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H Hanafusa

Publications and source records attributed to H Hanafusa.

At least 91 records · Page 5Linked to original sources
PubMed1994

Expression of the v-crk oncogene product in PC12 cells results in rapid differentiation by both nerve growth factor- and epidermal growth factor-dependent pathways.

The transforming gene of the avian sarcoma virus CT10 encodes a fusion protein (p47gag-crk or v-Crk) containing viral Gag sequences fused to cellular sequences consisting primarily of Src homology regions 2 and 3 (SH2 and SH3 sequences). Here we report a novel function of v-Crk in the mammalian pheochromocytoma cell line, PC12, whereby stable expression of v-Crk induces accelerated differentiation, as assessed by induction of neurites following nerve growth factor (NGF) or basic fibroblast growth factor (bFGF) treatment compared with the effect in native PC12 cells. Surprisingly, however, these cells also develop extensive neurite processes after epidermal growth factor (EGF) stimulation, an event which is not observed in native PC12 cells. Following EGF or NGF stimulation of the v-CrkPC12 cells, the v-Crk protein itself became tyrosine phosphorylated within 1 min. Moreover, in A431 cells or TrkA-PC12 cells, which overexpress EGF receptors and TrkA, respectively, a GST-CrkSH2 fusion protein was indeed capable of binding these receptors in a phosphotyrosine-dependent manner, suggesting that v-Crk can directly couple to receptor tyrosine kinase pathways in PC12 cells. In transformed fibroblasts, v-Crk binds to specific tyrosine-phosphorylated proteins of p130 and paxillin. Both of these proteins are also complexed to v-Crk in PC12 cells, as evidenced by their coprecipitation with v-Crk in detergent lysates, suggesting that common effector pathways may occur in both cell types. However, whereas PC12 cellular differentiation can occur solely by overexpression of the v-Src or oncogenic Ras proteins, that induced by v-Crk requires a growth factor stimulatory signal, possibility in a two-step process.

Animals↗
PubMed1994

Specific motifs recognized by the SH2 domains of Csk, 3BP2, fps/fes, GRB-2, HCP, SHC, Syk, and Vav.

Src homology 2 (SH2) domains provide specificity to intracellular signaling by binding to specific phosphotyrosine (phospho-Tyr)-containing sequences. We recently developed a technique using a degenerate phosphopeptide library to predict the specificity of individual SH2 domains (src family members, Abl, Nck, Sem5, phospholipase C-gamma, p85 subunit of phosphatidylinositol-3-kinase, and SHPTP2 (Z. Songyang, S. E. Shoelson, M. Chaudhuri, G. Gish, T. Pawson, W. G. Haser, F. King, T. Roberts, S. Ratnofsky, R. J. Lechleider, B. G. Neel, R. B. Birge, J. E. Fajardo, M. M. Chou, H. Hanafusa, B. Schaffhausen, and L. C. Cantley, Cell 72:767-778, 1993). We report here the optimal recognition motifs for SH2 domains from GRB-2, Drk, Csk, Vav, fps/fes, SHC, Syk (carboxy-terminal SH2), 3BP2, and HCP (amino-terminal SH2 domain, also called PTP1C and SHPTP1). As predicted, SH2 domains from proteins that fall into group I on the basis of a Phe or Tyr at the beta D5 position (GRB-2, 3BP2, Csk, fps/fes, Syk C-terminal SH2) select phosphopeptides with the general motif phospho-Tyr-hydrophilic (residue)-hydrophilic (residue)-hydrophobic (residue). The SH2 domains of SHC and HCP (group III proteins with Ile, Leu, of Cys at the beta D5 position) selected the general motif phospho-Tyr-hydrophobic-Xxx-hydrophobic, also as predicted. Vav, which has a Thr at the beta D5 position, selected phospho-Tyr-Met-Glu-Pro as the optimal motif. Each SH2 domain selected a unique optimal motif distinct from motifs previously determined for other SH2 domains. These motifs are used to predict potential sites in signaling proteins for interaction with specific SH2 domain-containing proteins. The Syk SH2 domain is predicted to bind to Tyr-hydrophilic-hydrophilic-Leu/Ile motifs like those repeated at 10-residue intervals in T- and B-cell receptor-associated proteins. SHC is predicted to bind to a subgroup og these same motifs. A structural basis for the association of Csk with Src family members is also suggested from these studies.

Amino Acid Sequence↗
PubMed1993

src-specific immune regression of Rous sarcoma virus-induced tumors.

