Showing papers by "Hiroyuki Mano published in 1994"

A novel signaling molecule, p130, forms stable complexes in vivo with v-Crk and v-Src in a tyrosine phosphorylation-dependent manner.

Abstract: p47v-crk (v-Crk), a transforming gene product containing Src homology (SH)-2 and -3 domains, induces an elevated level of tyrosine phosphorylation of several cellular proteins. Among these proteins, a 125-135 kDa protein (p130) shows marked phosphorylation at tyrosines and tight association with v-Crk, suggesting a direct signal mediator of v-Crk. Here we report the molecular cloning of rat p130 by immunoaffinity purification. The p130 is a novel SH3-containing signaling molecule with a cluster of multiple putative SH2-binding motifs of v-Crk. Immunochemical analyses revealed that p130 is highly phosphorylated at tyrosines during transformation by p60v-src (v-Src), as well as by v-Crk, forming stable complexes with these oncoproteins. The p130 behaves as an extremely potent substrate of kinase activity included in the complexes and it is a major v-Src-associated substrate of the Src kinase by partial peptidase mapping. Subcellular fractionation demonstrated that the cytoplasmic p130 could move to the membrane upon tyrosine phosphorylation. The p130 (designated Cas for Crk-associated substrate) is a common cellular target of phosphorylation signal via v-Crk and v-Src oncoproteins, and its unique structure indicates the possible role of p130Cas in assembling signals from multiple SH2-containing molecules.

Generation of the AML1-EVI-1 fusion gene in the t(3;21)(q26;q22) causes blastic crisis in chronic myelocytic leukemia.

Abstract: The t(3;21)(q26;q22) translocation, which is one of the consistent chromosomal abnormalities found in blastic crisis of chronic myelocytic leukemia (CML), is thought to play an important role in the leukemic progression of CML to an acute blastic crisis phase. The AML1 gene, which is located at the translocation breakpoint of the t(8;21)(q22;q22) translocation found in acute myelocytic leukemia, was also rearranged by the t(3;21)(q26;q22) translocation. Screening of a cDNA library of the t(3;21)-carrying leukemic cell line cells (SKH1) resulted in the isolation of two potentially complete AML1-EVI-1 chimeric cDNAs of 6 kb. Two species of AML1-EVI-1 fusion transcripts of 8.2 and 7.0 kb were detected in SKH1 cells. These cells expressed the 180 kDa AML1-EVI-1 fusion protein containing an N-terminal half of AML1 including a runt homology domain which is fused to the entire zinc finger EVI-1 protein. The AML1-EVI-1 fusion transcript was consistent in all three cases of the t(3;21)-carrying leukemia examined by RNA-based PCR. These findings strongly suggest that the t(3;21) translocation results in the formation of a new class of chimeric transcription factor which could contribute to the leukemic progression of CML through interference with cell growth and differentiation.

Tec kinase associates with c-kit and is tyrosine phosphorylated and activated following stem cell factor binding.

Abstract: Stem cell factor (SCF) plays a crucial role in hematopoiesis through its interaction with the receptor tyrosine kinase c-kit. However, the signaling events that are activated by this interaction and involved in the control of growth or differentiation are not completely understood. We demonstrate here that Tec, a cytoplasmic, src-related kinase, physically associates with c-kit through a region that contains a proline-rich motif, amino terminal of the SH3 domain. Following SCF binding, Tec is tyrosine phosphorylated and its in vitro kinase activity is increased. Tyrosine phosphorylation of Tec is not detected in the response to other cytokines controlling hematopoiesis, including colony-stimulating factor-1 (CSF-1), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-3 (IL-3). Conversely, the cytoplasmic kinase JAK2 is activated by IL-3 but not by SCF stimulation. The activation of distinct cytoplasmic kinases may account for the synergy seen in the actions of SCF and IL-3 on hematopoietic stem cells.

The C-terminal SH3 domain of the mouse c-Crk protein negatively regulates tyrosine-phosphorylation of Crk associated p130 in rat 3Y1 cells.

