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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The v-src oncogene encodes a nonreceptor tyrosine kinase. When this gene was expressed in the myeloblastic cell line 32Dcl3, it was found to abrogate interleukin-3 (IL-3) dependence of this cell line and to block its ability to terminally differentiate into granulocytes in response to granulocyte colony-stimulating factor (GCSF). In contrast, a highly related tyrosine kinase gene, v-fgr, fails to render this cell line IL-3 independent for growth or to block its ability to undergo terminal differentiation in the presence of GCSF. The active structural domains of v-src that are responsible for the abrogation of IL-3 dependence of myeloid cells and the mechanisms by which v-src transforms these cells are at present unclear. To identify the domains in v-src which are responsible for this activity, we constructed several chimeric recombinants between the v-src and the related Src family member v-fgr by replacing portions of v-src with corresponding domains of v-fgr. These chimeric DNAs were transfected into 32Dcl3 cells and examined for their abilities to render this cell line IL-3 independent. Our results show that only chimeras containing both the SH3 and the SH2 domains of v-src were capable of rendering the 32Dcl3 cell line IL-3 independent. To understand the possible mechanisms underlying the IL-3-independent growth of v-src-transformed 32Dcl3 cells, we examined the phosphorylation status of JAK-1, JAK-2, and JAK-3 kinases in the v-src- and v-fgr-transformed 32Dcl3 cells. Our results show that none of the JAK kinases are constitutively phosphorylated by v-src or v-fgr. We then examined the phosphorylation status of the STAT (signal transducers and activators of transcription) family of transcription factors. Our results show that STAT1, STAT3, and STAT5 exist in a constitutively phosphorylated state in v-src-transformed 32Dcl3 cells, while such constitutive phosphorylation is not seen in v-fgr-transformed cell lines. Our results also show that STAT3 coimmunoprecipitates with v-Src, suggesting that the activation of STAT3 occurs due to direct association with v-Src. However, STAT1 and STAT5, which also exist in a constitutively phosphorylated state in v-src-transformed 32Dcl3 cells, do not coimmunoprecipitate with v-Src, suggesting that these proteins either interact weakly with v-Src or are phosphorylated by a mechanism distinctive from that of STAT3.
Mol Cell Biol 1997 Jun
PMID:Abrogation of interleukin-3 dependence of myeloid cells by the v-src oncogene requires SH2 and SH3 domains which specify activation of STATs. 915 28

Cytokine receptors activate multiple signal transduction pathways, resulting in the induction of specific target genes. We have recently identified a hematopoietic cell-specific immediate-early gene, DUB-1, that encodes a growth-regulatory deubiquitinating enzyme. The DUB-1 gene contains a 112-bp enhancer element that is specifically induced by the beta c subunit of the interleukin-3 (IL-3) receptor. To investigate the mechanism of DUB-1 induction, we examined the effects of dominant-negative forms of JAK kinases, STAT transcription factors, and Raf-1 in transient transfection assays. In Ba/F3 cells, IL-3 induced a dose-dependent activation of DUB-1-luciferase (luc) and GAS-luc reporter constructs. A dominant-negative form of JAK2 (truncated at amino acid 829) inhibited the induction of DUB-1-luc and GAS-luc by IL-3. A dominant-negative form of STAT5 (truncated at amino acid 650) inhibited the induction of GAS-luc but not DUB-1-luc. A dominant-negative form of Raf-1 inhibited the induction of DUB-1-luc but had no effect on the induction of GAS-luc by IL-3. The requirement for JAK2 in the stimulation of the DUB-1 enhancer was further supported by the suppression of DUB-1 induction in Ba/F3 cells stably expressing the dominant-negative JAK2 polypeptide. We hypothesize that IL-3 activates a JAK2/Raf-1 signaling pathway that is required for DUB-1 induction and is independent of STAT5.
Mol Cell Biol 1997 Jun
PMID:JAK2 is required for induction of the murine DUB-1 gene. 915 35