We have developed an oncogene-specific tumor regression system in chickens. Injection s.c. of chicken wing webs with either rASV1702 or rASV157, mutants of Rous sarcoma virus (RSV) that express nonmyristolyated src product containing novel N-terminal domains, results in noninvasive fibrosarcomas that regress fully. The ability of challenge infections with wild-type RSV to form tumors is suppressed. This protective effect was shown to be specific for determinants encoded or induced by v-src (I. Gelman and H. Hanafusa, J. Virol., 61: 2461-2468, 1989). In the current study, we used SC chickens, inbred for major histocompatibility complex Class I haplotype B2/B2, to investigate whether this protection results from active immunity. Preinoculation of chickens with either replication-defective rASV1702 virus or non-virus-producing syngeneic chicken embryo fibroblasts expressing 1702src conferred protection against challenge infections with RSV. Thus, viremia was not required for this protection. Splenic lymphocytes from rASV1702-infected donors could transfer protective immunity against RSV tumor challenge to naive chickens. These lymphocytes were cytotoxic in vitro against RSV- or rASV1702-infected SC-chicken embryo fibroblasts, but not against SC-chicken embryo fibroblasts infected with helper virus, suggesting a specificity for src-encoded or -induced determinants. In contrast, splenic lymphocytes from RSV-infected chickens transferred protective immunity poorly and exhibited low in vitro cytotoxic potential for src determinants, suggesting possible suppression mechanisms. Finally, murine cell lines expressing 157src or 1702src produced tumors in nude mice that failed to regress. Thus, although cells expressing 157src or 1702src are inherently tumorigenic, the tumors they induce most likely regress due to immune mechanisms. These results suggest that 1702src and 157src induce src-specific tumor Ag that potently prime an oncogene-specific protective cellular immunity.

Animals↗
PubMed1993

Activation of a histone H1 kinase by tyrosine phosphorylation in v-src-transformed fibroblasts.

Using anti-phosphotyrosine immunoaffinity chromatography, we have searched for serine/threonine kinases that are directly regulated by tyrosine phosphorylation in v-src-transformed rat 3Y1 fibroblasts. Tyrosine phosphoprotein preparations from v-src-transformed cells contain a kinase activity that phosphorylates histone H1 in vitro on serine residues and this activity is present at a 20-fold greater level than that in parental cell preparations. This activity elutes from a MonoQ FPLC column as a single peak and gel filtration chromatography suggests that the kinase has a molecular mass of approximately 55 kDa. Tyrosine phosphatase treatment inactivates the histone H1 kinase and this result indicates that the specific activity of the kinase is regulated by tyrosine phosphorylation. Experiments with cells transformed with a temperature-sensitive mutant of the v-src oncogene demonstrate that the tyrosine phosphorylation of the histone H1 kinase is an early event in v-src transformation. The kinase is distinct from known cdc2 family members that contain the PSTAIR motif, because the kinase can be separated almost completely from these proteins by immunoprecipitation with an antibody against p34cdc2. The profile of antibody reactivity and sensitivity to modulators of protein kinases suggests that this activity is distinct from known second messenger-regulated kinases and from previously characterized MAP kinases.

Amino Acid Sequence↗
PubMed1993

Transformation-associated cytokine 9E3/CEF4 is chemotactic for chicken peripheral blood mononuclear cells.

9E3/CEF4, which is released from transformed chicken embryo fibroblasts (CEF), is a member of the platelet factor 4 family of inflammatory proteins and may be the avian homolog of interleukin-8. Since the function of 9E3/CEF4 is unknown, we examined the effect of the protein on mitogenicity and chemotaxis, as well as its expression, in fibroblasts and peripheral blood cells. 9E3/CEF4 mRNA was expressed in chicken peripheral blood monocytes, and its expression was stimulated by incubation of the monocytes with lipopolysaccharide or phorbol myristic acetate. Boyden double-membrane analysis of chemotaxis showed that 9E3/CEF4 was chemotactic for chicken peripheral blood mononuclear cells, as well as for heterophils. Untransformed CEF and CEF transformed with Rous sarcoma virus also migrated to 9E3/CEF4 protein, as measured by Boyden single-membrane analysis. 9E3/CEF4 was slightly mitogenic for CEF, causing a doubling of [3H]thymidine uptake when added to serum-starved CEF.9E3/CEF4 was found associated not only with the cell and in the culture medium of Rous sarcoma virus-transformed CEF but also with the extracellular matrix. The in vivo role of 9E3/CEF4 may be involved with chemotaxis and metastasis, rather than with direct stimulation of mitogenicity.

Animals↗
PubMed1993

A 31-amino-acid N-terminal extension regulates c-Crk binding to tyrosine-phosphorylated proteins.

