Biomedical subjects

R Jove

Publications and source records attributed to R Jove.

At least 55 records · Page 3Linked to original sources

PubMed1998

Stat3 activation by Src induces specific gene regulation and is required for cell transformation.

While signal transducers and activators of transcription (STATs) were originally discovered as intracellular effectors of normal signaling by cytokines, increasing evidence also points to a role for STAT transcription factors in oncogenesis. Previous studies have demonstrated that one STAT family member, Stat3, possesses constitutively elevated tyrosine phosphorylation and DNA-binding activity in fibroblasts stably transformed by the Src oncoprotein. To determine if this Stat3 activation by Src could induce Stat3-mediated gene expression, luciferase reporter constructs based on synthetic and authentic promoters were transfected into NIH 3T3 cells. Activation of endogenous cellular Stat3 by the Src oncoprotein induced gene expression through a Stat3-specific binding element (TTCCCGAA) of the C-reactive protein gene promoter. A naturally occurring splice variant of human Stat3 protein, Stat3beta, with a deletion in the C-terminal transactivation domain abolished this gene induction in a dominant negative manner. Expression of Stat3beta did not have any effect on a reporter construct based on the c-fos serum response element, which is not dependent on Stat3 signaling, indicating that Stat3beta does not nonspecifically inhibit other signaling pathways or Src function. Transfection of vectors expressing Stat3beta together with Src blocked cell transformation by Src as measured in a quantitative focus formation assay using NIH 3T3 cells. By contrast, Stat3beta had a much less pronounced effect on focus formation induced by the Ras oncoprotein, which does not activate Stat3 signaling. In addition, three independent clones of NIH 3T3 cells stably overexpressing Stat3beta were generated and characterized, demonstrating that Stat3beta overexpression does not have a toxic effect on cell viability. These Stat3beta-overexpressing clones were shown to be deficient in Stat3-mediated signaling and refractory to Src-induced cell transformation. We conclude that Stat3 activation by the Src oncoprotein leads to specific gene regulation and that Stat3 is one of the critical signaling pathways involved in Src oncogenesis. Our findings provide evidence that oncogenesis-associated activation of Stat3 signaling is part of the process of malignant transformation.

3T3 Cells↗

PubMed1998

Elevated c-Src protein expression is an early event in colonic neoplasia.

The molecular events regulating the development and progression of colonic neoplasia are currently being delineated. Recent studies have implicated c-Src protein kinase activation as an early event in the malignant transformation of colonic epithelial cells. However, increased c-Src activity has also been reported in colon carcinomas as well as in metastatic hepatic and extrahepatic colon carcinomas. To further investigate the potential role of c-Src in the progression of colonic neoplasia, we analyzed c-Src levels by immunohistochemistry in 27 colorectal resection specimens. Mouse monoclonal antibody to c-Src protein was applied to 3-micron sections from formalin-fixed, paraffin-embedded tissues using the avidin-biotin-peroxidase method. The combination of adenomatous (AD) and adjacent carcinomatous mucosa (CA) specimens were present in 20 of 27 patients. In 15 cases, synchronous metastatic (MT) lesions were available for evaluation. Strong c-Src expression was evident in 95% of AD (n = 20), in contradistinction to 32% of MT (n = 19) and 14% of CA (n = 22). Weak-to-moderate c-Src expression was seen in adjacent normal colonic mucosa (NM) in 96% of cases. Signed rank test univariate analysis revealed a statistically significant difference in c-Src expression between NM/AD (p = 0.0001), NM/CA (p = 0.0001), NM/MT (p = 0.0006), AD/CA (p = 0.0001), and AD/MT (p = 0.0002). No significant correlation between levels of c-Src expression and patient survival, tumor size, histologic grade, or tumor configuration was observed using the Cox's Regression Model. Similar results were obtained by analysis of c-Src protein levels and c-Src kinase activity as measured by Western blot and in vitro kinase assays of representative cases. Our results indicate that: (a) elevated c-Src expression is an important early event during colorectal carcinogenesis; (b) its activation may be involved in tumor progression in a subset of colonic carcinomas; and (c) additional molecular events are necessary for invasion to occur.

Adult↗

PubMed1997

Activation of c-Src by receptor tyrosine kinases in human colon cancer cells with high metastatic potential.