Several different Janus kinases (JAKs) and signal transducers and activation of transcription (STATs) have been implicated in mediating the biological responses induced by PRL, based on their ligand-dependent tyrosine phosphorylation and activation. However, these criteria alone do not prove that a particular JAK or STAT is essential for signal transduction. We have used mutant cell lines defective in JAK1, JAK2, or STAT1 to examine their roles in PRL-dependent signaling. JAK2 is absolutely required for PRL-dependent phosphorylation of the receptor, activation of STATs, and induction of beta-lactoglobulin. Wild type, but not kinase-negative JAK2, restores all responses to PRL in JAK2-defective cells, suggesting that JAK2 function, not merely the protein, is required. In contrast, JAK1, which is phosphorylated in response to PRL, is not required for any of these functions. Although STAT1 homodimers do form in response to PRL, no defect in PRL-dependent signaling is apparent when STAT1 is missing, suggesting that STAT5, which is strongly activated in response to PRL, is primarily responsible for driving the expression of PRL-responsive genes.
Mol Endocrinol 1997 Jul
PMID:JAK2 and STAT5, but not JAK1 and STAT1, are required for prolactin-induced beta-lactoglobulin transcription. 921 64

We have investigated the possible involvement of the MAPK pathway in the growth hormone(GH)-induced activation of one of the members of signal transducers and activators of transcription, STAT5, by using the MAPK kinase (MEK) inhibitor PD98059. PD98059 treatment of Chinese hamster ovarian cells, stably transfected with the GH receptor (CHOA cells), abolished the GH-induced MAPK activity. PD98059 decreased the amount of GH-induced STAT5 in nuclear extract with DNA-binding capacity. Furthermore, GH dependent transcription of a STAT5 regulated reporter gene was inhibited by PD98059. The MEK inhibitor did not reduce GH-stimulated nuclear translocation of STAT5. We also investigated if PD98059 differentially influences the activation of the two STAT5 homologs, STAT5a and STAT5b, which differ mainly at the C-terminal end, one of the differences being the presence of a possible MAPK phosphorylation site in STAT5a. Expression plasmids for these transcription factors were transfected into CHOA cells together with a reporter gene. GH-stimulated fold induction of transcription was reduced by PD98059 in STAT5a but not in STAT5b overexpressing cells. A MAPK phosphorylation site-mutated version of STAT5a was also transfected into CHOA cells. GH-stimulated fold induction of cotransfected reporter gene was not reduced by PD98059 in cells overexpressing mutant STAT5a. The above data show that the MAPK pathway is required for the full activation of one of the STAT5 isoforms (STAT5a).
Mol Cell Endocrinol 1997 Oct 20
PMID:Mitogen-activated protein kinase kinase inhibition decreases growth hormone stimulated transcription mediated by STAT5. 940 63

Granulocyte-macrophage colony-stimulating factor (GM-CSF) induces various functions, including the proliferation and differentiation of a broad range of hematopoietic cells. We previously reported that at least two distinct pathways are involved in human GM-CSF receptor signaling; both require the box 1 region of the common beta subunit (beta c). This region is essential for the activation of JAK2, which is necessary for all the biological functions of GM-CSF. The activation of JAK2 by GM-CSF leads to rapid tyrosine phosphorylation of cellular proteins, including the beta c. However, the significance of beta c phosphorylation with regard to the regulation of signaling molecules and the expression of GM-CSF functions is less well understood. Here we investigated the role of the cytoplasmic tyrosine residues of the beta c by using a series of beta c mutants expressed in murine BA/F3 cells. A mutant beta c with all eight cytoplasmic tyrosines converted to phenylalanine (Fall) activated JAK2 but not SHP-2, MAPK cascades, STAT5, or the c-fos promoter in BA/F3 cells, and it did not effectively induce proliferation. Adding back each tyrosine to Fall revealed that Tyr577, Tyr612, and Tyr695 are involved in the activation of SHP-2, MAPK cascades, and c-fos transcription, while every tyrosine, particularly Tyr612, Tyr695, Tyr750, and Tyr806, facilitated STAT5 activation. Impaired growth was also restored, at least partly, by any of the tyrosines. These results provide evidence that beta c tyrosines possess distinct yet overlapping functions in activating multiple signaling pathways induced by GM-CSF.
Mol Cell Biol 1998 Feb
PMID:Definition of the role of tyrosine residues of the common beta subunit regulating multiple signaling pathways of granulocyte-macrophage colony-stimulating factor receptor. 944 70

