Diacylglycerol kinases are involved in cell signaling, either as regulators of diacylglycerol levels or as intracellular signal‐generating enzymes. However, neither their role in signal transduction nor their biochemical regulation has been elucidated. Hepatocyte growth factor (HGF), upon binding to its tyrosine kinase receptor, activates multiple signaling pathways stimulating cell motility, scattering, proliferation and branching morphogenesis. Herein we demonstrate that: (i) the enzymatic activity of α‐diacylglycerol kinase (αDgk) is stimulated by HGF in epithelial, endothelial and αDgk‐transfected COS cells; (ii) cellular expression of an αDgk kinase‐defective mutant inhibits activation of endogenous αDgk acting as dominant negative; (iii) specific inhibition of αDgk prevents HGF‐induced cell movement of endothelial cells; (iv) HGF induces the association of αDgk in a complex with Src, whose tyrosine kinase activity is required for αDgk activation by HGF; (v) Src wild type stimulates αDgk activity in vitro; and (vi) αDgk can be tyrosine phosphorylated in intact cells.

Activation of the HGF receptor, encoded by the c-MET protooncogene (Met receptor), triggers motility, matrixinvasion and branching morphogenesis in epithelial cells. It has recently been shown that the Met receptor interacts with Gab-1, an IRS-like adaptor protein, via the docking site (Y 1349 VHVNATY 1356 VNV) known to bind Grb2 and multiple SH2-containing signal transducers. Here we show that Gab1 is the major phosphorylation-substrate of the Met receptor and of its oncogenic variant Tpr-Met. A series of point mutations in the docking site established a direct correlation between the ability to recruit and phosphorylate Gab1 and the transforming potential. Interestingly, the mutations of either Y 1356 or N 1358 abolished the binding of both Grb2 and Gab1 in intact cells. Furthermore, peptides designed to block either the SH2 or the SH3 domains of Grb2 interfered with the receptor-Gab1 interaction. These data indicate that Gab1 coupling to the Met receptor requires binding of Grb2 and correlates with the transforming potential of Tpr-Met.

nef is a human immunodeficiency virus (HIV) gene encoding a 27-kDa myristoylated protein with structural features of a signal transducing molecule, but whose functions are largely unknown. We studied the interactions of Nef with the signal transduction pathways triggered by the platelet-derived growth factor (PDGF) receptor. The association of phosphatidylinositol (PI) 3-kinase with the activated receptor was severely impaired by nef expression. Conversely, PDGF-induced receptor tyrosine phosphorylation, binding to phospholipase C-gamma and to Ras-GAP were not modified. Microtubule-associated protein kinase activation and intracellular calcium influx in response to PDGF were either unaffected or only slightly enhanced. Nef significantly reduced the proliferative response to the growth factor, while the chemotactic response was unchanged. These data show that Nef affects selectively the PI 3-kinase signaling pathway and suggest that this interference results in some of the HIV adverse effects on host cell functions.

The assumption that genes encoding tyrosine kinase receptors could play a role in human cancers has been confirmed by the identification of oncogenic mutations in the kinase domain of RET and KIT. Recently, homologous residues were found mutated in MET, in papillary renal carcinomas (PRCs). The link coupling these genetic lesions to cellular transformation is still unclear. MET PRC mutations result in increased kinase activity and-in some instances, i.e., M1250T substitution-in changes in substrate specificity. A direct correlation occurs between the transforming potential of MET PRC mutants and their ability to constitutively associate with signal transducers through two phosphorylated tyrosines (Y 1349 VHVNATY 1356 VNV) located in the receptor tail. Substitution of these ''docking tyrosines'' with phenylalanines leaves unaffected the altered properties of the kinase but abrogates transformation and invasiveness in vitro. Uncoupling the receptor from signal transducers with a tyrosine-phosphorylated peptide derivative (Y p VNV) inhibits invasive growth induced by MET PRC mutants. These data indicate that constitutive receptor coupling to downstream signal transducers is a key mechanism in neoplastic transformation driven by mutated MET and suggest a therapeutic strategy to target neoplastic diseases associated with this oncogene.

