Cas-L (pp105), a Crk-associated substrate (p130Cas )-related protein, was first identified as a 105-kDa protein that is tyrosine-phosphorylated following ␤1 integrin cross-linking in T cells. Cas-L contains possible multiple binding sites for the Src homology (SH) 2 domains of various signaling molecules, and appears to be involved in signal transduction through phosphorylated tyrosine-mediated protein-protein interaction. Since Cas-L is preferentially expressed in lymphocytes, it is conceivable that Cas-L plays an important role in lymphocytespecific signals. Here, we show the involvement of Cas-L in the T cell receptor (TCR)/CD3 signaling pathway. Cas-L is transiently phosphorylated following CD3 cross-linking, and tyrosine-phosphorylated Cas-L binds to Crk and C3G. Furthermore, a Cas-L mutant that lacks the SH3 domain, the binding site for focal adhesion kinase (FAK), is also tyrosine-phosphorylated upon CD3 cross-linking, but not upon ␤1 integrin crosslinking, suggesting that FAK is not involved in CD3-dependent Cas-L phosphorylation. Taken together, the present study indicates a novel signaling pathway mediated by tyrosine-phosphorylated Cas-L upon the TCR/CD3 stimulation. T cell receptor (TCR)1 -antigen binding induces gene expression, cytokine production, and cell proliferation in T lymphocytes (1). These TCR-dependent signals are mediated by tyrosine phosphorylation of various proteins including CD3␦, CD3⑀, CD3␥, Shc, Vav,. These signaling molecules appear to be involved in the phosphorylated tyrosinemediated protein-protein interaction and the recruitment of the other signaling molecules containing Src homology (SH) 2 domains (3). The recruitment of these signaling molecules is essential to induce various signals to the downstream events such as the activation of mitogen-activated protein kinases, Ca 2ϩ influx, and transcriptional activation of various genes (1).Thus, protein tyrosine phosphorylation plays a key role during the initial phase of TCR-mediated T cell activation. However, the essential phosphorylated molecule and the precise function of each phosphorylated molecule for these signaling pathwayshave not yet been clarified. Cas-L (pp105) was first identified as a 105-kDa protein that is tyrosine-phosphorylated upon ␤1 integrin cross-linking (4). Cas-L belongs to Cas-family along with p130Cas and Efs/Sin (5-7). The three proteins share the structural characteristics of an N-terminal SH3 domain, followed by multiple (8 to 15) YXXP motifs, which are putative binding sites for the Crk SH2 domain, and a conserved YDYVHL sequence that possibly binds to the Src SH2 domain. (5-7). Cas-L was shown to associate with the C-terminal domain of focal adhesion kinase (FAK), a cytoplasmic tyrosine kinase that is localized to focal adhesions (5,8). Recently, we reported that the conserved YDYVHL sequence of Cas-L was phosphorylated by FAK following ␤1 integrin cross-linking and that an Src family tyrosine kinase is recruited to the phosphorylated YDYVHL sequence and causes further tyrosine phosphorylation of ...

