We have developed a generalized approach, using two hybrid interactions, to isolate Ha-Ras effector loop mutations that separate the ability of Ha-Ras to interact with different downstream effectors. These mutations attenuate or eliminate Ha-ras(G12V) transformation of mammalian cells, but retain complementary activity, as demonstrated by synergistic induction of foci of growth-transformed cells, and by the ability to activate different downstream components. The transformation defect of Ha-ras(G12V, E37G) is rescued by a mutant, raf1, that restores interaction. These results indicate that multiple cellular components, including Raf1, are activated by Ha-Ras and contribute to Ha-Ras-induced mammalian cell transformation.

We used a Saccharomyces cerevisiae genetic system to detect the physical interaction of RAS and RAF oncoproteins. We also observed interaction between RAS and byr2, a protein kinase implicated as a mediator of the Schizosaccharomyces pombe rasl protein. Interaction with RAS required only the N-terminal domains of RAF or byr2 and was disrupted by mutations in either the guanine nucleotidebinding or effector-loop domains of RAS. We observed interaction between MEK (a kinase that phosphorylates mitogenactivated protein kinases) and the catalytic domain of RAF. RAS and MEK also interacted but only when RAF was overexpressed.The ras genes encode guanine nucleotide-binding proteins and were first identified as oncogenes of acutely transforming RNA tumor viruses (reviewed in ref. 1). Subsequently, mutated RAS genes were found in many human tumors, providing the first evidence of a common genetic defect in cancer. RAS proteins participate in signal transduction pathways regulating cell proliferation and differentiation, but their precise biochemical functions are unknown. In mammals a variety of extracellular agonists, such as insulin, platelet-derived growth factor, and nerve growth factor, that act through proteintyrosine kinase receptors require RAS to exert their effects (2-7). These agonists activate a set ofprotein-serine/threonine kinases known as mitogen-activated protein kinases (MAP kinases) reviewed in ref. 8. Activation of the MAP kinases by these factors requires RAS (5-7), and RAS can itself activate the MAP kinases in cells (5-7) and in complex cell-free systems (9, 10).The RAF oncogene is a strong candidate to encode a downstream effector for RAS in mammalian cells. (i) Activated RAF can bypass the cellular requirement for RAS function (3,4). (ii) Dominant negative mutations of RAF can block transformation induced by RAS (11). (iii) Hyperphosphorylated RAF kinase is observed in cells treated with agonists that activate RAS and in cells containing the activated RAS oncogene itself (7, 12). (iv) The RAF kinase can activate the MAP kinase kinase known as MEK (13,14).RAS proteins are widely conserved in eukaryotes. In many respects, the function of rasl, the Schizosaccharomyces pombe RAS homolog, resembles the function of RAS in vertebrates. Genetic evidence indicates that rasl activates byr2, a protein kinase involved in sexual differentiation (15). Overexpression of byr2 can bypass defects resulting from the loss of rasl, and expression of the N-terminal putative regulatory domain of byr2 appears to interfere with rasl signaling. Genetic studies place byr2 upstream of byrl, a homolog of mammalian MEK and Saccharomyces cerevisiae STE7 protein kinases, each of which is implicated in the activation of protein kinases of the MAP kinase familyThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.(16-18). byr2 is itself a homolog of S. cerevisiae STEll...

Osteoprotegerin ligand (OPGL) targets osteoclast precursors and osteoclasts to enhance differentiation and activation, however, little is known about OPGL effects on osteoclast survival. In vitro, the combination of OPGL ؉ colony-stimulating factor-1 (CSF-1) is required for optimal osteoclast survival. Ultrastructurally, apoptotic changes were observed in detached cells and culture lysates exhibited elevated caspase 3 activity, particularly in cultures lacking CSF-1. DEVD-FMK (caspase 3 inhibitor) partially protected cells when combined with OPGL, but not when used alone or in combination with CSF-1. CSF-1 maintained NF-B activation and increased the expression of bcl-2 and bcl-X L mRNA, but had no effect on JNK activation. In contrast, OPGL enhanced both NF-B and JNK kinase activation and increased the expression of c-src, but not bcl-2 and bcl-X L mRNA. These data suggest that aspects of both OPGL's and CSF-1's signaling/survival pathways are required for optimal osteoclast survival. In mice, a single dose of OPG, the OPGL decoy receptor, led to a >90% loss of osteoclasts because of apoptosis within 48 hours of exposure without impacting osteoclast precursor cells. Therefore, OPGL is essential, but not sufficient, for osteoclast survival and endogenous CSF-1 levels are insufficient to maintain osteoclast viability in the absence of OPGL. Osteoclasts mediate the resorption component of bone modeling and remodeling, which together are pivotal to the formation and maintenance of the mammalian skeleton. These specialized members of the monocyte-macrophage family arise from hematopoietic precursors with the location and magnitude of their activity guided by cells that surface the bone matrix. The bone lining cells, which include osteoblasts, endosteal, and periosteal lining cells, seem to mediate the local, systemic, physiological, and/or pathological stimuli impinging on them and consequently provide molecular signals that eventuate in osteoclast-mediated bone resorption.

