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The Delta/Serrate/LAG-2 (DSL) domain containing proteins are considered to be ligands for Notch receptors. However, the physical interaction between DSL proteins and Notch receptors is poorly understood. In this study, we cloned a cDNA for mouse Jagged1 (mJagged1). To identify the receptor interacting with mJagged1 and to gain insight into its binding characteristics, we established two experimental systems using fusion proteins comprising various extracellular parts of mJagged1, a "cell" binding assay and a "solid-phase" binding assay. mJagged1 physically bound to mouse Notch2 (mNotch2) on the cell surface and to a purified extracellular portion of mNotch2, respectively, in a Ca(2+)-dependent manner. Scatchard analysis of mJagged1 binding to BaF3 cells and to the soluble Notch2 protein demonstrated dissociation constants of 0.4 and 0.7 nM, respectively, and that the number of mJagged1-binding sites on BaF3 is 5,548 per cell. Furthermore, deletion mutant analyses showed that the DSL domain of mJagged1 is a minimal binding unit and is indispensable for binding to mNotch2. The epidermal growth factor-like repeats of mJagged1 modulate the affinity of the interaction, with the first and second repeats playing a major role. Finally, solid-phase binding assay showed that Jagged1 binds to Notch1 and Notch3 in addition to Notch2, suggesting that mJagged1 is a ligand for multiple Notch receptors.

Leukocyte tyrosine kinase (LTK) is a receptor tyrosine kinase that belongs to the insulin receptor family. LTK is mainly expressed in pre B cells and brain. Previously we cloned the full-length cDNA of human LTK, but no ligands have so far been identified, and hence, very little is known about the physiological role of LTK. To analyze the function of the LTK kinase, we constructed chimeric receptors composed of the extracellular domain of epidermal growth factor receptor and the transmembrane and the cytoplasmic domains of LTK and established cell lines that stably express these chimeric molecules. When cultured in medium containing EGF, growth of these cell lines was stimulated, and these fusion proteins became autophosphorylated and associated with Shc in vivo in a ligand-dependent manner. By treatment with EGF, Shc was associated with the Grb2/Ash-Sos complex. Our analyses demonstrate that LTK associates with Grb2/Ash through an internal adaptor, Shc, depending on a ligand stimulation. The LTK binding site for Shc was tyrosine 862 at the carboxyl-terminal domain and to a lesser extent tyrosine 485 at the juxtamembrane domain. Both of them are located in NP/AXY motif which is consistent with binding sites for Shc. These findings demonstrate that LTK can activate the Ras pathway in a ligand-dependent manner and that at least one of the functions of this kinase is involved in the cell growth.

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 p130(Cas) 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.

The related adhesion focal tyrosine kinase (RAFTK) is tyrosine-phosphorylated following beta1 integrin or B cell antigen receptor stimulation in human B cells. Two substrates that are tyrosine-phosphorylated following integrin ligation in B cells are p130(Cas) and the Cas family member human enhancer of filamentation 1 (HEF1), both of which can associate with RAFTK. In this report we observed that RAFTK was involved in the phosphorylation of these two proteins. While a catalytically active RAFTK was required for both p130(Cas) and HEF1, phosphorylation of p130(Cas), but not of HEF1, was dependent on an intact autophosphorylation site (Tyr402) on RAFTK. To determine if RAFTK phosphorylated p130(Cas) and HEF1 directly or through an intermediate, we assayed the ability of RAFTK and of a Tyr402 mutant to phosphorylate purified HEF1 and p130(Cas) domains. RAFTK was able to phosphorylate the substrate domains of both p130(Cas) and HEF1, but only the C-terminal domain of p130(Cas). Furthermore, Tyr402, which mediates the binding of RAFTK to c-Src kinase, was required for the phosphorylation of the C-terminal domain of p130(Cas). These data suggest that RAFTK itself is sufficient for HEF1 phosphorylation, whereas a cooperation between RAFTK and Src kinases is required for the complete phosphorylation of p130(Cas).

