistinguishable. The distinct tyrosine residue requirements suggest that GAP and P13K bind different regions of the PDGFR. This possibility was also supported by the observation that the antibody to the PDGFR kinase insert Y751 region that blocks association of P13K had only a minor effect on the in vitro binding of GAP. In addition, highly purified PI3K and GAP associated in the absence of other cellular proteins and neither cooperated nor competed with each other's binding to the PDGFR. Taken together, these studies indicate that GAP and P13K bind directly to the PDGFR and have discrete binding sites that include portions of the kinase insert domain.

Once thought to be a remnant of cell division, the midbody (MB) has recently been shown to have roles beyond its primary function of orchestrating abscission. Despite the emerging roles of post-abscission MBs, how MBs accumulate in the cytoplasm and signal to regulate cellular functions remains unknown. Here, we show that extracellular post-abscission MBs can be internalized by interphase cells, where they reside in the cytoplasm as a membrane-bound signaling structure that we have named the MBsome. We demonstrate that MBsomes stimulate cell proliferation and that MBsome formation is a phagocytosis-like process that depends on a phosphatidylserine/integrin complex, driven by actin-rich membrane protrusions. Finally, we show that MBsomes rely on dynamic actin coats to slow lysosomal degradation and propagate their signaling function. In summary, MBsomes may sometimes serve as intracellular organelles that signal via integrin and EGFR-dependent pathways to promote cell proliferation and anchorage-independent growth and survival.

Ligand-stimulated autophosphorylation of the platelet-derived growth factor receptor (PDGFR) (3 subunit creates a number of binding sites for SH2-containing proteins. One of the PDGFR-associated proteins is a 64-kDa protein of unknown identity and function. We present data indicating that the 64-kDa protein that associates with the activated PDGFR is Syp (also called SH-PTP2, PTP-1D, or SH-PTP3), the ubiquitously expressed 64-kDa SH2-containing proteintyrosine phosphatase. Phosphorylation of Tyr-1009 in the C terminus of the PDGFR is required for the stable association of Syp, suggesting that phosphorylation of this residue creates a binding site for the Syp SH2 domains. Although Syp stably associates with the PDGFR, this event is not required for PDGF-stimulated tyrosine phosphorylation of Syp. These data raise the interesting possibility that protein-tyrosine phosphatases contribute to the intracellular relay of biological signals originating from receptor tyrosine kinases such as the PDGFR.

When expressed in PC12 cells, the platelet-derived growth factor ␤ receptor (␤PDGF-R) mediates cell differentiation. Mutational analysis of the ␤PDGF-R indicated that persistent receptor stimulation of the Ras/Raf/mitogenactivated protein (MAP) kinase pathway alone was insufficient to sustain PC12 cell differentiation. PDGF receptor activation of signal pathways involving p60 c-src or the persistent regulation of phospholipase C␥ was required for PC12 cell differentiation. ␤PDGF-R regulation of phosphatidylinositol 3-kinase, the GTPase-activating protein of Ras, and the tyrosine phosphatase, Syp, was not required for PC12 cell differentiation. In contrast to overexpression of oncoproteins involved in regulating the MAP kinase pathway, growth factor receptor-mediated differentiation of PC12 cells requires the integration of other signals with the Ras/Raf/MAP kinase pathway.The platelet-derived growth factor receptor (PDGF-R) is a transmembrane polypeptide encoding an intrinsic tyrosine kinase in its intracellular domain. Two distinct PDGF-R genes encode either an ␣ (7, 42, 47) or a ␤ (6, 19, 73) subunit. Binding of PDGF (22) induces dimerization (23) and trans phosphorylation of the ␤PDGF-R on specific tyrosine residues (35). The phosphorylated receptor initiates a series of intracellular signals which ultimately lead to cell growth (12), differentiation (20), and chemotaxis (70) depending on the cellular context.A number of tyrosines on the intracellular domain of the ␤PDGF-R are phosphorylated upon activation of the receptor and serve as recognition sites for proteins which contain Src homology 2 (SH2) domains (57). For example, the ␤PDGF-R has been shown to associate with Src family tyrosine kinases p60 c-src , p59 fyn , and p62 c-yes (39) via juxtamembrane tyrosines 579 and 581, which are in vivo phosphorylation sites (50). The SH2 domain-containing proteins p46 and p52 (Shc proteins) bind to the ␤PDGF-R at multiple phosphotyrosines including tyrosine 581 and indirectly via association with other tyrosinephosphorylated proteins (74). Phosphorylation of tyrosines 740 and 751 is critical for association of the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3-K) with the ␤PDGF-R (2, 8, 29-31). Phosphorylated tyrosine 751 also binds Nck via its SH2 binding domain (52). The GTPase-activating protein (GAP) of Ras is tyrosine phosphorylated in response to PDGF and binds to the receptor at phosphorylated 33,49). The SH2-containing phosphotyrosine phosphatase, Syp, associates with phosphorylated tyrosine 1009 (34, 41). In addition, there is evidence which suggests that the adaptor protein, Grb2, associates with the ␤PDGF-R via tyrosine 716 (1) and indirectly through Syp (44). Phosphorylated tyrosine 1009 may also influence the binding of phospholipase C␥ (PLC␥) to tyrosine 1021 (28, 59). Mutational analysis of the ␤PDGF-R has demonstrated that phosphorylated tyrosines 740 and 751, which mediate association with PI3-K, and tyrosine 1021, which mediates association with PLC␥, are necessary for the transducti...

