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Characterization of the Kinase Activity Essential for Tyrosine Phosphorylation of P130cas in ®Broblasts

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Characterization of the Kinase Activity Essential for Tyrosine Phosphorylation of P130cas in ®Broblasts Oncogene (1997) 14, 1419 ± 1426 1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00 Characterization of the kinase activity essential for tyrosine phosphorylation of p130Cas in ®broblasts Ryuichi Sakai1,4, Tetsuya Nakamoto2, Keiya Ozawa1, Shin-ichi Aizawa3 and Hisamaru Hirai2 1Molecular Biology Division, Jichi Medical School, 3311-1 Yakushiji, Minamikawachi-machi, Kawachi-gun, Tochigi 329-04; 2The Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113; 3Department of Morphogenesis, Institute of Molecular Embryology and Genetics, Kumamoto University, School of Medicine, 2-2-1 Honjo, Kumamoto 860, Japan The cellular transformation by v-Src or v-Crk induces and Gish, 1992). Each SH2 region binds to speci®c sets tyrosine phosphorylation of a common substrate mole- of phosphotyrosine-containing proteins by recognizing cule, p130Cas (Cas), which tightly binds these oncopro- a phosphotyrosine in the context of several adjacent teins in vivo. From its structure, Cas is deduced to be an amino acids (Moran et al., 1990; Muller et al., 1992; ideal substrate for Src family kinases and Abl kinase. Songyang et al., 1993). In non-receptor type tyrosine The tyrosine kinase activity associated with Cas was kinases, such as Src, Fps and Abl, the SH2 regions are analysed using mouse variant ®broblasts lacking at least located immediately at N-terminal to kinase domains. one of tyrosine kinases. In normal ®broblasts, Cas is Deletion or substitution of SH2 regions of activated associated with a signi®cant level of tyrosine kinase variants of Src and Fps often impairs the catalytic and activity which eciently phosphorylates Cas in vitro. The transforming activities of these kinases (Kitamura and Cas-associated tyrosine kinase activity was remarkably Yoshida, 1983; Sadowski et al., 1986; Raymond and elevated in Csk7/7 cells, which resulted in hyperphos- Parsons, 1987) and particular mutations within the phorylation of cellular Cas. The associated kinase SH2 region of Src induce host-dependent transforming activity was slightly increased in Src7/7 cells whereas phenotypes (DeCue et al., 1987; Hirai and Varmus, not signi®cantly changed in Abl7/7 nor Fak7/7 cells. On 1990). Furthermore, there is a signi®cant similarity the contrary, the Cas-associated kinase activity was between the substrate speci®city of these tyrosine remarkably decreased in Fyn7/7 cells. At the same time, kinases and the binding speci®city of their SH2 association of Cas with Fyn kinase in vitro was most regions, which was demonstrated by the experiment obviously detected in normal ®broblasts as well as using degenerate synthetic peptides (Pawson, 1995; Csk7/7 cells. Transient expression of v-Crk induced Songyang et al., 1995). elevation of the Cas-associated kinase activity in all of Thus speci®c protein tyrosine phosphorylation these cell lines except the primary culture of Fyn7/7 induced by each of tyrosine kinases is believed to be ®broblasts. These results indicate that Fyn kinase plays a critical machinery of cellular signal transduction. A an essential role in v-Crk-mediated phosphorylation of substantial amount of recent information is obtained Cas. regarding main target substrates of phosphorylation directly attacked by tyrosine kinases. For example, Keywords: p130Cas; tyrosine phosphorylation; Src phosphorylation of PLC-g (Wahl et al., 1988), GAP homology 2; signal transduction (Ellis et al., 1990), Nck (Park and Rhee, 1992; Li et al., 1992; Meisenhelder and Hunter, 1992), Vav (Bustelo et al., 1992; Margolis et al., 1992), Shc (Pelicci et al., 1992), Syp/SH-PTP2 (Feng et al., 1993), and cortactin Introduction (p80/p85) (Wu and Parsons, 1993) is observed during cellular transformation or by various growth factor Numbers of signaling molecules have been identi®ed in stimuli. Each of these signaling molecules may receive the signal transduction pathway from the membrane to several kinds of upstream signals and transduce a the nucleus. As a manner of the signal transfer between common set of signals which regulate cellular growth these molecules, phosphorylation of cellular proteins and dierentiation in a phosphorylation-dependent has been highlighted. Especially, the critical roles of manner. It is, though, still technically dicult to tyrosine phosphorylation in the signal transduction are identify the tyrosine kinase responsible for phosphor- certi®ed by the fact that many receptors for growth ylation of a particular substrate. factors and oncoproteins responsible for malignant We have recently cloned a novel kinase substrate, transformation are proved as tyrosine kinases