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Src-mediated activation of alpha-diacylglycerol kinase is required for hepatocyte growth factor-induced cell motility

2000; Springer Nature; Volume: 19; Issue: 17 Linguagem: Inglês

10.1093/emboj/19.17.4614

1460-2075

Santina Cutrupi,

Liver Disease Diagnosis and Treatment

Article1 September 2000free access Src-mediated activation of α-diacylglycerol kinase is required for hepatocyte growth factor-induced cell motility Santina Cutrupi Santina Cutrupi Department of Medical Sciences, University Amedeo Avogadro of Piemonte Orientale, Novara, Italy Department of Genetics, Biology and Biochemistry, University of Torino, Torino, Italy Search for more papers by this author Gianluca Baldanzi Gianluca Baldanzi Department of Medical Sciences, University Amedeo Avogadro of Piemonte Orientale, Novara, Italy Department of Genetics, Biology and Biochemistry, University of Torino, Torino, Italy Search for more papers by this author Daniela Gramaglia Daniela Gramaglia Institute for Cancer Research and Treatment (IRCC), University of Torino, Torino, Italy Search for more papers by this author Antonella Maffè Antonella Maffè Institute for Cancer Research and Treatment (IRCC), University of Torino, Torino, Italy Search for more papers by this author Dick Schaap Dick Schaap Division of Cellular Biochemistry, The Netherlands Cancer Institute, 1066 CX, Amsterdam, The Netherlands Search for more papers by this author Enrico Giraudo Enrico Giraudo Institute for Cancer Research and Treatment (IRCC), University of Torino, Torino, Italy Search for more papers by this author Wim J. van Blitterswijk Wim J. van Blitterswijk Division of Cellular Biochemistry, The Netherlands Cancer Institute, 1066 CX, Amsterdam, The Netherlands Search for more papers by this author Federico Bussolino Federico Bussolino Department of Genetics, Biology and Biochemistry, University of Torino, Torino, Italy Institute for Cancer Research and Treatment (IRCC), University of Torino, Torino, Italy Search for more papers by this author Paolo M. Comoglio Paolo M. Comoglio Institute for Cancer Research and Treatment (IRCC), University of Torino, Torino, Italy Search for more papers by this author Andrea Graziani Corresponding Author Andrea Graziani Department of Medical Sciences, University Amedeo Avogadro of Piemonte Orientale, Novara, Italy Department of Genetics, Biology and Biochemistry, University of Torino, Torino, Italy Department of Medical Sciences, University Amedeo Avogadro, v. Solaroli 17, 28100 Novara, Italy Search for more papers by this author Santina Cutrupi Santina Cutrupi Department of Medical Sciences, University Amedeo Avogadro of Piemonte Orientale, Novara, Italy Department of Genetics, Biology and Biochemistry, University of Torino, Torino, Italy Search for more papers by this author Gianluca Baldanzi Gianluca Baldanzi Department of Medical Sciences, University Amedeo Avogadro of Piemonte Orientale, Novara, Italy Department of Genetics, Biology and Biochemistry, University of Torino, Torino, Italy Search for more papers by this author Daniela Gramaglia Daniela Gramaglia Institute for Cancer Research and Treatment (IRCC), University of Torino, Torino, Italy Search for more papers by this author Antonella Maffè Antonella Maffè Institute for Cancer Research and Treatment (IRCC), University of Torino, Torino, Italy Search for more papers by this author Dick Schaap Dick Schaap Division of Cellular Biochemistry, The Netherlands Cancer Institute, 1066 CX, Amsterdam, The Netherlands Search for more papers by this author Enrico Giraudo Enrico Giraudo Institute for Cancer Research and Treatment (IRCC), University of Torino, Torino, Italy Search for more papers by this author Wim J. van Blitterswijk Wim J. van Blitterswijk Division of Cellular Biochemistry, The Netherlands Cancer Institute, 1066 CX, Amsterdam, The Netherlands Search for more papers by this author Federico Bussolino Federico Bussolino Department of Genetics, Biology and Biochemistry, University of Torino, Torino, Italy Institute for Cancer Research and Treatment (IRCC), University of Torino, Torino, Italy Search for more papers by this author Paolo M. Comoglio Paolo M. Comoglio Institute for Cancer Research and Treatment (IRCC), University of Torino, Torino, Italy Search for more papers by this author Andrea Graziani Corresponding Author Andrea Graziani Department of Medical Sciences, University Amedeo Avogadro of Piemonte Orientale, Novara, Italy Department of Genetics, Biology and Biochemistry, University of Torino, Torino, Italy Department of Medical Sciences, University Amedeo Avogadro, v. Solaroli 17, 28100 Novara, Italy Search for more papers by this author Author Information Santina Cutrupi1,2, Gianluca Baldanzi1,2, Daniela Gramaglia3, Antonella Maffè3, Dick Schaap4, Enrico Giraudo3, Wim J. van Blitterswijk4, Federico Bussolino2,3, Paolo M. Comoglio3 and Andrea Graziani 1,2,5 1Department of Medical Sciences, University Amedeo Avogadro of Piemonte Orientale, Novara, Italy 2Department of Genetics, Biology and Biochemistry, University of Torino, Torino, Italy 3Institute for Cancer Research and Treatment (IRCC), University of Torino, Torino, Italy 4Division of Cellular Biochemistry, The Netherlands Cancer Institute, 1066 CX, Amsterdam, The Netherlands 5Department of Medical Sciences, University Amedeo Avogadro, v. Solaroli 17, 28100 Novara, Italy *Corresponding author. E-mail: [email protected] The EMBO Journal (2000)19:4614-4622https://doi.org/10.1093/emboj/19.17.4614 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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. Introduction Diacylglycerol kinase (Dgk) phosphorylates diacylglycerol (DG) to generate phosphatidic acid (PA). The role of PA in cell signaling in intact cells still awaits elucidation. Dgk enzymes, by metabolizing DG, may also be involved in modulating activation of PKC. However, in vitro, PA regulates the enzymatic activity of a number of signaling molecules including type I phosphatidylinositol-4- phosphate 5-kinase (PI4P 5-kinase), n-chimerin, RhoGDI/Rac dissociation and NADPH oxidase (reviewed in Topham and Prescott, 1999). Furthermore, the view of PA as second messenger is supported by the discovery of phospholipase D (PLD) as an effector of small G proteins ARF and Ral (Jiang et al., 1995; Exton, 1997). To date, nine distinct Dgk isoforms have been cloned in mammals, all encoding soluble proteins, which reversibly associate to the membrane or to the nucleus (reviewed in Topham and Prescott, 1999). All isoforms share a catalytic domain preceded by two zinc fingers. However, the sequence of each isoform contains distinct regulatory domains. Class I Dgks, the α-, β- and γ-isoforms, share a pair of E-F hand calcium-binding domains preceding the zinc fingers. αDgk is abundant in T lymphocytes, but is also expressed in endothelial and epithelial cells, fibroblasts and oligodendrocytes (Schaap et al., 1990; A.Graziani, unpublished observations). Despite the wealth of information on their structure, the biological functions and biochemical regulation of Dgk enzymes remain to be elucidated. αDgk is activated by interleukin-2 (IL-2) in T cells, where it is required for IL-2-induced G1–S phase transition (Flores et al., 1996, 1999). In Drosophila, specific deletion of either a retinal specific Dgk or CDP-DG synthase results in the failure to resynthesize phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] from DG during sustained phosphatidylinositol (PI) turnover, leading to retinal degeneration (Hurley, 1995). Hepatocyte growth factor (HGF), through binding to its tyrosine kinase receptor, induces a range of biological responses, including scattering of epithelial cells, proliferation, motility and branching morphogenesis of both epithelial and endothelial cells, and invasiveness of carcinoma cells (Montesano et al., 1991; Rubin et al., 1993). In vivo, HGF is required for early development of liver, placenta and limb muscles, and is involved in kidney and liver regeneration (reviewed in Birchmeier and Gherardi, 1998). Furthermore, HGF induces angiogenesis in vivo in infarcted myocardium (Aoki et al., 2000). Signal transduction of HGF