Recent data suggest that signal transduction may have a critical role in the development and regulation of the metastatic phenotype. Here, we investigated the role of c-Src activation in the process of human colon cancer metastasis to the liver. Our data, derived from two different sets of human colon cancer cell line metastatic variants, suggest that not only do highly-metastatic cells display constitutively elevated c-Src protein kinase activity when compared to poorly metastatic cells, but also that receptor tyrosine kinases participate in the ligand-activation of c-Src above basal levels. Specifically, the epidermal growth factor receptor (EGFR), p185HER2/Neu and the hepatocyte growth factor receptor (c-Met) appear to be linked to the process because they preferentially activate c-Src in highly-metastatic cells. EGFR was found to associate with c-Src in colon cancer cells and specific inhibitors of the EGFR resulted in a reduction of c-Src activity to basal levels. In addition, c-Src transfectants displayed partially-activated EGFRs, suggesting a feedback role for c-Src in the regulation of the EGFR. p185HER2/Neu was also identified in immunocomplexes of c-Src following ligand activation of the EGFR, but only in highly-metastatic cells. Collectively, these observations suggest a paradigm whereby c-Src interacts with multiple cell-surface growth factors in a catalytic fashion for the development of tumor cells with metastatic potential.

Colonic Neoplasms↗

PubMed1997

Constitutive activation of the Janus kinase-STAT pathway in T lymphoma overexpressing the Lck protein tyrosine kinase.

The Lck protein, a Src family tyrosine kinase, plays a critical role in T cell maturation and activation. Dysregulation of Lck expression or Lck kinase activity has been implicated in T cell leukemias from mice to humans, although the mechanism underlying Lck-mediated oncogenesis is still largely unclear. We report here that both DNA binding activities and tyrosine phosphorylation of STAT3 and STAT5, but not STAT1, are constitutively enhanced in the mouse T cell lymphoma LSTRA, which is a well-characterized cell line that overexpresses Lck protein and exhibits high levels of Lck kinase activity. Furthermore, Janus kinase 1 (jak1) and Jak2 protein tyrosine kinases are constantly activated in these cells, as determined by their autophosphorylation in an in vitro kinase assay and increased levels of tyrosine phosphorylation on immunoblots. Therefore, like Src-transformed cells, Lck-overexpressing LSTRA cells also exhibit constitutive activation of distinct Jak and STAT proteins.

3T3 Cells↗

PubMed1997

Role of epidermal growth factor receptor and STAT-3 activation in autonomous proliferation of SUM-102PT human breast cancer cells.

This report describes the isolation and characterization of a new human breast cancer cell line, SUM-102PT, obtained from a minimally invasive human breast carcinoma. SUM-102PT cells have a near diploid karyotype, and early-passage cells had minor chromosomal abnormalities including a 5, 12 and a 6, 16 reciprocal translocation. The cells were isolated and have been continually cultured in three defined media, one of which contains exogenous epidermal growth factor (EGF). SUM-102PT cells have also been carried in an EGF-free medium supplemented with progesterone. All SUM-102PT cells require EGF receptor (EGFR) activation for continuous growth, because incubation of the cells with EGFR-neutralizing antibodies or with EGFR kinase inhibitors blocks growth of these cells. Southern analysis indicates that the EGFR gene is not amplified in these cells; however, these cells express high levels of EGFR mRNA. Thus, SUM-102PT is representative of a class of human breast cancers characterized by high level EGFR expression in the absence of gene amplification. SUM-102PT cells cultured in EGF-free, progesterone-containing medium express high levels of constitutively active EGFR. Conditioned medium from SUM-102PT cells contains an EGF-like mitogen that binds to a heparin-agarose affinity matrix with high affinity. Northern analysis for various EGF family members indicates that SUM-102PT cells synthesize heparin binding (HB)-EGF mRNA. HB-EGF protein is detectable on the surface of these cells by immunohistochemistry, and SUM-102PT cells are killed by diphtheria toxin, which acts by binding to HB-EGF. Furthermore, HB-EGF antibodies partially neutralize the mitogenic activity of the conditioned medium. Thus, EGFR activation in SUM-102PT cells is mediated, at least in part, by autocrine/juxtacrine stimulation by HB-EGF. SUM-102PT cells also express constitutively active STAT-3 homodimers. Constitutively tyrosine-phosphorylated STAT-3 homodimers were also detected in another breast cancer cell line, MDA468, which has an EGFR amplification and also has constitutive EGFR activity. Thus, SUM-102PT is a new human breast cancer cell line that expresses activated EGFR as a result of an autocrine/juxtacrine interaction with HB-EGF which, in turn, results in activation of STAT-3.