The Src family tyrosine kinases and their substrates are involved in cell-cell and cell-matrix interactions. We found that in PC12h cells, an increase of cell density enhanced the tyrosine phosphorylation levels of several intracellular proteins including p130(cas). Because it is a possible substrate for Src family kinases, we measured pp60(c-src) activity and found that it was higher in high density cultures than in low density cultures. This phenomenon was also observed in PC12 (the parental cell line of the PC12h subclone), Balb/c 3T3, Swiss 3T3, and Hela cells. One of the possible mechanisms regulating the kinase activity of pp60(c-src) is the phosphorylation and dephosphorylation of its negative regulatory site located at its C terminus. However, the tyrosine phosphorylation level of the regulatory site did not change depending on cell density. Subcellular fractionation showed that in high density culture, pp60(c-src) was translocated from detergent-soluble to detergent-insoluble fractions. These results suggest that cell-cell interaction might induce the activation of pp60(c-src) without changing its tyrosine phosphorylation levels.

Engagement of beta1 integrins in terminally differentiated human B cell lines, such as ARH-77, leads to prominent tyrosine phosphorylation of the p130 Crk-associated substrate (Cas). Cas regulates the assembly of several SH2 and SH3 domain-containing proteins into signaling complexes, which are potentially involved in the propagation of downstream signals. We demonstrate here that immunoprecipitated Cas from beta1 integrin-stimulated ARH-77 cells was associated with tyrosine kinase and phosphatase activities and that integrin ligation led to the recruitment of at least p59(Fyn) tyrosine kinase and SHP2 tyrosine phosphatase in Cas immune complexes. Cotransfection studies in COS-7 cells further indicated that Fyn/Cas physical interaction and Fyn-mediated Cas phosphorylation required amino acids 638-889 in the C-terminal region of Cas. This sequence contains both c-Src SH2 and SH3 domain-binding motifs. In vitro binding studies using glutathione S-transferase fusion proteins derived from the SH2 or SH3 domains of Fyn suggested that both Fyn domains can participate in Fyn/Cas interaction. These data implicate Fyn and SHP2 as potential modulators of Cas signaling complexes in B cells.

Many growth factors including epidermal growth factor (EGF) induce tyrosine phosphorylation of the c-Cbl proto-oncogene product, whose function, however, remains unclear. Recently, Sli-1, a Caenorhabditis elegans homologue of c-Cbl, was found to be a negative regulator of let-23-mediated vulval induction pathway, suggesting that c-Cbl may negatively regulate EGF receptor (EGFR)-mediated signaling. In this study, by an antisense RNA approach, we examined the effects of expression level of c-Cbl on EGFR signaling and showed that overexpression of c-Cbl reduces and antisense repression of c-Cbl enhances autophosphorylation of EGF receptors and activation of the JAK-STAT pathway. However, in contrast to the Sli-1 protein, the expressed amount of c-Cbl does not affect activation of the Ras pathway, suggesting that the EGFR-mediated signaling pathways are differently regulated by c-Cbl among nematodes and mammals.

The AML1 gene encodes DNA-binding proteins that contain the runt homology domain and is found at the breakpoints of t(8;21), t(3;21), and t(12;21) translocations associated with myelogenous leukemias. AML1 heterodimerizes with PEBP2beta/CBFbeta, resulting in the enhanced affinity with DNA. The runt homology domain is responsible for binding with DNA and heterodimerizing with PEBP2beta/CBFbeta. AML1 is suggested to perform a pivotal role in myeloid cell differentiation, whereas it can cause neoplastic transformation when overexpressed in fibroblasts. In this study, we demonstrated that the reducing reagent, dithiothreitol (DTT), markedly enhances the DNA binding of AML1 expressed in COS7 cells. Oxidation by diamide or modification by N-ethylmaleimide of the free sulfhydryl residues inhibited the interaction of AML1 with DNA. The diamide effect was reversible with excess of DTT, whereas DTT could not restore the DNA binding of AML1 treated with N-ethylmaleimide. Site-directed mutagenesis of the amino acid residue 72, a highly conserved cysteine in the runt homology domain of AML1, to serine almost completely abolished DNA binding without altering the interaction with PEBP2beta/CBFbeta. This substitution also impaired transactivation through the consensus DNA sequence and transformation of fibroblasts induced by AML1b. These data indicate an essential role of the conserved cysteine residue in DNA binding of AML1, and it is possible that the redox state of AML1 could contribute to the regulation of its function.