Abstract: We have isolated the mouse c-crk cDNA from a mouse liver cDNA library. It encodes 304 amino acids and consists mainly of SH2/SH3 regions. In Northern blot analysis, the mouse c-crk mRNA is expressed ubiquitously in every tissue and organ, suggesting that the c-Crk protein may be a common signal transducing molecule among tissues. In contrast to the v-Crk protein, which has a single SH3 domain, the c-Crk protein contains two, the more N-terminal SH3(1) domain and the C-terminal SH3(2) domain. To elucidate functions of these SH3 domains, we have constructed two c-crk mutants, B-crk and D-crk, which lack the SH3(2) and the SH3(1) domain, respectively. These mutants were expressed in rat 3Y1 cells, and examined for their transforming ability in terms of morphological phenotypes and for tyrosine phosphorylation profiles of cells expressing the mutant proteins. Morphological alteration and increased tyrosine phosphorylation of 130-140 kDa proteins, the major component of which is the Crk-associated p130, were observed in cells expressing B-Crk as well as those expressing v-Crk, but little in cells expressing c-Crk even at a similar level of expression. Although a highly tyrosine-phosphorylated form of the p130 was coimmunoprecipitated with c-Crk as well as B-Crk, the relative level of tyrosine phosphorylation of the p130, which is normalized to the amount of Crk protein immunoprecipitated, was 10 to 20 times higher in B-Crk-expressing cells than in c-Crk- or D-Crk-expressing cells. The present results indicate that the SH3(2) domain of mouse c-Crk protein negatively regulates tyrosine phosphorylation of the p130, and that lack of the SH3(2) domain in B-Crk and v-Crk may contribute, at least partly, to their morphological alteration or transforming ability through increasing tyrosine phosphorylation of the p130.

Molecular Cloning and Analysis of the Human Tec Protein-Tyrosine Kinase

Abstract: Mouse Tec is a non-receptor type protein-tyrosine kinase and is highly expressed in many hematopoietic cell lines. To investigate the roles of the Tec kinase in the human hematopoietic system, we isolated cDNAs encoding the human Tec kinase. The human tec cDNAs can encode a peptide of 631 amino acid residues with a calculated molecular mass of 73,624. The predicted human Tec protein is highly homologous to those of the members of the Tec family including mouse Tec type IV (94% homology), mouse Tsk/Itk (60%), and human Btk (57%). The homology between human Tec and other members of the Tec family can be observed not only in the Src homology 3 (SH3), SH2, and kinase domains, but also in the N-terminal unique domain. Northern blot analysis demonstrated that the major transcripts of tec could be detected at 2.6 kb and 3.6 kb in a wide range of human hematopoietic cell lines including myeloid, B-, and T-cell lineages. Interestingly, high expression of the tec gene could be detected in all of the three patients examined with myelodysplastic syndrome. The human tec gene was mapped by fluorescence in situ hybridization (FISH) to chromosome 4p12.

Tec protein-tyrosine kinase directly associates with lyn protein-tyrosine kinase through its n-terminal unique domain

Abstract: Most of non-receptor-type protein-tyrosine kinases share common structures, such as N-terminal unique domains, Src homology region (SH)-2 domains, SH-3 domains and kinase domains. Although vast effort has brought some information about the in vivo roles of SH-2, -3 and kinase domains, little is still understood about the function of N-terminal unique domain. By utilizing the glutathione S-transferase (GST)-fusion system, we have investigated the role of N-terminal unique domain of the Tec protein-tyrosine kinase in a mouse IL-3-dependent myeloid cell line. We could reveal that the C-terminal half of the Tec N-terminal unique domain (NTec2 region) can bind to a set of tyrosine-phosphorylated cellular proteins in vitro in an IL-3-dependent manner. Surprisingly, p56/53Lyn constitutively binds to the NTec2 region. Among the NTec2-bound Lyn proteins, only the p56 form seems to be inducibly tyrosine-phosphorylated in response to IL-3. Binding domain of Lyn to the NTec2 region was localized to its SH-3 domain. Tec was also shown to make a stable complex with Lyn in vivo. This is the first report demonstrating the direct association between distinct cytoplasmic protein-tyrosine kinases, especially through N-terminal unique domain.

Molecular analysis of the t(1;19)(q23;p13) translocation observed in adult leukemias

Abstract: We report three cases of adult leukemias with the t(1;19)(q23;p13) translocation, two with acute lymphoblastic leukemia (ALL:L2) and one with megakaryoblastic crisis of chronic myelocytic leukemia. Only one patient with ALL showed the same E2A/PBX1 fusion transcripts as those observed in childhood ALLs with the t(1;19) by RNA-based polymerase chain reaction. This patient survived for 8 months only from the time of his diagnosis. On the other hand, the other two cases were negative for the E2A/PBX1 fusion and one of them with ALL remains in remission. Our observations suggest that there could be heterogeneity in the molecular events among adult leukemias carrying the t(1;19) and that the E2A/PBX1 fusion formation might also be associated with an adverse treatment outcome in adult leukemias as in childhood leukemias.