Adenocarcinoma↗

PubMed1997

The hsp90-binding antibiotic geldanamycin decreases Raf levels and epidermal growth factor signaling without disrupting formation of signaling complexes or reducing the specific enzymatic activity of Raf kinase.

We have expressed the mitogenic signaling proteins Src, Ras, Raf-1, Mek (MAP kinase kinase), and Erk (MAP kinase) in baculovirus-infected Sf9 insect cells in order to study a potential role for the chaperone hsp90 in formation of multiprotein complexes. One such complex obtained by immunoadsorption with anti-Ras antibody of cytosol prepared from cells simultaneously expressing Ras, Raf, Mek, and Erk contained Ras, Raf, and Erk. To detect directly the protein-protein interactions involved in forming multiprotein complexes, we combined cytosols from single infections in vitro in all possible combinations of protein pairs. We detected complexes between Ras.Raf, Ras.Src, Raf.Mek, and Raf.Src, but no complex containing Erk was obtained by mixing cytosols. Thus, cellular factors appear to be required for assembly of the Erk-containing multiprotein complex. One cellular factor thought to be involved in signaling protein complex formation is the chaperone hsp90, and we show that Src, Raf, and Mek are each complexed with insect hsp90. Treatment of Sf9 cells with geldanamycin, a benzoquinone ansamycin that binds to hsp90 and disrupts its function, did not decrease coadsorption of either Raf or Erk with Ras, although it did decrease the level of cytosolic Raf. To study geldanamycin action, we treated rat 3Y1 fibroblasts expressing v-Raf and showed that the antibiotic blocked assembly of Raf.hsp90 complexes at an intermediate stage of assembly where Raf is still bound to the p60 and hsp70 components of the assembly mechanism. As in Sf9 cells, Raf levels decline with geldanamycin treatment of 3Y1 cells. To determine if geldanamycin affects mitogenic response, we treated HeLa cells with epidermal growth factor (EGF) and showed that geldanamycin treatment decreased EGF signaling and decreased the level of Raf protein without affecting the EGF-mediated increase in Raf kinase activity. We conclude that hsp90 is not required for forming complexes between the mitogenic signaling proteins or for Raf kinase activity and that EGF signaling is decreased indirectly by geldanamycin because the antibiotic increases degradation of Raf and perhaps other components of the signaling pathway.

Animals↗

PubMed1997

Constitutive activation of JAK1 in Src-transformed cells.

We have previously found that the signal transducer and activator of transcription (Stat) 3 is constitutively activated in cells stably transformed by the v-Src oncoprotein. While activation of Stat proteins has also been observed following epidermal growth factor or platelet-derived growth factor stimulation, Stat3 activation is more commonly associated with signaling through cytokine receptors and activation of the Janus family tyrosine kinases JAK1 or JAK2. We therefore investigated whether JAK1 or JAK2 were activated in Src-transformed cells. In three v-Src-transformed fibroblast cell lines (NIH3T3, Balb/c, and 3Y1), JAK1 displayed increased tyrosyl phosphorylation compared to non-transformed cells. The level of tyrosyl phosphorylation of JAK1 was significantly greater in NIH3T3 cells transformed by expression of v-Src or high levels of a constitutively active mutant of c-Src (Y527F) than in cells overexpressing the less transforming normal c-Src. Enzymatic activity of JAK1 was assessed using autophosphorylation assays. In anti-JAK1 immunoprecipitates from v-Src-transformed NIH3T3 cells, a protein with the same migration as JAK1 showed substantially increased levels of 32P incorporation compared to immunoprecipitates from non-transformed cells. Similar results were obtained using anti-JAK2 immunoprecipitates; however, the level of JAK2 tyrosyl phosphorylation and 32P incorporation in anti-JAK2 immunoprecipitates were markedly lower than in anti-JAK1 immunoprecipitates. We conclude that JAK1, and possibly JAK2, are constitutively activated in Src-transformed cells, raising the possibility that Janus family kinases contribute to the constitutive activation of Stat3 previously observed in these cells and/or other properties of Src-transformed cells.