occurs through receptor recruitment and activation of several intracellular signaling transducers (Graziani et al., 1991, 1993; Ponzetto et al., 1994; Weidner et al., 1996). Membrane and receptor recruitment of Gab1 and Grb2, activation of Ras and PI 3-kinase are all required for HGF-induced cell migration, tubulogenesis and proliferation (Ponzetto et al., 1994; Derman et al., 1995; Royal and Park, 1995; Rahimi et al., 1996; Weidner et al., 1996; Khwaja et al., 1998; Maroun et al., 1999). Moreover, activation of Rac by HGF is required for cell motility (Derman et al., 1995; Royal and Park, 1995), while activation of both Src and Rho is required for tyrosine phosphorylation of focal adhesion and formation of stress fibers (Rahimi et al., 1998; Fukata et al., 1999; Royal et al., 2000). The involvement of Dgk proteins in receptor-tyrosine kinase signaling has never been reported. Herein we show that (i) αDgk is activated upon HGF stimulation; (ii) specific inhibition of αDgk through a dominant-negative mutant impairs HGF-induced cell movement; and (iii) upon HGF stimulation αDgk associates in a complex with Src, which mediates its activation. Results HGF activates αDgk We measured in vitro the enzymatic activity of αDgk protein specifically immunoprecipitated from lysates of either control or HGF-stimulated epithelial (GTL 16) and porcine endothelial (PAE) cells, and from COS-7 cells transiently expressing recombinant αDgk. Dgk activity was assayed by incubating the immunoprecipitates in the presence of exogenous DG and [γ-32P]ATP. Dgk activity was 3- to 6-fold higher in immunoprecipitates from HGF-stimulated GTL 16, PAE and αDgk-transfected COS cells than in those from unstimulated cells (Figure 1). The immunoprecipitates contained a single 86 kDa protein, which was identified as αDgk by western blotting with αDgk antibodies. All cell lines express HGF receptor, which becomes tyrosine phosphorylated upon HGF treatment (S.Cutrupi and A.Graziani, unpublished observations; Naldini et al., 1991). Figure 1.HGF activates αDGK in endothelial, epithelial and αDgk-transfected COS cells. Quiescent GTL 16, PAE and COS cells transfected with either empty vector or αDgk were stimulated with HGF (250 U/ml, 15 min). After lysis in detergent-containing buffer, αDgk was immunoprecipitated with a mix of anti-αDgk monoclonal antibodies. The immunocomplexes were analyzed for Dgk enzymatic activity (upper panel) and for αDgk protein by western blotting with anti-αDgk antibodies (lower panel). Download figure Download PowerPoint Catalytically inactive αDgk acts as a dominant-negative mutant In order to prove the involvement of αDGK in HGF signaling, we generated a dominant-negative mutant of αDGK. Many enzymes, such as SHP2, Src and Raf (Schaap et al., 1993b; Mohamed and Swope, 1999; Inagaki et al., 2000), are specifically inhibited by expression of their catalytically inactive mutants, which act as dominant negative. Substitution of Gly355 with Asp in the conserved kinase domain of ζDgk results in a kinase-inactive enzyme (Topham et al., 1998). Thus, we performed the homologous mutation in αDgk by substituting Gly433 with Asp, generating a catalytically inactive αDgk (αDgkK−) (data not shown). myc-tagged αDgkK− was subcloned in PINCOS retrovirus, which also expresses green fluorescent protein (GFP). The retrovirus obtained was used to infect PAE cells (Grignani et al., 1998). The efficiency of infection, measured as GFP expression by FACS analysis, was ∼80% (data not shown). To verify that αDgkK− acts as dominant negative, we evaluated the Dgk activity of anti-αDgk immunoprecipitates obtained from PAE cells infected with either PINCOS/myc-αDgkK− or the empty retrovirus (Figure 2). αDgk antibodies detect both endogenous and infected αDgk, which can however be recognized by SDS– PAGE as the mutant enzyme migrates more slowly for the presence of myc tag. Indeed, the expression of myc-αDgkK− inhibited the activation of endogenous endothelial αDgk induced by HGF stimulation of PAE cells (Figure 2). This experiment demonstrates that the kinase-inactive αDgk mutant acts as dominant negative. Figure 2.