The Philadelphia chromosome (Ph) translocation generates a chimeric tyrosine kinase oncogene, BCR/ABL, which causes chronic myelogenous leukemia (CML) and a type of acute lymphoblastic leukemia (ALL). In primary samples from virtually all patients with CML or Ph+ALL, the CRKL adapter protein is tyrosine phosphorylated and physically associated with p210(BCR/ABL). CRKL has one SH2 domain and two SH3 domains and is structurally related to c-CRK-II (CRK) and the v-Crk oncoprotein. We have previously shown that CRKL, but not the related adapter protein c-CRK, is tyrosine phosphorylated in cell lines transformed by BCR/ABL, and that CRKL binds to BCR/ABL through the CRKL-SH3 domains. Furthermore, the CRKL-SH2 domain has been shown to bind one or more cellular proteins, one of which is p120(CBL). Here we demonstrate that another cellular protein linked to BCR/ABL through the CRKL-SH2 domain is p130(CAS). p130(CAS) was found to be tyrosine phosphorylated and associated with CRKL in BCR/ABL expressing cell lines and in samples obtained from CML and ALL patients, but not in samples from controls. In both normal and BCR/ABL transformed cells, p130(CAS) was detected in focal adhesion-like structures, as was BCR/ABL. In normal cells, the focal adhesion proteins tensin, p125(FAK), and paxillin constitutively associated with p130(CAS). However, in BCR/ABL transformed cells, the interaction between p130(CAS) and tensin was disrupted, while the associations between p130(CAS), p125(FAK), and paxillin were unaffected. These results suggest that the BCR/ABL oncogene could alter the function of p130(CAS) in at least three ways: tyrosine phosphorylation, inducing constitutive binding of CRKL to a domain in p130(CAS) containing Tyr-X-X-Pro motifs (substrate domain), and disrupting the normal interaction of p130(CAS) with the focal adhesion protein tensin. These alterations in the structure of signaling proteins in focal adhesion like structures could contribute to the known adhesion abnormalities in CML cells.

p130Cas is a major tyrosine-phosphorylated protein that tightly binds v-Crk in v-crk-transformed cells and v-Src in v-src-transformed cells. The "substrate domain" of p130Cas contains 15 possible Src homology (SH) 2-binding motifs, most of which conform to the binding motif for the Crk SH2 domain. Another region near its C terminus contains possible binding motifs for the Src SH2 domain and proline-rich sequences that are candidates for SH3-binding sites. Using GST fusion proteins, we revealed that both SH2 and SH3 domains of Src bind p130Cas, whereas v-Crk binds p130Cas through its SH2 domain. We located the binding site of p130Cas for the Src SH3 domain at the sequence RPLPSPP in the region near its C terminus. Mutations within this sequence or at Tyr762 of p130Cas caused a significant reduction in the association of p130Cas with Src, and no association was detected when both of them were deleted. The kinase activity in v-Crk-transformed cells was also associated with p130Cas through this region. On the other hand, the deletion of the substrate domain abolished the binding with v-Crk. The association through the C-terminal region of p130Cas with Src kinase may facilitate effective hyperphosphorylation of tyrosine residues in the substrate domain of p130Cas, resulting in the binding of SH2-containing molecules to p130Cas.