3T3 Cells↗

PubMed1997

Activation of STAT transcription factors by herpesvirus Saimiri Tip-484 requires p56lck.

Signal transducers and activators of transcription (STATs) relay signals from activated cell surface receptors directly to the nucleus. Previously, a protein required for T-cell transformation by the DNA tumor virus herpesvirus saimiri (HVS) and designated tyrosine kinase interacting protein (Tip-484) was shown to interact with and dramatically upregulate the activity of p56lck. p56lck is a nonreceptor tyrosine kinase that is essential for signaling by the T-cell receptor and also interacts with the CD4, CD8, and interleukin-2 receptors. The present data show activation of STAT1 and -3 by Tip-484. STAT1 and -3 were also found to complex with glutathione S-transferase-Tip-484 only in the presence of p56lck, and STAT3 was shown to be phosphorylated by the Tip-484-p56lck multiprotein complex in vitro. Infection of T cells with HVS or expression of recombinant Tip-484 significantly increased the DNA-binding activity of the STAT1 and STAT3 transcription factors in nuclear extracts and also increased the phosphorylation of STAT3 in vivo. This is the first report of STAT activation by a DNA tumor virus protein. Moreover, these studies demonstrate that p56lck is required for STAT activation by Tip-484.

Animals↗

PubMed1997

Activation of extracellular signal-regulated kinase (ERK) by mitogenic stimuli is repressed in v-Src-transformed cells.

Stimulation of mitogenic signaling pathways results in transient activation of the extracellular signal-regulated kinase (ERK) subfamily of mitogen-activated protein kinases (MAPK) in normal cells. We demonstrate here that activation of ERKs in response to serum or phorbol ester stimulation was markedly repressed in three different rodent fibroblast cell lines stably transformed by v-Src. Activation of the MAPK/ERK kinase (MEK) was also repressed in v-Src-transformed cells, indicating that the repression occurs upstream of ERK. Consistent with repression occurring predominantly at the level of MEK, the phosphatase inhibitor orthovanadate could restore ERK activation to a limited extent in some but not all v-Src-transformed cell lines. A similar repression of ERK activation was observed in v-Ras- and v-Raf-transformed cells. In addition, ERK activity was not constitutively elevated in exponentially growing cells transformed by v-Src, v-Ras, or v-Raf as compared with normal cells. These results establish that the ERK activation pathway is repressed in rodent fibroblasts stably transformed by viral oncoproteins that chronically stimulate receptor tyrosine kinase signaling pathways. Furthermore, our findings suggest that elevated ERK activity above basal levels is not required for maintaining cell transformation by these oncoproteins. Taken together, these results indicate that ERK signaling pathways are subject to negative feedback regulation upstream of ERK as a consequence of oncogenic transformation.

3T3 Cells↗

PubMed1997

Constitutive activation of Stat3 in fibroblasts transformed by diverse oncoproteins and in breast carcinoma cells.

Signal transducers and activators of transcription (STATs) were originally identified as key components of signaling pathways involved in mediating responses to IFNs. Previous studies showed that the Src oncoprotein constitutively activates one STAT family member, Stat3. In this study, we investigated STAT activation in a panel of rodent fibroblast cell lines stably transformed by diverse viral oncoproteins. Using a temperature-sensitive mutant of v-Src, we determined that Stat3 is activated within 15 min of shift from nonpermissive to permissive temperature for cell transformation. This finding indicates that v-Src tyrosine kinase activity is required for Stat3 activation and suggests that Stat3 is proximal to signaling initiated by Src. In addition, Stat3 activation is induced by another nonreceptor tyrosine kinase, v-Fps; by polyoma virus middle T antigen, which activates Src family kinases; and by v-Sis, which acts as a ligand for the platelet-derived growth factor receptor. In contrast SV40 large T antigen, which transforms cells through different mechanisms, and the v-Ras and v-Raf oncoproteins, which lie in signaling pathways downstream of tyrosine kinases, do not activate Stat3. We did not detect significant activation of Stat1, Stat5, or Stat6 in fibroblasts transformed by the viral oncoproteins investigated. Moreover, Stat3 is activated in response to epidermal growth factor (EGF) but not heregulins in immortalized normal human breast epithelial cells. Because constitutive activation of c-Src and EGF receptor kinases is associated with the progression of breast cancer, we examined activation of STATs in human cell lines derived from breast carcinomas. We detected constitutive activation of Stat3 in five of nine breast carcinoma cell lines but not in normal breast epithelial cells. Furthermore, experiments with an EGF receptor-specific inhibitor indicated that the constitutive activation of Stat3 in these breast carcinoma cell lines is not necessarily dependent on signaling through the EGF receptor, although EGF stimulation further increases Stat3 activation. Taken together, our results demonstrate that selective activation of Stat3 is a common event during oncogenic transformation that directly or indirectly involves activation of specific tyrosine kinase signaling pathways.