Abnormal erythropoietin (EPO)-independent cell growth is induced after infection of erythroid progenitor cells with a polycythemic strain of Friend virus (FVp). Binding of its Env-related glycoprotein (gp55) to the EPO receptor (EPOR) mimics the activation of the EPOR with EPO. We investigated the gp55-EPOR signaling in erythroblastoid cells from mice infected with FVp and in cells of FVp-induced or gp55-transgenic-mouse-derived erythroleukemia cell lines, comparing it with the EPO-EPOR signaling in EPO-responsive erythroblastoid cells. While the Janus protein tyrosine kinase JAK2 and the transcription factor STAT5 became tyrosine phosphorylated with the EPO stimulation in EPO-responsive erythroblastoid cells from anemic mice, JAK1 and STAT5 were constitutively tyrosine phosphorylated in all of these FVp gp55-induced erythroblastoid or erythroleukemic cells. Moreover, this constitutively tyrosine-phosphorylated STAT5 was unable to bind to its specific DNA sequences and did not translocate to the nucleus. Nuclear translocation and DNA binding of this STAT5 species required EPO stimulation. These findings clearly indicate that the FVp gp55-EPOR signaling is distinct from the EPO-EPOR signaling and suggest that STAT5 may not play an essential role in the transmission of the cell growth signals in FVp gp55-induced erythroleukemia cells.
Mol Cell Biol 1998 Mar
PMID:Erythropoietin and Friend virus gp55 activate different JAK/STAT pathways through the erythropoietin receptor in erythroid cells. 948 32

We have previously described a 160-bp enhancer (BCE-1) in the bovine beta-casein gene that is activated in the presence of prolactin and extracellular matrix (ECM). Here we report the characterization of the enhancer by deletion and site-directed mutagenesis, electrophoretic mobility shift analysis, and in vivo footprinting. Two essential regions were identified by analysis of mutant constructions: one binds C/EBP-beta and the other binds MGF/STAT5 and an as-yet-unidentified binding protein. However, no qualitative or quantitative differences in the binding of these proteins were observed in electrophoretic mobility shift analysis using nuclear extracts derived from cells cultured in the presence or absence of ECM with or without prolactin, indicating that prolactin- and ECM-induced transcription was not dependent on the availability of these factors in the functional cell lines employed. An in vivo footprinting analysis of the factors bound to nuclear chromatin in the presence or absence of ECM and/or prolactin found no differences in the binding of C/EBP-beta but did not provide definitive results for the other factors. Neither ECM nor prolactin activated BCE-1 in transient transfections, suggesting that the chromosomal structure of the integrated template may be required for ECM-induced transcription. Further evidence is that treatment of cells with inhibitors of histone deacetylase was sufficient to induce transcription of integrated BCE-1 in the absence of ECM. Together, these results suggest that the ECM induces a complex interaction between the enhancer-bound transcription factors, the basal transcriptional machinery, and a chromosomally integrated template responsive to the acetylation state of the histones.
Mol Cell Biol 1998 Apr
PMID:Characterization of BCE-1, a transcriptional enhancer regulated by prolactin and extracellular matrix and modulated by the state of histone acetylation. 952 90

After birth, the endocrine actions of insulin-like growth factor (IGF)-I and -II become increasingly important. In postnatal animals, most of circulating IGFs occur in 150-kDa complexes formed by association of an acid-labile subunit (ALS) with complexes of IGF and IGF-binding protein-3. ALS is synthesized almost exclusively in liver. GH stimulates the transcription of the ALS gene, resulting in increased hepatic mRNA and circulating ALS levels. To map the GH response element, a series of 5'-deletion fragments of the mouse ALS promoter (nt -2001 to -49, A(+1)TG) were inserted in the luciferase reporter plasmid pGL3 and transfected into the H4-II-E rat hepatoma cell line. GH stimulated the activity of promoter fragments with 5'-ends between nucleotide (nt) -2001 and nt -653 by 1.9- to 2.7-fold. This stimulation was abolished by deletion of the region located between nt -653 and nt -483. This region contains two sites, ALS-GAS1 and ALS-GAS2, that resemble the gamma-interferon activated sequence (GAS). Mutation of the ALS-GAS1 site, but not of the ALS-GAS2 site, eliminated the response to GH when assessed in the context of a GH-responsive promoter fragment, indicating that ALS-GAS1 was necessary for GH induction. Three tandem copies of ALS-GAS1 were sufficient to confer GH inducibility to the minimal promoter of the thymidine kinase gene. In electrophoretic mobility shift assays, ALS-GAS1 formed a specific, GH-dependent protein-DNA complex with nuclear extracts from H4-II-E cells. Using antibodies directed against members of the family of signal transducers and activators of transcription (STAT), this complex was shown to be composed of STAT5a and STAT5b. Identical results were obtained when transfections and mobility shift assays were performed in primary rat hepatocytes in which the endogenous ALS gene is expressed. Thus, the transcriptional activation of the mouse ALS gene by GH is mediated by the binding of STAT5 isoforms to a single GAS-like element.
Mol Endocrinol 1998 May
PMID:Binding of STAT5a and STAT5b to a single element resembling a gamma-interferon-activated sequence mediates the growth hormone induction of the mouse acid-labile subunit promoter in liver cells. 960 30