by the p130Cas (Cas) which contains a cluster of multiple molecular cloning techniques. Recent studies suggest putative SH2-binding motifs and another signaling that a domain called Src homology 2 (SH2) domain region called Src homology 3 (SH3) domain (Sakai et has a signi®cant function to transduce the signal in a al., 1994a). The cellular transformation by v-Src or v- tyrosine phosphorylation-dependent manner (Pawson Crk induces tyrosine phosphorylation of Cas and stable association of Cas with these oncoproteins in vivo, suggesting a critical role of Cas in these oncogenic Correspondence: H Hirai signal transduction and cellular transformation 4Present address: Program in Molecular Biology and Cancer, Samuel (Matsuda et al., 1990; Birge et al., 1992; Sakai et al., Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada 1994a,b). v-Crk was ®rst identi®ed as a regulator of Received 22 July 1996; revised 13 November 1996; accepted 13 tyrosine kinases and causes elevation of tyrosine kinase November 1996 activity which results in transformation of ®broblasts Tyrosine kinase associated with p130Cas RSakaiet al 1420 (Mayer et al., 1988). It is still unclear, however, what a 1 2 3 b kind of tyrosine kinases is regulated by v-Crk and how 3Y1 3Y1-Crk SR-3Y1 the regulation takes place. Because tyrosine phosphor- ylation of Cas is the most obvious change found during 205 — the transformation of ®broblasts by v-Crk, it is quite 117 — important to identify the tyrosine kinase(s) responsible for the phosphorylation of Cas to elucidate the 80 — mechanism of cellular transformation and kinase regulation by v-Crk. 49 — Tyrosine phosphorylation of Cas is also observed in some biological events such as integrin-mediated cell Figure 1 Phosphorylation of p130Cas in vivo.(a) 3Y1-Crk (lane adhesion (Nojima et al., 1995; Petch et al., 1995). Since 2) and SR-3Y1 cells (lane 3) as well as normal 3Y1 cells (lane 1) Cas is predicted to be an ideal target molecule for are labeled 3 h in vivo by orthophosphate, immunoprecipitated by anti-Cas antibody and electrophoresed. Elevated tyrosine several SH2-containing tyrosine kinases including Src phosphorylation of Cas in these cells can be detected as shifts family kinases and Abl judging from their substrate of bands towards apparently higher molecular weight. The speci®cities and SH2-binding speci®cities (Pawson, position of phosphorylated p130 is indicated by a square bracket 1995; Mayer et al., 1995), it might act as a cellular at both ends. Molecular weights are given in kilodaltons. (b) The binding partner of these tyrosine kinases. To know the bands corresponding to phosphorylated Cas were cut out from the gel of (a), digested by TPCK trypsin and mapped two- biological role of Cas in cellular signal transduction, it dimensionally. The origins of electrophoresis were marked by is critical to identify the tyrosine kinase associated with close circles Cas. That is also important for understanding how tyrosine kinases share their roles in signal transduction network. Detection of the Cas-associated kinase (CASK) activity In this work, we characterized and analysed the in normal NIH3T3 cells kinase activity associated with Cas. We utilized variants of mouse ®broblasts from embryos lacking various To identify the cellular kinases responsible for the tyrosine kinases as well as normal mouse ®broblasts for phosphorylation of Cas, we analysed tyrosine kinase analysis of kinase(s) responsible for tyrosine phosphor- activity associated with Cas in the variants of mouse ylation of Cas. The eects of transient expression of v- ®broblasts. We used mutant ®broblasts lacking various Crk on the tyrosine kinase activity were also analysed in tyrosine kinases derived from embryos of gene- these cells for identi®cation of the tyrosine kinase disrupted mice. The complexes associated with Cas involved in transformation by v-Crk. were precipitated from these cell lysates using anti-Cas antiserum and the in vitro kinase assays of the complexes were performed with or without an exogenous substrate, poly[Glu-Tyr]. The kinase activ- Results ities involved in the complexes were analysed by the level of phosphorylation of Cas or of poly[Glu-Tyr]. Phosphopeptide mapping of phosphorylated Cas In normal NIH3T3 cells, a distinct tyrosine kinase To analyse the substrate speci®cities of the kinases activity associated with Cas was observed (Figure 2a, responsible for phosphorylation of p130Cas (Cas) in lanes 1 and 5). The kinase activity eciently transformation of ®broblasts, tyrosine-phosphorylation phosphorylated Cas and poly[Glu-Tyr] in vitro.We patterns of trypsin-digested fragments of Cas were tentatively designate this kinase activity as the CASK compared between cells transformed by v-Crk and v- (Cas-associated Kinase) activity. Cas itself was the Src using two-dimensional phosphopeptide
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