We describe a protein kinase, Shkl, from the fission yeast Schizosaccharomyces pombe, which is structurally related to the Saccharomyces cerevisiae Ste2O and mammalian p65PAK protein kinases. We provide genetic evidence for physical and functional interaction between Shkl and the Cdc42 GTP-binding protein required for normal cell morphology and mating in S. pombe. We further show that expression of the STE20 gene complements the shki null mutation and that Shkl is capable of signaling to the pheromone-responsive mitogen-activated protein kinase cascade in S. cerevisiae. Our results lead us to propose that signaling modules composed of small GTP-binding proteins and protein kinases related to Shkl, Ste2O, and p65PAK, are highly conserved in evolution and participate in both cytoskeletal functions and mitogen-activated protein kinase signaling pathways.ras genes are highly conserved in evolution and encode small GTP-binding proteins that regulate cell growth and differentiation in a broad spectrum of eukaryotic organisms (1). The fission yeast Schizosaccharomyces pombe possesses a single known ras homolog, rasl, the product of which is required for at least two distinct cellular functions. First, Rasl is required for sexual differentiation-namely, conjugation and sporulation-which is induced by starvation and by peptide mating pheromones that bind to serpentine receptors (2-4). Rasl functions upstream of a mitogen-activated protein (MAP) kinase (MAPK) In this report, we describe a protein kinase, Shk,1, and provide evidence that it mediates functions of the Rasl/Cdc42 signaling complex in S. pombe. Shkl is highly related in structure to the Ste2O kinase, which is required for sexual response in Saccharomyces cerevisiae (19,20), and to the mammalian Cdc42/Racl-binding kinase, p65PAK (Pak) (21).Our results lead us to propose that signaling pathways mediated by small GTP-binding proteins and protein kinases related to Shkl are conserved in evolution and participate in regulation of the cytoskeleton and MAPK modules.MATERIALS AND METHODS Microbial Manipulation and Analysis. S. pombe strains SP870 (h9O ade6-210 leul-32 ura4-D18) and SP66 (h9o ade6-216 leul-32) were provided by D. Beach (Cold Spring Harbor Laboratory). SP870D (h90 ade6-210/ade6-210 leul-32/leul-32 ura4-D18/ura4-D18) is a spontaneous diploid derived from SP870 (V. Jung, personal communication). SP206U (h90 ade6-210/ade6-210 leul-32/leul-32 ura4-D18/ura4-D18 shk1::ura4/shk1) was constructed by transformation of SP870D with an Ec1136II-Msc I fragment of shkl::ura4 from plasmid pBSSHK1::URA4. SP206UA (h90 ade6-210/ade6-210 leul-32/leul-32 ura4-D18/ ura4-D18 shk1::ura4:.ADE2/shk1+) was constructed by transforming SP206U with a Not I fragment of ura4:.ADE2 obtained from pVIN (22). SP42N17 (h90 ade6-210 leul-32 ura4::adh1-cdc42[T17N]-ADE2) was constructed by transforming the S.Abbreviations: MAPK, mitogen-activated protein kinase; MAPKK, MAPK kinase; MAPKKK, MAPKK kinase; GST, glutathione Stransferase.

The growth of solid tumors is dependent on the continued stimulation of endothelial cell proliferation and migration resulting in angiogenesis. The angiogenic process is controlled by a variety of factors of which the vascular endothelial growth factor (VEGF) pathway and its receptors play a pivotal role. Small-molecule inhibitors of VEGF receptors (VEGFR) have been shown to inhibit angiogenesis and tumor growth in preclinical models and in clinical trials. A novel nicotinamide, AMG 706, was identified as a potent, orally bioavailable inhibitor of the VEGFR1/Flt1, VEGFR2/kinase domain receptor/Flk-1, VEGFR3/Flt4, platelet-derived growth factor receptor, and Kit receptors in preclinical models. AMG 706 inhibited human endothelial cell proliferation induced by VEGF, but not by basic fibroblast growth factor in vitro, as well as vascular permeability induced by VEGF in mice. Oral administration of AMG 706 potently inhibited VEGF-induced angiogenesis in the rat corneal model and induced regression of established A431 xenografts. AMG 706 was well tolerated and had no significant effects on body weight or on the general health of the animals. Histologic analysis of tumor xenografts from AMG 706-treated animals revealed an increase in endothelial apoptosis and a reduction in blood vessel area that preceded an increase in tumor cell apoptosis. In summary, AMG 706 is an orally bioavailable, well-tolerated multikinase inhibitor that is presently under clinical investigation for the treatment of human malignancies. (Cancer Res 2006; 66(17): 8715-21)