Crk proteins are Src homology (SH) 2/SH3-containing adapter proteins that can mediate the formation of signaling complexes. We show that engaging the B cell antigen receptor (BCR) on the RAMOS B cell line caused both Crk-L and Crk II to associate with several tyrosine-phosphorylated proteins. We identified two of these phosphoproteins as Cas and Cbl and showed that both bound to the Crk SH2 domain after BCR engagement. BCR ligation also increased the amount of Crk proteins in the particulate fraction of the cells and induced the formation of Crk.Cas and Crk.Cbl complexes in the particulate fraction. We propose that tyrosine phosphorylation of membrane-associated Cas and Cbl creates binding sites for the Crk SH2 domain and recruits Crk complexes to cellular membranes. Thus, Crk proteins may participate in BCR signaling by using their SH2 domains to direct the interactions and subcellular localization of proteins that bind to their SH3 domains. In RAMOS cells, we found that the SH3 domains of Crk-L and Crk II bound C3G. Since C3G activates Rap, a negative regulator of the Ras pathway, Crk proteins may participate in regulation of Ras signaling by the BCR.

Leukocyte tyrosine kinase (LTK) is a receptor tyrosine kinase, which belongs to the insulin receptor family and is mainly expressed in pre-B cells and brain. In this study, we show that LTK utilizes insulin receptor substrate-1 (IRS-1) and Shc as major two substrates and possesses two NPXY motifs for them separately, tyrosine 485 of one NPXY motif at the juxtamembrane domain for IRS-1 and tyrosine 862 of another NPXY motif at the carboxyl-terminal domain for Shc. By using Ba/F3 cells expressing epidermal growth factor receptor-LTK chimeric receptors containing a mutation at each NPXY site, we showed that while both NPXY motifs equally contribute to activation of the Ras pathway and generation of mitogenic signals, only tyrosine 485 of LTK transmits cell survival signals. These data suggest that IRS-1 possesses anti-apoptotic function at least in LTK signaling. Moreover, our data indicate that the survival signaling pathway of LTK is distinct from the Ras pathway and the p70(S6) kinase pathway. Our results provide a useful insight in understanding the distinctive roles of Shc and IRS-1 in the signal transduction system of the insulin receptor family, and this anti-apoptotic function of IRS-1 may explain the survival effects of insulin, IGF-1, and interleukin 4.

Complementary DNA (cDNA) clones encoding a transcription factor S-II were isolated and characterized. The primary structure of S-II was determined by nucleotide sequence analysis of these clones. The predicted primary structure was consistent with the model that we proposed previously from the results of biochemical analyses of S-II. Using these clones as probes, we analyzed the mRNA for S-II. RNA blot analysis demonstrated the presence of four species of mRNA that hybridized with S-II cDNA in Ehrlich ascites tumor cells. This is the first evidence of polymorphism of mRNA encoding a transcription factor of RNA polymerase II. The results of analysis of the genomic structure suggested that the polymorphism of mRNA may be due to alternative splicing, or differences in initiation or termination of transcription.

The MEN gene (also called ELL) encodes an RNA polymerase II elongation factor that has been implicated in t(11;19)(q23;p13.1) translocation in myeloid leukemias. The function of another elongation factor, elongin, is known to be inhibited by VHL tumor suppressor protein in vitro, suggesting the possible relationship of aberrant transcriptional elongation to oncogenesis. We overexpressed the MEN protein in Rat1 fibroblasts to evaluate its transforming activity. MEN-overexpressing cells acquired the capacity for anchorage-independent growth. In addition, the growth factor requirement was decreased in these cells. However, cells expressing a deletion mutant of MEN lacking the lysine-rich region did not exhibit such biological abilities. c-Fos protein expression and AP-1 activity were elevated in the MEN-expressing cells, which might be part of the mechanism responsible for the transformation. The c-fos mRNA, the expression of which is known to be regulated partly at the stage of transcriptional elongation, appeared earlier in the MEN-expressing cells than in cells transfected with an empty vector or the deletion mutant lacking the lysine-rich region after stimulation with epidermal growth factor. The RNA polymerase II elongation factor MEN may play an important role in the regulation of cell proliferation.