STAT (signal transducers and activators of transcription) proteins are transcription factors which are activated by phosphorylation on tyrosine residues upon stimulation by cytokines. Seven members of the STAT family are known, including the closely related STAT5A and STAT5B, which are activated by various cytokines. Except for prolactin-dependent beta-casein production in mammary gland cells, the biological consequences of STAT5 activation in various systems are not clear. We applied PCR-driven random mutagenesis and a retrovirus-mediated expression screening system to identify constitutively active forms of STAT5. By this strategy, we have identified a constitutively active STAT5 mutant which has two amino acid substitutions; one is located upstream of the putative DNA binding domain (H299R), and the other is located in the transactivation domain (S711F). The mutant STAT5 was constitutively phosphorylated on tyrosine residues, localized in the nucleus, and was transcriptionally active. Expression of the mutant STAT5 partially dispenses with interleukin 3 (IL-3) as a growth stimulant of IL-3-dependent cell lines. Further analyses of the mutant STAT5 have demonstrated that both of the mutations are required for nuclear localization, efficient transcriptional activation, and induction of IL-3-independent growth of an IL-3-dependent cell line, Ba/F3, and have indicated that a molecular basis for the constitutive activation is the stability of the phosphorylated form of the mutant STAT5.
Mol Cell Biol 1998 Jul
PMID:Identification and characterization of a constitutively active STAT5 mutant that promotes cell proliferation. 963 71

Thrombopoietin (TPO) is a hematopoietic growth factor that plays fundamental roles is both megakaryopoiesis and thrombopoiesis through binding to its receptor, c-mpl. Although TPO has been shown to activate various types of intracellular signaling molecules, such as the Janus family of protein tyrosine kinases, signal transducers and activators of transcription (STATs), and ras, the precise mechanisms underlying TPO-induced proliferation and differentiation remain unknown. In an effort to clarify the mechanisms of TPO-induced proliferation and differentiation, c-mpl was introduced into F-36P, a human interleukin-3 (IL-3)-dependent erythroleukemia cell line, and the effects of TPO on the c-mpl-transfected F-36P (F-36P-mpl) cells were investigated. F-36P-mpl cells were found to proliferate and differentiate at a high rate into mature megakaryocytes in response to TPO. Dominant-negative (dn) forms of STAT1, STAT3, STAT5, and ras were inducibly expressed in F-36P-mpl cells, and their effects on TPO-induced proliferation and megakaryocytic differentiation were analyzed. Among these dn molecules, both dn ras and dn STAT5 reduced TPO- or IL-3-induced proliferation of F-36P-mpl cells by approximately 30%, and only dn ras could inhibit TPO-induced megakaryocytic differentiation. In accord with this result, overexpression of activated ras (H-rasG12V) for 5 days led to megakaryocytic differentiation of F-36P-mpl cells. In a time course analysis on H-rasG12V-induced differentiation, activation of the ras pathway for 24 to 28 h was required and sufficient to induce megakaryocytic differentiation. Consistent with this result, the treatment of F-36P-mpl cells with TPO was able to induce prolonged activation of ras for more than 24 h, whereas IL-3 had only a transient effect. These results suggest that prolonged ras activation may be involved in TPO-induced megakaryocytic differentiation.
Mol Cell Biol 1998 Jul
PMID:Involvement of prolonged ras activation in thrombopoietin-induced megakaryocytic differentiation of a human factor-dependent hematopoietic cell line. 963 12


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