Integrin-ligand binding induces the tyrosine phosphorylation of various proteins including focal adhesion kinase (pp125 FAK ) and Crk-associated substrate (Cas). FAK is activated and autophosphorylated by the ligation of integrins, although the substrate of FAK has not been revealed. We show here that p130Cas and Cas-L are FAK substrates. FAK directly phosphorylates Cas proteins primarily at the YDYVHL sequence that is conserved among all Cas proteins. Furthermore, the phosphorylated YDYVHL sequence is a binding site for Src family protein-tyrosine kinases, and the recruited Src family kinase phosphorylates the other tyrosine residues within Cas. The Cas-L YDYVHL sequence is phosphorylated upon integrin-ligand binding, and this integrin-mediated tyrosine phosphorylation is inhibited by the cotransfection of the FAK COOH-terminal domain that does not contain a kinase domain. These findings strongly suggest that FAK initiates integrin-mediated tyrosine phosphorylation of Cas proteins; then, Src family tyrosine kinases, which are recruited to phosphorylated Cas and FAK, further phosphorylate Cas proteins.Integrin-ligand binding induces various biological and biochemical signals in addition to cell adhesion (1-4). We and others showed the costimulatory effect of the ligation of ␤1 integrin in CD3-dependent T cell proliferation, indicating that integrin-ligand binding can promote cell proliferation (5, 6). To reveal the mechanism of the integrin-mediated biological signals, numbers of laboratories have studied the role of protein tyrosine phosphorylation in various cell types (7-10). However, how proteins are tyrosine phosphorylated upon integrin stimulation is currently unknown. Since integrin themselves are not a tyrosine kinase, a tyrosine kinase(s) that is functionally linked to integrins is essential for the integrin signaling pathway.One of the prime candidates for this tyrosine kinase is focal adhesion kinase, pp125 Cas is a 120 -130-kDa protein that was first identified as a highly tyrosine-phosphorylated protein in both v-Src-and v-Crk-transformed fibroblasts (25-27). Subsequently, a 105-110-kDa p130Cas -related protein (Cas-L or HEF1) was identified as a 105-kDa protein that is tyrosine phosphorylated upon the ligation of ␤1 integrins in T lymphocytes (28,29). Furthermore, an 83-kDa Cas-related protein, Efs (or Sin), was reported as the binding proteins for the Src homology 3 (SH3) domains of Fyn and Src (30,31). All Cas proteins contain one SH3 domain in the NH 2 -terminal region, and they all contain multiple putative binding sites for Src homology 2 (SH2) domains, including 8 -15 binding sites for the Crk SH2 domain. All three Cas proteins also contain a YDYVHL sequence, which is a potential binding site for the Src SH2 domain (31, 32). We and others reported that both p130 Cas and Cas-L were tyrosine phosphorylated upon the ligation of integrins and that tyrosine-phosphorylated Cas proteins bind to the SH2 domain-containing proteins Crk, Nck, and SHP-2 (28, 33, 34). Furthermore, Cas proteins bind direc...

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We found clear differences in clinical features between early- and late-onset AN groups.

Expression of the gamma chain, which is shared among functional receptor complexes for interleukin (IL)-2, IL-4 and IL-7, was examined with hematopoietic cells in mouse thymus and spleen by flow cytometry. The gamma chain was expressed in cell populations from the spleen. Stimulation with concanavalin A and lipopolysaccharide caused fluctuation in expression of the gamma chain in T and B cells, respectively. T lineage cells developing in the adult thymus expressed the gamma chain. Fetal thymus at day 15 contained mostly immature thymocytes, which also expressed the gamma chain. These results demonstrate that the gamma chains is widely expressed in T lineage cells, probably indicating that the gamma chain plays a role not only in the proliferation of mature hematopoietic cells but also in the development of immature cells through signal transduction as a common receptor subunit for multiple cytokines.

The "touchdown" polymerase chain reaction (PCR) technique has been applied to analyze expression of the neuron-specific protein, PGP9.5, and tyrosine hydroxylase (TH) genes for detection of minimal residual neuroblastoma cells in bone marrow and peripheral blood. PGP9.5 and TH gene products were not detected in any normal samples (n = 72) examined. However, in patients more than 1 year of age with stage III and IV neuroblastoma PGP9.5 mRNA was detected in six of seven bone marrow samples and in four of eight peripheral blood samples, and TH mRNA in four of seven and three of eight, respectively. The detection sensitivity was up to 10(-6) to 10(-7) micrograms of total cellular RNA for PGP9.5 and 10(-4) micrograms for TH. Among forty bone marrow specimens from nineteen patients with neuroblastoma both PGP9.5 and TH mRNAs were detected in six, and only PGP9.5 mRNA was detected in ten. Since detection of PGP9.5 and TH gene transcripts by the "touchdown" PCR was highly specific and sensitive, it might be most informative at present to carry out both PGP9.5 and TH mRNA assays for minimal residual neuroblastoma cells in blood and bone marrow.