αDgk kinase-inactive mutant acts as dominant negative. Quiescent PAE cells infected with either PINCOS or PINCOS/myc-αDgkK− retrovirus were stimulated with HGF (250 U/ml, 15 min). After lysis in detergent-containing buffer, both endogenous and recombinant αDgk were immunoprecipitated with a mix of specific anti-αDgk monoclonal antibodies. The immunocomplexes were analyzed for Dgk enzymatic activity (upper panel) and for αDgk proteins, both endogenous and myc-αDgkK−, by western blotting with anti-αDgk antibodies (lower panel). Download figure Download PowerPoint Inhibition of αDgk impairs HGF-induced cell motility We investigated the role of αDgk in HGF signaling by inhibiting it in intact cells by either expression of a dominant-negative αDgk mutant or cell treatment with a specific inhibitor, R59949 [3-(2-{4-[bis-(4-fluorophenyl)methylene]-1-piperidinyl}ethyl)-2,3-dihydro-2-thioxo-4(1H) quinazolinone]. R59949 is a specific and powerful inhibitor of αDgk, while other tested Dgk isoforms are either not or poorly inhibited by it (Jiang et al., 2000). Cell treatment with 1 μM R59949 inhibited αDgk activity immunoprecipitated from HGF-stimulated cells (data not shown). The αDgk dominant-negative mutant inhibited by ∼50% the migration of endothelial cells induced by HGF in a chemotaxis assay (Figure 3A). The expression of infected αDgk, either dominant negative or wild type, and of endogenous αDgk is shown in Figure 3B. The expression of wild-type αDgk in PAE cells resulted in a slow increase, though not statistically significant, of cell migration following HGF stimulation. Moreover, HGF-induced chemotaxis of endothelial cells was also inhibited to a similar extent by 1 μM R59949 (Figure 3). The inhibition by R59949 was completely overridden by the overexpression of wild-type myc-αDgk. Together these two observations indicate that R59949 inhibits HGF-induced cell migration by acting specifically on αDgk, making it a suitable and convenient reagent to investigate the role of αDgk in cell signaling. Interestingly, the inhibition of αDgk by R59949 did not affect the migration of endothelial cells induced by 10% fetal calf serum (FCS), suggesting that αDgk is involved in a pathway that is specifically required for HGF signaling, but dispensable for serum-induced signaling. Figure 3.αDgk dominant-negative mutant and R59949 inhibit HGF-induced cell motility of PAE cells. (A) Quiescent PAE cells infected with either PINCOS (open boxes), PINCOS/myc-αDgk (hatched boxes) or PINCOS/myc-αDgkK− (dark boxes) retrovirus were induced to migrate in a Boyden chamber assay with 250 U/ml HGF or 10% FCS in the presence or absence of 1 μM R59949. The graph shows a representative experiment of three. Each value is the mean ± SE of triplicates. The differences between HGF-stimulated PINCOS versus HGF-stimulated PINCOS/myc-αDgkK−, HGF-stimulated PINCOS in the presence versus absence of 1 μM R59949 and HGF-stimulated R59949-treated PINCOS versus PINCOS/myc-αDgk are statistically significant (t-test, p <1%). (B) Quiescent PAE cells infected with either PINCOS, PINCOS/myc-αDgk or PINCOS/myc-αDgkK− were solubilized in Laemmli buffer, 50 μg of total proteins were separated by 10% SDS–PAGE and analyzed by western blotting with αDgk antibodies. Download figure Download PowerPoint Taken together, these data demonstrate by both a molecular and a pharmacological approach that activation of αDgk is required for a full chemotactic response to HGF. HGF induces the association of αDgk in a complex with Src We investigated the mechanisms of activation of αDgk by HGF. The sequence of αDgk does not feature any SH2 or PTP domain, suggesting that it is not docked to tyrosine kinase receptors. Indeed, no Dgk activity was found to associate with HGF receptor following HGF stimulation (data not shown). As enhanced synthesis of PA had been reported in v-Src-transformed fibroblasts and a Dgk activity was found to co-purify with v-Src (Sugimoto et al., 1984), we explored the hypothesis that Src may be involved in the activation of αDgk. We and others have previously shown