The Crk-associated substrate p130(Cas) (Cas) and the recently described human enhancer of filamentation 1 (HEF1) are two proteins with similar structure (64% amino acid homology), which are thought to act as "docking" molecules in intracellular signaling cascades. Both proteins contain an N-terminal Src homology (SH), three domain and a cluster of SH2 binding motifs. Here we show that ligation of either beta1 integrin or B cell antigen receptor (BCR) on human tonsillar B cells and B cell lines promoted tyrosine phosphorylation of HEF1. In contrast, Cas tyrosine phosphorylation was observed in certain B cell lines but not in tonsillar B cells, indicating a more general role for HEF1 in B cell signaling. Interestingly, pretreatment of tonsillar B cells with cytochalasin B dramatically reduced both integrin- and BCR-induced HEF1 phosphorylation, suggesting that some component of the BCR-mediated signaling pathway is closely linked with a cytoskeletal reorganization. Both HEF1 and Cas were found to complex with the related adhesion focal tyrosine kinase (RAFTK), and when tyrosine phosphorylated, with the adapter molecule CrkL. In addition, the two molecules were detected in p53/56(Lyn) immunoprecipitates, and Lyn kinase was found to specifically bind the C-terminal proline-rich sequence of Cas in an in vitro binding assay. These associations implicate HEF1 and Cas as important components in a cytoskeleton-linked signaling pathway initiated by ligation of beta1 integrin or BCR on human B cells.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) and erythropoietin (Epo) are hematopoietic growth factors that regulate proliferation and differentiation of hematopoietic cells. They elicit and control a cascade of biochemical events, the earliest of which is tyrosine phosphorylation of several cellular proteins. Grb2/Ash is composed of SH2 and SH3 domains. The SH2 domain binds to tyrosine-phosphorylated proteins, and the SH3 domains bind to proteins containing proline-rich regions. It is considered that Grb2/Ash functions as an adapter protein linking tyrosine kinases and Ras in downstream of receptors for growth factors in fibroblasts. However, the mechanisms of signal transduction through Grb2/Ash and the roles of proteins associated with Grb2/Ash remain to be determined in hematopoietic cells. By means of the binding experiments using the glutathione S-transferase fusion protein including the full-length Grb2/Ash, we have found that Shc and unidentified 130- and 135-kDa proteins are associated with Grb2/Ash and that they are tyrosine phosphorylated by treatment with GM-CSF or Epo in a human leukemia cell line, UT-7. We have purified the 130-kDa protein (pp130) using the glutathione S-transferase-Grb2/Ash affinity column. The amino acid sequence analysis of the three peptides derived from the in situ protease digestion of the purified pp130 showed that the pp130 was identical to the human c-cbl proto-oncogene product (c-Cbl). c-Cbl constitutively binds to the SH3 domain of Grb2/Ash both in vitro and in vivo but not to the SH2 domain of Grb2/Ash, and the binding of Grb2/Ash to c-Cbl or Sos was not altered by GM-CSF stimulation. Moreover, c-Cbl (pp130) becomes tyrosine phosphorylated rapidly and transiently depending on GM-CSF or Epo stimulation. These findings strongly suggest that c-Cbl is implicated in the signal transduction of GM-CSF or Epo in hematopoietic cells and that c-Cbl is involved in another signaling pathway different from the Ras signaling pathway.

gp130 is a signal-transducing subunit of receptors for the interleukin-6 (IL-6)-related cytokine subfamily including IL-6, leukemia inhibitory factor, oncostatin M, IL-11, and ciliary neurotrophic factor, indicating that gp130-mediated signals are involved in the immune response, hematopoiesis, inflammation, and endocrine and nervous system activity. We previously showed that gp130 stimulation rapidly activates Jak, Btk, and Tec tyrosine kinases, all of which constitutively associate with gp130. To further elucidate intracellular signal transduction through gp130, we examined the possible involvement of another nonreceptor tyrosine kinase, p92c-fes (Fes). We showed that gp130 stimulation rapidly induced tyrosine phosphorylation of Fes and actually activated its kinase activity in hematopoietic lineage cells. Furthermore, Fes associated with gp130 independently of ligand stimulation like Jak, Btk, and Tec tyrosine kinases. These results indicate that multiple nonreceptor tyrosine kinases are involved in the gp130-mediated signal transduction pathway. Because both gp130 and Fes are expressed not only in hematopoietic lineage cells but also in heart and nerve cells, Fes may play a role in signal transduction through gp130 in these tissues.