Overproduction of v-Crk, but not of c-Crk, in chicken embryo fibroblasts results in cell transformation. The transforming activity of v-Crk mutants correlates with their ability to cause increased tyrosine phosphorylation of specific cellular proteins, a property that depends on the binding of v-Crk to phosphotyrosine residues via its SH2 domain. In this study, proteins translated in rabbit reticulocyte lysates were used to analyze interactions between Crk derivatives and tyrosine-phosphorylated proteins, particularly the epidermal growth factor (EGF) receptor. The results demonstrate that the binding affinity of c-Crk is much lower than that of v-Crk, despite the fact that both proteins contain identical SH2 domains. Moreover, a 31-amino-acid N-terminal extension of c-Crk, resulting from upstream translational initiation at a CUG codon, significantly increases the ability of the resulting protein to bind to phosphotyrosine-containing proteins. Of those 31 amino acids, 24 can be found in the 27-amino-acid region between Gag and Crk sequences in v-Crk, and removal of this region results in a protein with lower affinity toward the EGF receptor. In addition, fusion of Gag to the amino terminus of c-Crk yields a protein with a binding activity that is lower than that of v-Crk but significantly higher than that of c-Crk without the fusion. These data suggest that sequences N terminal to the Crk SH2 regulate binding activity to tyrosine-phosphorylated proteins and that the amino acids encoded immediately 5' to the c-Crk initiator AUG specifically increase binding affinity. In contrast, deletion of one or two SH3 domains of c-Crk proteins did not change their affinity for the EGF receptor. These results were confirmed in vivo by using A431-derived cell lines overproducing either the chicken c-Crk protein or c-Crk with the 31-amino-acid N-terminal extension. Furthermore, the in vivo experiments suggest that binding of Crk proteins to the stimulated EGF receptor results in Crk phosphorylation and subsequent loss of binding affinity.

Amino Acid Sequence↗
PubMed1993

Inactivation of c-Yes tyrosine kinase by elevation of intracellular calcium levels.

We have previously shown that the c-Src tyrosine kinase is activated four- to fivefold when cultured keratinocytes differentiate following the elevation of intracellular calcium levels. In contrast to c-Src, another Src family tyrosine kinase, c-Yes, was rapidly inactivated in these same cells, despite its marked similarity in structure and enzymatic activity to c-Src. The inactivation of c-Yes was independent of the protein kinase C pathway, which is usually activated by elevation of intracellular calcium levels. The protein levels of c-Src and c-Yes were not altered, but the phosphotyrosine content of both proteins was greatly reduced. As has been demonstrated for c-Src, in vitro dephosphorylation of c-Yes by incubation with protein tyrosine phosphatases also resulted in its activation, not inactivation. In vitro reconstitution experiments showed that c-Yes can be inactivated by preincubation with a Ca(2+)-supplemented cell extract and that this inhibition was reversed by the addition of EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid]. Gradient sedimentation of cell lysates showed that in cells treated with calcium and ionophore, c-Yes formed complexes with two distinct cellular proteins, whereas similar complexes were not seen in c-Src immunoprecipitates. One of these two proteins has the ability to inhibit c-Yes kinase activity in vitro. Finally, the Ca(2+)-dependent inactivation of c-Yes was observed in kidney tubular cells and fibroblasts, suggesting that the Ca(2+)-dependent regulation of c-Yes tyrosine kinase is not unique to keratinocytes. We postulate that c-Yes is inactivated through a Ca2+ -dependent association with cellular proteins, which seems to override its activation resulting from tyrosine dephosphorylation.

CSK Tyrosine-Protein Kinase↗
PubMed1993

Identification and characterization of a high-affinity interaction between v-Crk and tyrosine-phosphorylated paxillin in CT10-transformed fibroblasts.