that HGF activates Src in several cell lines (Ponzetto et al., 1994; Rahimi et al., 1998). Indeed, Dgk activity co-precipitated specifically with Src in anti-Src immunoprecipitates from HGF-stimulated GTL 16 and PAE cell lysates. Dgk activity did not co-purify with Src from unstimulated cells, nor was it detected in control immunoprecipitates. The Dgk activity co-precipitated with Src in PAE cells was inhibited by R59949, suggesting that it is the α isoform (Figure 4A). We have been able to detect both αDgk activity and protein in anti-Src immunopreciptates from HGF-stimulated COS cells (Figure 4B). Neither Dgk activity nor αDgk protein was detected in control immunoprecipitates. To provide further proof of the identity of the Src-associated Dgk, we have transiently expressed αDgk in COS cells. The formation of the Src–αDgk complex was observed by analyzing both anti-Src and anti-αDgk immunocomplexes with anti-αDgk and anti-Src antibodies, respectively. The Src–αDgk complex was detected only in immunoprecipitates obtained from HGF-stimulated cells, and was not detected in control immunoprecipitates obtained in the absence of either anti-Src or anti-αDgk antibodies (Figure 4C). Figure 4.HGF induces association of αDGK with Src. Quiescent cells were stimulated with HGF (250 U/ml, 15 min) and lysed in detergent-containing buffer. (A) Src was immunoprecipitated with anti-Src antibodies from either GTL 16 or PAE cell lysates. Control and anti-Src immunocomplexes were analyzed for Dgk enzymatic activity (where indicated R59949 was added in the reaction mix) (upper panel) and for Src protein by western blotting with anti-Src antibodies (lower panel). (B) Src was immunoprecipitated with anti-Src antibodies from COS cell lysates. Control and anti-Src immunocomplexes were analyzed for Dgk enzymatic activity (upper panel), for αDgk protein by western blotting with αDgk antibodies (central panel), and for Src protein by western blotting with anti-Src antibodies (lower panel). (C) Either αDgk or Src was immunoprecipitated with anti-αDgk or anti-Src antibodies from lysates of αDgk-transfected COS cells. Control, anti-αDgk and anti-Src immunocomplexes were analyzed by western blotting with anti-αDgk and anti-Src antibodies. Download figure Download PowerPoint In summary, these experiments demonstrate that the αDgk–Src complex is formed upon HGF stimulation, suggesting that indeed Src tyrosine kinase may mediate the HGF-induced activation of αDgk. Src function is necessary for HGF-induced αDgk activation In order to elucidate the role of Src in the mechanism of activation of αDgk, we investigated the effect of specific inhibition of Src tyrosine kinase activity on the HGF-induced activation of αDgk. Src was inhibited either by transient expression of a Src kinase-inactive mutant or by cell treatment with PP1 ([4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]-pyrimidine), a specific inhibitor of Src tyrosine kinase activity (Hanke et al., 1996). Upon overexpression, the Src kinase-deficient mutant acts as dominant negative by inhibiting both tyrosine phosphorylation of protein substrates and cellular responses induced by activation of endogenous Src (Barone and Courtneidge, 1995; Mukhopadhyay et al., 1995; Mohamed and Swope, 1999). Transient co-expression of the Src kinase-deficient mutant with αDgk in COS cells significantly reduced activation of αDgk by HGF (Figure 5A), while it does not affect αDgk activity of unstimulated cells. In these experiments, αDgk activity was measured in whole-cell lysates in the presence of exogenous DG and ATP substrates. Under these experimental conditions, endogenous Dgk activity in untransfected cells was negligible compared with transfected αDgk. Similarly, treatment of cells with 1 μM PP1 inhibited HGF-induced activation of αDgk transiently expressed in COS cells (Figure 5B). The same concentration of PP1 did not inhibit αDgk activity in vitro or HGF receptor tyrosine phosphorylation (data not shown). In summary, inhibition of Src tyrosine kinase activity by both a molecular and a pharmacological approach inhibits the