Binding of platelet-derived growth factor (PDGF) to the PDGF receptor (PDGFR) beta subunit triggers receptor tyrosine phosphorylation and the stable association of a number of signal transduction molecules, including phospholipase C gamma (PLC gamma), the GTPase activating protein of ras (GAP), and phosphatidylinositol-3 kinase (PI3K). Previous reports have identified three PDGFR tyrosine phosphorylation sites in the kinase insert domain that are important for stable association of GAP and PI3K. Two of them, tyrosine (Y) 740, and Y-751 are required for the stable association of PI3K, while Y-771 is required for binding of GAP. Here we present data for two additional tyrosine phosphorylation sites, Y-1009 and Y-1021, that are both in the carboxy-terminal region of the PDGFR. Characterization of PDGFR mutants in which these phosphorylation sites are substituted with phenylalanine (F) indicated that Y-1021 and Y-1009 were required for the stable association of PLC gamma and a 64-kDa protein, respectively. An F-1009/F-1021 double mutant selectively failed to bind both PLC gamma and the 64-kDa protein, whereas all of the carboxy-terminal mutants bound wild-type levels of GAP and PI3K. The carboxy terminus encodes the complete binding site for PLC gamma, since a phosphorylated carboxy-terminal fusion protein selectively bound PLC gamma. To determine the biological consequences of failure to associate with PLC gamma, we measured PDGF-dependent inositol phosphate production and initiation of DNA synthesis. The PDGFR mutants that failed to associate with PLC gamma were not able to mediate the PDGF-dependent production of inositol phosphates. Since tyrosine phosphorylation of PLC gamma enhances its enzymatic activity, we speculated that PDGFR mutants that failed to activate PLC gamma were unable to mediate its tyrosine phosphorylation. Surprisingly, the F-1021 receptor mediated readily detectable levels of PDGF-dependent PLC gamma tyrosine phosphorylation. Thus, the production of inositol phosphates requires not only PLC gamma tyrosine phosphorylation but also its association with the PDGFR. Comparison of the mutant PDGFRs' abilities to initiate PDGF-dependent DNA synthesis indicated that failure to associate with PLC gamma and produce inositol phosphates diminished the mitogenic response by 30%. In contrast, preventing the PDGFR from binding the 64-kDa protein did not compromise PDGF-triggered DNA synthesis at saturating concentrations of PDGF. Thus, it appears that phosphorylation of the PDGFR at Y-1021 is required for the stable association of PLC gamma to the receptor's carboxy terminus, the production of inositol phosphates, and initiation of the maximal mitogenic response.

Cancers are the group of diseases, which arise because of the uncontrolled behavior of some of the genes in our cells. There are possibilities of gene amplifications, overexpressions, deletions and other anomalies which might lead to the development and spread of cancer. One of the most dangerous ways to the cancers is the mutations of the genes. The mutated genes can start unstoppable proliferation of cells, their uncontrolled motility, protection from apoptosis, the DNA mutation enhancement as well as other anomalies, leading to the cancer. This review focuses on the genes, which are frequently mutated in various cancers and are known to be important in the advance and progression of colorectal cancer and melanoma, namely KRAS, NRAS and BRAF.