3T3 Cells↗

PubMed1997

Overexpression of normal c-Src in poorly metastatic human colon cancer cells enhances primary tumor growth but not metastatic potential.

Whereas genetic paradigms are now defined for the development of human colon cancer, little is known regarding the mechanisms that regulate development of the metastatic phenotype. Recent reports have indirectly linked the expression and activation of c-Src to the process of human colon cancer metastasis. Whereas v-Src, a highly activated mutational derivative of c-Src, has been shown to induce metastasis, normal c-Src has not been tested for this property. We hypothesized that c-Src overexpression in the milieu of a poorly metastatic cancer cell might permit the development of a highly metastatic cell. Two poorly metastatic human colon cancer cell lines were stably transfected with expression vectors encoding normal human c-Src. Clones producing 4-10-fold more c-Src than controls were injected s.c. and intrasplenically into the nude mouse to assess primary tumor growth and liver metastatic potential. Whereas metastatic potential was unaffected, primary tumor growth in vivo was significantly enhanced by c-Src overexpression. No effects on rates of tumor cell proliferation were seen in vitro. Our findings suggest that normal c-Src may be necessary but is insufficient for the induction of the metastatic phenotype.

Cell Division↗

PubMed1996

N-terminal sequences contained in the Src homology 2 and 3 domains of p120 GTPase-activating protein are required for full catalytic activity toward Ras.

The p120 GTPase-activating protein (GAP) is a negative regulator of Ras, which has a central role in signal transduction pathways that control cell proliferation. p120 GAP accelerates the conversion of activated Ras-GTP to its inactive form, Ras-GDP, thereby inhibiting mitogenic signaling. To examine potential contributions of p120 N-terminal sequences to regulation of its C-terminal catalytic domain, we constructed deletion mutants lacking defined regions, including the variable hydrophobic region as well as the Src homology 2 (SH2) and 3 (SH3) domains. These mutant proteins were expressed in infected Sf9 insect cells from recombinant baculoviruses and assayed in vitro for their ability to stimulate the intrinsic GTPase activity of purified Ras. While deletion of the variable hydrophobic region had no effect on p120 GAP activity, deletion of the entire SH2/SH3/SH2 region severely impaired catalytic activity toward Ras. Deletion of individual SH2 and SH3 domains within this region partially inhibited p120 GAP activity. Moreover, p120 N-terminal sequences enhanced the Ras GTPase-stimulating activity of the neurofibromin GAP-related domain. These results demonstrate that sequences in the SH2/SH3/SH2 region of p120 GAP are required for full catalytic activity toward Ras. Together with earlier findings that the p120 GAP SH2 domains mediate interactions with several GAP-associated proteins, our results suggest multiple roles for the N-terminal sequences in regulating p120 GAP catalytic activity and mitogenic signaling pathways. In addition, our results raise the possibility that SH2 domain point mutations in p120 GAP detected in some basal cell carcinomas reduce catalytic activity toward Ras and thereby contribute to oncogenesis.

Animals↗

PubMed1995

Two SH2 domains of p120 Ras GTPase-activating protein bind synergistically to tyrosine phosphorylated p190 Rho GTPase-activating protein.

p120 GTPase-activating protein (GAP) is a negative regulator of Ras that functions at a key relay point in signal transduction pathways that control cell proliferation. Among other proteins, p120 GAP associates with p190, a GAP for the Ras-related protein, Rho. To characterize the p120.p190 interaction further, we used bacterially expressed glutathione S-transferase fusion polypeptides to map the regions of p120 necessary for its interactions with p190. Our results show that both the N-terminal and the C-terminal SH2 domains of p120 are individually capable of binding p190 expressed in a baculovirus/insect cell system. Moreover, the two SH2 domains together on one polypeptide bind synergistically to p190, and this interaction is dependent on tyrosine phosphorylation of p190. In addition, mutation of the highly conserved Arg residues in the critical FLVR sequences of both SH2 domains of full-length p120 reduces binding to tyrosine-phosphorylated p190. The dependence on p190 phosphorylation for complex formation with p120 SH2 domains observed in vitro is consistent with analysis of the native p120.p190 complexes formed in vivo. These findings suggest that SH2-phosphotyrosine interaction is one mechanism by which the cell regulates p120.p190 association and thus may be a means for coordinating the Ras- and Rho-mediated signaling pathways.