Overexpressed or activated hepatocyte growth factor receptor, encoded by the MET proto-oncogene, was found in the majority of colorectal carcinomas (CRCs), whose stepwise progression to malignancy requires transcriptional activation of b-catenin. We here demonstrate that a functional crosstalk between Met and b-catenin signaling sustains and increases CRC cell invasive properties. Hepatocyte growth factor (HGF) stimulation prompts b-catenin tyrosine phosphorylation and dissociation from Met, and upregulates b-catenin expression via the phosphatidylinositol 3-kinase pathway in conditions that mimic those found by the invading and metastasizing cells. Additionally, a transcriptionally active form of b-catenin, known to be oncogenic, enhances Met expression. Furthermore, HGF treatment increases the activity of the b-catenin-regulated T-cell factor transcription factor in cells expressing the wild-type or the oncogenic b-catenin. In the mirror experiments, either Met or b-catenin knocking down also reduces their protein level. In biological assays, b-catenin knocking down abrogates the HGF-induced motile phenotype, whereas active b-catenin fosters ligand-independent cell scattering. Met and b-catenin also cooperate in promoting entry into the cell cycle and in protecting cells from apoptosis. In conclusion, Met and b-catenin pathways are mutually activated in CRC cells. This might generate a selfamplifying positive feedback loop resulting in the upregulation of the invasive growth properties of CRC cells.

The HIV-1 nef gene, essential for AIDS pathogenesis, encodes a 27-kDa protein (Nef) whose biochemical and biological functions are unclear. It has been suggested that Nef expression contributes to the T cell depletion observed during the disease by promoting their apoptosis. We report that in CD4+ human lymphoblastoid cell lines transfected with the nef cDNA obtained from three different HIV-1 strains, expression of the Nef protein enhances and accelerates the response to four unrelated apoptotic agents (staurosporine, anisomycin, camptothecin, and etoposide) but not to an anti-Fas agonist Ab. Nef reduces the expression of the anti-apoptotic proteins Bcl-2 and Bcl-XL and induces a striking enhancement of apoptotic hallmarks, including mitochondrial depolarization, exposure of phosphatidylserine on the cell surface, activation of caspase-3, and cleavage of the caspase target poly(ADP-ribose) polymerase. Interestingly, the peptide Z-Val-Ala-DL-Asp-fluoromethylketone (a broad-spectrum caspase inhibitor) reduces, but does not abolish, phosphatidylserine exposure, suggesting that Nef also activates a caspase-independent apoptotic pathway. Surprisingly, Nef expression increases DNA degradation but without causing oligonucleosomal fragmentation. An increased apoptotic response and down-modulation of Bcl-2/Bcl-XL following Nef expression are observed also in NIH-3T3 fibroblasts. These data show that Nef enhances programmed cell death in different cell types by affecting multiple critical components of the apoptotic machinery independently from the Fas pathway.

Cancer of unknown primary origin (CUP) defines metastatic disease of unknown origin, accounting for 3–5% of all cancers. Growing evidence demonstrates that inappropriate execution of a genetic program named “invasive growth,” driven by the MET oncogene, is implicated in the metastatic process. MET activation in cancers is mainly consequent to overexpression, whereas mutations are rarely found. We reasoned that the occurrence of MET somatic mutations might sustain premature occult dissemination of cancer cells, such as that observed in CUPs. We sequenced MET in genomic DNA obtained from 47 early metastatic cancers. By extensive immunohistochemical analysis a primary site was afterward postulated in 24 patients, whereas 23 cases remained of unknown primary (CUPs). MET somatic mutations were found in seven cases, all belonging to the CUP cohort. Mutational incidence (30%) was thus significantly higher than the expected one (4%), in the absence of high mutational background. Several nucleotide changes were novel and clustered either in the kinase domain or in the extracellular semaphorin domain. Mutated receptors were functional and sustained the transformed phenotype, suggesting that MET activating mutations are genetic markers associated with the CUP syndrome. Hum Mutat 31:1–7, 2010. © 2010 Wiley‐Liss, Inc.

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