Control of myeloid differentiation and survival by Stats.

Hematopoiesis involves a complex array of growth factors that regulate the survival and proliferation of immature progenitors, influence differentiation commitment, and modulate end-stage cell functions. This mini-review is focused on the role of Stat activation in the development of myeloid cells in response to hematopoietic cytokines. Much of the evidence implicating Stats in these cellular processes comes from studies of mutant cytokine receptors selectively uncoupled from Stat activation, dominant-inhibitory Stat mutants, and mice with targeted disruptions of Stat genes. Together these approaches provide strong evidence that Stat activation, particularly of Stat3 and Stat5, plays an important role in myeloid differentiation and survival. Oncogene (2000).

The nonreceptor protein-tyrosine kinase c-Fes is involved in fibroblast growth factor-2-induced chemotaxis of murine brain capillary endothelial cells.

Fibroblast growth factor-2 (FGF-2)-induced migration of endothelial cells is involved in angiogenesis in vivo. However, signal transduction pathways leading to FGF-2-induced chemotaxis of endothelial cells are largely unknown. Previous studies have shown that the cytoplasmic protein-tyrosine kinase c-Fes is expressed in vascular endothelial cells and may influence angiogenesis in vivo. To investigate the contribution of c-Fes to FGF-2 signaling, we expressed wild-type or kinase-inactive human c-Fes in the murine brain capillary endothelial cell line, IBE (Immortomouse brain endothelial cells). Wild-type c-Fes was tyrosine-phosphorylated upon FGF-2-stimulation in transfected cells, whereas kinase-inactive c-Fes was not. Overexpression of wild-type c-Fes promoted FGF-2-independent tube formation of IBE cells. Tube formation was not observed with endothelial cells expressing kinase-inactive c-Fes, indicating a requirement for c-Fes kinase activity in this biological response. Expression of kinase-defective c-Fes suppressed endothelial cell migration following FGF-2 treatment, suggesting that activation of endogenous c-Fes may be required for the chemotactic response. Expression of either wild-type c-Fes or the kinase-inactive mutant did not affect the tyrosine phosphorylation FRS2, Shc, or phospholipase C-gamma, nor did it influence the kinetics of mitogen-activated protein kinase activation. These results implicate c-Fes in FGF-2-induced chemotaxis of endothelial cells through signaling pathways not linked to mitogenesis.

Affinity of Src family kinase SH3 domains for HIV Nef in vitro does not predict kinase activation by Nef in vivo.

Nef is an HIV accessory protein required for high-titer viral replication and AIDS progression. Previous studies have shown that the SH3 domains of Hck and Lyn bind to Nef via proline-rich sequences in vitro, identifying these Src-related kinases as potential targets for Nef in vivo. Association of Nef with Hck causes displacement of the intramolecular interaction between the SH3 domain and the SH2-kinase linker, leading to kinase activation both in vitro and in vivo. In this study, we investigated whether interaction with Nef induces activation of other Src family kinases (Lyn, Fyn, Src, and Lck) following coexpression with Nef in Rat-2 fibroblasts. Coexpression with Nef induced Hck kinase activation and fibroblast transformation, consistent with previous results. In contrast, coexpression of Nef with Lyn was without effect, despite equivalent binding of Nef to full-length Lyn and Hck. Furthermore, Nef was found to suppress the kinase and transforming activities of Fyn, the SH3 domain of which exhibits low affinity for Nef. Coexpression with Nef did not alter c-Src or Lck tyrosine kinase or transforming activity in this system. Differential modulation of Src family members by Nef may produce unique downstream signals depending on the profile of Src kinases expressed in a given cell type.

Inhibition of the prenylation of K-Ras, but not H- or N-Ras, is highly resistant to CAAX peptidomimetics and requires both a farnesyltransferase and a geranylgeranyltransferase I inhibitor in human tumor cell lines.

The farnesyltransferase (FTase) inhibitor FTI-277 is highly effective at blocking oncogenic H-Ras but not K-Ras4B processing and signaling. While inhibition of processing and signaling of oncogenic K-Ras4B is more sensitive to the geranylgeranyltransferase I (GGTase I) inhibitor GGTI-286 than it is to FTI-277 in K-Ras4B-transformed NIH3T3 cells, the sensitivity of K-Ras as well as H- and N-Ras to the CAAX peptidomimetics in human tumor cell lines is not known. Here, we report that a panel of five human carcinoma cell lines from pancreatic, pulmonary, and bladder origins all express H-, N-, and K-Ras, and their respective prenylation sensitivities to the FTase and GGTase I inhibitors is variable. In all of the cell lines investigated, the prenylation of N-Ras was highly sensitive to FTI-277, and in two of the cell lines, N-Ras showed slight sensitivity to GGTI-298, an analog of GGTI-286. Although the prenylation of H-Ras was also sensitive to FTI-277, complete inhibition of H-Ras processing even at high concentrations of FTI-277 and/or GGTI-298 was never achieved. The prenylation of K-Ras, on the other hand, was highly resistant to FTI-277 and GGTI-298. Most significantly, treatment of human tumor cell lines with both inhibitors was required for inhibition of K-Ras prenylation. In one cell line, the human lung adenocarcinoma A-549, prenylation of K-Ras was highly resistant even when co-treated with both inhibitors. Furthermore, soft agar experiments demonstrated that in all the human tumor cell lines tested inhibition of K-Ras prenylation was not necessary for inhibition of anchorage-independent growth. In addition, although GGTI-298 had very little effect on soft agar growth, the combination of FTI-277 and GGTI-298 resulted in significant growth inhibition. Therefore, the results demonstrate that while FTI-277 inhibits N-Ras and H-Ras processing in the human tumor cell lines evaluated, inhibition of K-Ras processing requires both an FTase inhibitor as well as a GGTase I inhibitor, and that inhibition of human tumor growth in soft agar does not require inhibition of oncogenic K-Ras processing.