Animals↗

PubMed1995

Enhanced DNA-binding activity of a Stat3-related protein in cells transformed by the Src oncoprotein.

Cytokines and growth factors induce tyrosine phosphorylation of signal transducers and activators of transcription (STATs) that directly activate gene expression. Cells stably transformed by the Src oncogene tyrosine kinase were examined for STAT protein activation. Assays of electrophoretic mobility, DNA-binding specificity, and antigenicity indicated that Stat3 or a closely related STAT family member was constitutively activated by the Src oncoprotein. Induction of this DNA-binding activity was accompanied by tyrosine phosphorylation of Stat3 and correlated with Src transformation. These findings demonstrate that Src can activate STAT signaling pathways and raise the possibility that Stat3 contributes to oncogenesis by Src.

Animals↗

PubMed1995

Dissociation of mitogen-activated protein kinase activation from the oxidative burst in differentiated HL-60 cells and human neutrophils.

In human polymorphonuclear leukocytes (PMNs), mitogen-activated protein kinases (MAPKs), also known as extracellular signal-regulated kinases (Erks), are activated within minutes upon stimulation with either chemoattractant formyl-Met-Leu-Phe (fMLP) or phorbol 12-myristate 13-acetate (PMA). This activation of MAPKs coincides with the formation of superoxide anion, which occurs through the activation of a multiple-component NADPH oxidase pathway. MAPKs have thus been suggested to be involved in signal transduction leading to the oxidative burst. To investigate whether MAPK activation plays a central role in the oxidative burst, we evaluated the effect of cAMP on MAPK activation induced by fMLP and PMA. cAMP inhibits many PMN functional responses, including the oxidative burst, and has recently been shown to reduce growth factor- and PMA-induced MAPK activities in a variety of cells. We found that in differentiated, neutrophil-like HL-60 cells, while cAMP reduced PMA-induced MAPK activation, it had no effect on fMLP-induced MAPK activation. Despite the presence of unchanged levels of activated MAPKs, the fMLP-induced oxidative burst was substantially diminished by cAMP. By contrast, O2-production induced by PMA remained the same even though MAPK activation was inhibited. In PMNs, although the levels of O2-induced by either 10 ng/ml or 100 ng/ml PMA were similar, only 100 ng/ml could stimulate MAPK activation, suggesting that the oxidative burst could occur in the absence of detectable activation of MAPKs. As in HL-60 cells, cAMP inhibited the O2-production in fMLP-stimulated PMNs but had no effect on MAPK activity. These results demonstrate that, while MAPK activation coincides with PMN activation, it can be dissociated from the oxidative burst.

Calcium-Calmodulin-Dependent Protein Kinases↗

PubMed1995

A direct interaction between G-protein beta gamma subunits and the Raf-1 protein kinase.

Raf-1 is a serine/threonine protein kinase positioned downstream of Ras in the mitogen-activated protein kinase cascade. Using a yeast two-hybrid strategy to identify other proteins that interact with and potentially regulate Raf-1, we isolated a clone encoding the carboxyl-terminal half of the G beta 2 subunit of heterotrimeric G-proteins. In vitro, purified G beta gamma subunits specifically bound to a GST fusion protein encoding amino acids 1-330 of Raf-1 (Raf/330). Binding assays with truncation mutants of GST-Raf indicate that the region located between amino acids 136 and 239 is a primary determinant for interaction with G beta gamma. In competition experiments, the carboxyl terminus of beta-adrenergic receptor kinase (beta ARK) blocked the binding of G beta gamma to Raf/330; however, the Raf-1-binding proteins, Ras and 14-3-3, had no effect. Scatchard analysis of in vitro binding between Raf/330 and G beta gamma revealed an affinity of interaction (Kd = 163 +/- 36 nM), similar to that seen between G beta gamma and beta ARK (Kd = 87 +/- 24 nM). The formation of native heterotrimeric G alpha beta gamma complexes, as measured by pertussis toxin ADP-ribosylation of G alpha, could be disrupted by increasing amounts of Raf/330, with an EC50 of approximately 200 nM, in close agreement with the estimated binding affinity. In vivo complexes of Raf-1 and G beta gamma were isolated from human embryonic kidney 293-T cells transfected with epitope-tagged G beta 2. The identification and characterization of this novel interaction raises several possibilities for signaling cross-talk between growth factor receptors and those receptors coupled to heterotrimeric G-proteins.