signaling pathways triggered by HGF, leading to the activation of αDgk. Figure 5.Src kinase activity is required for HGF-induced activation of αDgk. (A) Quiescent COS cells transfected with either vector alone, αDgk or αDgk and SrcK− were stimulated with HGF (250 U/ml, 15 min). Whole-cell homogenates were assayed for Dgk activity (upper panel), Src protein by western blotting with anti-avian Src antibodies (central panel) and αDgk protein by western blotting with anti-αDgk antibodies (lower panel). (B) Quiescent COS cells transfected with either vector alone or myc-αDgk were stimulated with HGF (250 U/ml, 15 min) and, where indicated, treated with 1 μM PP1 (15 min). After lysis in detergent-containing buffer, αDgk was immunoprecipitated with anti-myc monoclonal antibodies. The immunocomplexes were analyzed for Dgk enzymatic activity (upper panel) or for myc-αDgk protein by western blotting with anti-myc antibodies (lower panel). Download figure Download PowerPoint In order to provide further proof of the role of Src in the activation of αDgk, we have investigated the ability of Src to activate αDgk in vitro. Cell extracts from COS cells overexpressing either Src or αDgk were mixed in vitro in the presence of 1 mM ATP. Following 10 min incubation, αDgk activity was measured from whole extracts as described above (Figure 6). Co-incubation of αDgk and Src extracts resulted in the net stimulation of αDgk activity compared with co-incubation of αDgk and control extracts. Conversely, no Dgk activity was detected following co-incubation of Src and control extracts, indicating that only transfected αDgk activity was detected under these experimental conditions. In the same assay, the Src kinase-deficient mutant did not activate αDgk. Furthermore, pre-incubation with ATP was required for αDgk activation by Src (data not shown). Taken together, these data suggest that Src activates αDgk in vitro and that its tyrosine kinase domain is required for activation. Figure 6.Src activates αDgk in vitro. COS cells transfected with either vector alone, myc-αDgk, Src or SrcK− were homogenized in the absence of detergent. Cell extracts were mixed as indicated in the presence of 1 mM ATP for 15 min, and were analyzed for αDgk activity (upper panel), for myc-αDgk protein by western blotting with anti-αDgk antibodies (central panel) and for Src protein by western blotting with anti-avian Src antibodies (lower panel). Download figure Download PowerPoint Tyrosine phosphorylation of αDgk Although the data presented herein indicate that Src tyrosine kinase activity is required for activation of αDgk by HGF, we could not detect any tyrosine phosphorylation of αDgk immunoprecipitated from HGF-stimulated cells. In order to investigate whether αDgk can indeed be phosphorylated on the tyrosine residue, we co-expressed αDgk in COS cells with Src. Upon co-expression with wild-type Src, myc-αDgk becomes phosphorylated on tyrosine, as detected by anti-phosphotyrosine western blotting of anti-myc immunoprecipitates (Figure 7B). The tyrosine-phosphorylated band was confirmed to be αDgk by western blotting using anti-myc antibodies. Neither transfection with the Src kinase-defective mutant nor that with empty vector resulted in αDgk tyrosine phosphorylation. Furthermore, anti-myc immunoprecipitates from cells transfected with Src alone did not contain any tyrosine-phosphorylated protein. However, tyrosine phosphorylation of αDgk did not correlate with its enzymatic activation as αDgk activity is not stimulated by co-expression with Src (data not shown). Figure 7.Tyrosine phosphorylation of αDgk. (A) Quiescent PAE cells infected with PINCOS/myc-αDgk were stimulated with either HGF (250 U/ml, 15 min), pervanadate (1 mM Na3VO4, 2 mM H2O2, 15 min), or both. After lysis in detergent-containing buffer, αDgk was immunoprecipitated with anti-myc monoclonal antibodies. The immunocomplexes were analyzed for Dgk enzymatic activity (upper panel), for tyrosine-phosphorylated proteins by western blotting with anti-phosphotyrosine (central panel) or for myc-αDgk protein by western blotting with anti-myc