Inhibition of Ras prenylation: a signaling target for novel anti-cancer drug design.

The cancer-causing activity of Ras requires the prenylation of a cysteine fourth from its carboxyl terminus. Rational design of peptidomimetics of the carboxyl terminal tetrapeptide prenylation site on Ras resulted in pharmacological agents capable of inhibiting Ras processing, selectively antagonizing oncogenic signaling and suppressing human tumor growth in mouse models without side effects. This mini-review describes the efforts of several groups to design, synthesize and evaluate the biological activities of farnesyltransferase and geranylgeranyltransferase I inhibitors. Among the important issues that will be discussed are the mechanism of action of these inhibitors and the potential mechanisms of resistance to inhibition of K-Ras farnesylation.

Disruption of oncogenic K-Ras4B processing and signaling by a potent geranylgeranyltransferase I inhibitor.

Prenylation of the carboxyl-terminal CAAX (C, cysteine; A, aliphatic acid; and X, any amino acid) of Ras is required for its biological activity. We have designed a CAAX peptidomimetic, GGTI-287, which is 10 times more potent toward inhibiting geranylgeranyltransferase I (GGTase I) in vitro (IC50 = 5 nM) than our previously reported farnesyltransferase inhibitor, FTI-276. In whole cells, the methyl ester derivative of GGTI-287, GGTI-286, was 25-fold more potent (IC50 = 2 microM) than the corresponding methyl ester of FTI-276, FTI-277, toward inhibiting the processing of the geranylgeranylated protein Rap1A. Furthermore, GGTI-286 is highly selective for geranylgeranylation over farnesylation since it inhibited the processing of farnesylated H-Ras only at much higher concentrations (IC50 > 30 microM). While the processing of H-Ras was very sensitive to inhibition by FTI-277 (IC50 = 100 nM), that of K-Ras4B was highly resistant (IC50 = 10 microM). In contrast, we found the processing of K-Ras4B to be much more sensitive to GGTI-286 (IC50 = 2 microM). Furthermore, oncogenic K-Ras4B stimulation inhibited potently by GGTI-286 (IC50 = 1 microM) but weakly by FTI-277 (IC50 = 30 microM). Significant inhibition of oncogenic K-Ras4B stimulation of MAP kinase by GGTI-286 occurred at concentrations (1-3 microM) that did not inhibit oncogenic H-Ras stimulation of MAP kinase. The data presented in this study provide the first demonstration of selective disruption of oncogenic K-Ras4B processing and signaling by a CAAX peptidomimetic. The higher sensitivity of K-Ras4B toward a GGTase I inhibitor has a tremendous impact on future research directions targeting Ras in anticancer therapy.

Ras CAAX peptidomimetic FTI-277 selectively blocks oncogenic Ras signaling by inducing cytoplasmic accumulation of inactive Ras-Raf complexes.

Ras-induced malignant transformation requires Ras farnesylation, a lipid posttranslational modification catalyzed by farnesyltransferase (FTase). Inhibitors of this enzyme have been shown to block Ras-dependent transformation, but the mechanism by which this occurs remains largely unknown. We have designed FTI-276, a peptide mimetic of the COOH-terminal Cys-Val-Ile-Met of K-Ras4B that inhibited potently FTase in vitro (IC50 = 500 pM) and was highly selective for FTase over geranylgeranyltransferase I (GGTase I) (IC50 = 50 nM). FTI-277, the methyl ester derivative of FTI-276, was extremely potent (IC50 = 100 nM) at inhibiting H-Ras, but not the geranylgeranylated Rap1A processing in whole cells. Treatment of H-Ras oncogene-transformed NIH 3T3 cells with FTI-277 blocked recruitment to the plasma membrane and subsequent activation of the serine/threonine kinase c-Raf-1 in cells transformed by farnesylated Ras (H-RasF), but not geranylgeranylated, Ras (H-RasGG). FTI-277 induced accumulation of cytoplasmic non-farnesylated H-Ras that was able to bind Raf and form cytoplasmic Ras/Raf complexes in which Raf kinase was not activated. Furthermore, FTI-277 blocked constitutive activation of mitogen-activated protein kinase (MAPK) in H-RasF, but not H-RasGG, or Raf-transformed cells. FTI-277 also inhibited oncogenic K-Ras4B processing and constitutive activation of MAPK, but the concentrations required were 100-fold higher than those needed for H-Ras inhibition. The results demonstrate that FTI-277 blocks Ras oncogenic signaling by accumulating inactive Ras/Raf complexes in the cytoplasm, hence preventing constitutive activation of the MAPK cascade.