Amino Acid Sequence↗

PubMed1995

The Ras GTPase-activating protein (GAP) is an SH3 domain-binding protein and substrate for the Src-related tyrosine kinase, Hck.

The Ras GTPase-activating protein (GAP) is a target for protein tyrosine kinases of both the receptor and cytoplasmic classes and may serve to integrate tyrosine kinase and Ras signaling pathways. In this report, we provide evidence that GAP is an SH3 domain-binding protein and substrate for the Src-related tyrosine kinase Hck, which has been implicated in the regulation of myeloid cell growth, differentiation, and function. Wild-type (WT) or kinase-inactive (K269E) mutant Hck proteins were co-expressed with bovine GAP using the baculovirus/Sf-9 cell system. GAP was readily phosphorylated on tyrosine by WT but not K269E Hck. GAP was present in WT Hck immunoprecipitates from the co-infected cells, indicative of Hck.GAP complex formation. Unexpectedly, GAP also associated with the kinase-inactive mutant of Hck, suggesting that tyrosine autophosphorylation of Hck is not required for complex formation. The WT and K269E forms of Hck also associated with GAP mutants lacking either the C-terminal catalytic domain (delta CAT) or the Src homology region (delta SH), indicating that these GAP domains are dispensable for complex formation. Recombinant GST fusion proteins containing the Hck, Src, Fyn, or Lck SH3 domains associated with full-length GAP, delta CAT, and delta SH, all of which share an N-terminal proline-rich region resembling an SH3-binding motif (PPLPPPPPQLP). Deletion of the highly conserved YXY sequence from the Hck SH3 domain abolished binding. GAP-SH3 interaction was also inhibited by the proline-rich peptide GFPPLPPPPPQLPTLG, which corresponds to N-terminal amino acids 129-144 of bovine GAP. An N-terminal deletion mutant of GAP lacking this proline-rich region did not bind to the Hck SH3 domain. These data implicate the Hck SH3 domain in GAP interaction, and suggest a general function for the SH3 domains of Src family kinases in recognition of GAP via its proline-rich N-terminal domain.

Amino Acid Sequence↗

PubMed1995

Functional mapping of the N-terminal regulatory domain in the human Raf-1 protein kinase.

Raf-1 is a serine/threonine kinase poised at a key relay point in mitogenic signal transduction pathways from the cell surface to the nucleus. Activation of the transforming potential of Raf-1 has been associated with N-terminal truncation and/or fusion to other proteins, suggesting that the Raf-1 N-terminal half harbors a negative regulatory domain. Seven internal deletion mutants that together scan the entire N-terminal half of human Raf-1 protein were generated to map functional regions in this regulatory domain. Effects of the deletion mutations on kinase activity of Raf-1 were evaluated using a baculovirus/insect cell overexpression system and an in vitro kinase assay with the known physiological substrate of Raf-1, mitogen-activated protein kinase kinase. Deletion of amino acids 276-323 in the unique sequence between conserved regions 2 and 3 leads to modest elevation of Raf-1 basal kinase activity, whereas deletion of amino acids 133-180 in conserved region 1 results in diminished kinase activity. Surprisingly, none of the Raf-1 N-terminal deletion mutants, including a truncated version that is transforming in rodent fibroblasts, exhibits greatly increased levels of basal kinase activity. In addition, while activation of Raf-1 kinase by Ras requires sequences in conserved region 1, only the C-terminal half containing the kinase domain of Raf-1 is required for activation by Src. These findings demonstrate that N-terminal deletions in Raf-1 do not necessarily result in constitutively elevated basal kinase activity and that the N-terminal regulatory domain is completely dispensable for Raf-1 activation by Src.

Amino Acid Sequence↗