antibodies (lower panel). (B) Growing COS cells, transfected as indicated with either empty vector, myc-αDgk, Src or SrcK, were lysed in detergent-containing buffer; myc-αDgk was immunoprecipitated by anti-myc antibodies. Immunocomplexes were analyzed for tyrosine-phosphorylated protein by western blotting with anti-phosphotyrosine antibodies (upper panel) and for myc-αDgk protein by western blotting with anti-myc antibodies (lower panel). Download figure Download PowerPoint We have also investigated the tyrosine phosphorylation of αDgk following treatment of PAE cells expressing myc-αDgk with pervanadate, a strong inhibitor of protein-tyrosine phosphatases (Posner et al., 1994). Cell treatment with pervanadate (1 mM Na3VO4, 2 mM H2O2) for 15 min resulted in a strong induction of cellular protein-tyrosine phosphorylation (data not shown). Indeed, αDgk immunoprecipitated from pervanadate-treated cells was phosphorylated on tyrosine, as detected by anti-phosphotyrosine western blotting (Figure 7A). Interestingly, tyrosine-phosphorylated αDgk featured a higher enzymatic activity, as measured in anti-myc immunoprecipitates. However, in the same experiment HGF stimulated αDgk activity to a similar extent without affecting its tyrosine phosphorylation. In summary, the results presented in Figure 7 show that tyrosine phosphorylation of αDgk in intact cells can be detected, albeit under extreme experimental conditions such as co-expression with Src or strong inhibition of tyrosine phosphatases. Whether tyrosine phosphorylation occurs in cell signaling, even at low stoichiometry, still remains to be elucidated. Furthermore, the role of putative tyrosine phosphorylation of αDgk is still elusive, as it does correlate with enzymatic activation following pervanadate treatment but not upon co-expression with Src. Discussion The data presented herein show for the first time that αDgk is activated upon stimulation of a tyrosine kinase receptor, that it plays a role in the transduction of HGF signaling leading to cell motility, and that it is activated through a mechanism that involves Src tyrosine kinase. It has long been known that PA is generated in response to growth factors and PI turnover agonists (Hodgkin et al., 1998). Similarly, the level of PA is increased in cells transformed by oncogenes (Sugimoto et al., 1984; Martin et al., 1997). However, the lack of direct demonstration of ligand- or oncogene-induced stimulation of Dgk activity has made the role of Dgk enzymes in signal transduction elusive. Although several lines of evidence suggest that growth factors and neurotransmitters may activate Dgk enzymes, in most of these studies Dgk enzymatic activities were assayed on crude extracts and without identification of Dgk isoforms (Topham and Prescott, 1999). Herein we demonstrate in endothelial and epithelial cells, as well as in αDgk-transfected cells, that αDgk enzymatic activity, measured in vitro in specific immunoprecipitates, is stimulated upon HGF-induced activation of its tyrosine kinase receptor. Similarly, αDgk was previously shown to be activated by IL-2 in a T-cell-derived cell line (Flores et al., 1996). The role of Dgk enzymes in cell signaling has been investigated. Pharmacological inhibition of αDgk in T cells impairs G1–S phase transition upon IL-2 stimulation (Flores et al., 1996, 1999), while on the contrary PKC-mediated nuclear translocation of ζDgk attenuates serum-induced cell growth (Topham et al., 1998). In order to investigate the role of αDgk in HGF signaling, we have generated an αDgk dominant-negative mutant. Substitution of Gly433 in the ATP binding site of αDgk with Asp results in an inactive enzyme, which, as predicted, inhibits HGF-induced activation of endogenous αDgk. Both the dominant-negative mutant and R59949 (αDgk inhibitor) impair HGF-induced cell movement of endothelial cells. Interestingly, we also provide evidence that R59949 acts specifically on αDgk as its action is overridden by overexpression of wild-type αDgk. These observations provide the first evidence for an involvement of activation of αDgk in tyrosin