The genome of avian sarcoma virus CT10 encodes a fusion protein in which viral Gag sequences are fused to cellular Crk sequences containing primarily Src homology 2 (SH2) and Src homology 3 (SH3) domains. Transformation of chicken embryo fibroblasts (CEF) with the Gag-Crk fusion protein results in the elevation of tyrosine phosphorylation on specific cellular proteins with molecular weights of 130,000, 110,000, and 70,000 (p130, p110, and p70, respectively), an event which has been correlated with cell transformation. In this study, we have identified the 70-kDa tyrosine-phosphorylated protein in CT10-transformed CEF (CT10-CEF) as paxillin, a cytoskeletal protein suggested to be important for organizing the focal adhesion. Tyrosine-phosphorylated paxillin was found to be complexed with v-Crk in vivo as evident from coimmunoprecipitation studies. Moreover, a bacterially expressed recombinant glutathione S-transferase (GST)-CrkSH2 fragment bound paxillin in vitro with a subnanomolar affinity, suggesting that the SH2 domain of v-Crk is sufficient for binding. Mapping of the sequence specificity of a GST-CrkSH2 fusion protein with a partially degenerate phosphopeptide library determined a motif consisting of pYDXP, and in competitive coprecipitation studies, an acetylated A(p)YDAPA hexapeptide was able to quantitatively inhibit the binding of GST-CrkSH2 to paxillin and p130, suggesting that it meets the minimal structural requirements necessary for the interaction of CrkSH2 with physiological targets. To investigate the mechanism by which v-Crk elevates the tyrosine phosphorylation of paxillin in vivo, we have treated normal CEF and CT10-CEF with sodium vanadate to inhibit protein tyrosine phosphatase activity. These data suggest that paxillin is involved in a highly dynamic kinase-phosphatase interplay in normal CEF and that v-Crk binding may interrupt this balance to increase the steady-state level of tyrosine phosphorylation. By contrast, the 130-kDa protein was not tyrosine phosphorylated upon vanadate treatment of normal CEF and only weakly affected in the CT10-CEF, suggesting that a different mechanism may be involved in its phosphorylation.

Amino Acid Sequence↗
PubMed1993

Morphological transformation, tumorigenicity and src-specific cytotoxic T-lymphocyte-mediated tumor immunity induced by murine 3T3 cells expressing src oncogenes encoding novel non-myristylated N-terminal domains.

We previously reported the development of a src-specific tumor regression system in chickens in which preinfection with rASV1702, a mutant of Rous sarcoma virus (RSV) encoding non-myristylated src product with a novel N-terminal domain, results in the immune suppression of challenge tumors induced by RSV. In order to adapt this system to the mouse, we have developed NIH3T3 and Balb/c3T3 (B3T3) cell lines that express 1702src, v-src, c-src, and other src variants, either by transfection or by infection with packaged recombinant Moloney virus (MLV) vectors. The sequence of the 1702 src cDNA, produced by reverse transcription-polymerase chain reaction (RT-PCR), confirmed the previously suggested 1702src N-terminal domain structure, fusing six amino acids from Pr76gag and 39 amino acids of env signal peptide sequence to Ala-76 of src. These cell lines were characterized for src expression and activity, cell compartmentalization of src product, tyrosine phosphorylation substrate specificity and transforming and tumorigenic potential. Based on these parameters, murine cell lines expressing 1702src were characteristically similar to chicken embryo fibroblasts (CEFs) infected with rASV1702. Finally, B3T3/1702src expressors, or membrane fractions of these cells, induced src-specific tumor protection in syngeneic mice against v-src-transformed tumor challenges. Splenic lymphocytes isolated from Balb/c mice inoculated with B3T3/1702src cells showed in vitro cytotoxicity against B3T3/v-src cells but not against untransformed B3T3 cells. Antibodies specific for Lyt2, mouse CD3 and H-2Dd blocked this cytotoxicity, whereas those specific for L3T4 did not, suggesting MHC class I-restricted, CD8-mediated cell killing. These data indicate that Balb/c3T3-expressed 1702src induces a cellular anti-tumor immunity based on src-specific tumor rejection antigens.

3T3 Cells↗
PubMed1992

Increased tyrosine kinase activity of c-Src during calcium-induced keratinocyte differentiation.

In cultured human epidermal keratinocytes, induction of differentiation by Ca2+ and ionophore treatment was found to result in rapid elevation of c-Src tyrosine kinase activity and inactivation of the c-Yes tyrosine kinase. Activation of c-Src kinase was accompanied by tyrosine dephosphorylation, which might be explained by a rapid increase in intracellular protein-tyrosine phosphatase activity. Ca(2+)-induced differentiation was also associated with altered tyrosine phosphorylation of several cellular proteins and correlated with a marked redistribution of intracellular phosphotyrosine from membrane and adhesion sites to the nucleus. Some of the c-Src protein was also found in the nucleus after Ca2+ treatment, and Ca(2+)-activated c-Src bound to three cellular proteins (120 kDa, 65 kDa, and 34 kDa). In agreement with these results, immunohistochemistry on human epidermis revealed an increase in c-Src expression and tyrosine phosphorylation in cells undergoing differentiation, which strongly suggests a possible role of non-receptor tyrosine kinases in epithelial cell maturation.

Calcium↗
PubMed1992

Crystal structure of the phosphotyrosine recognition domain SH2 of v-src complexed with tyrosine-phosphorylated peptides.

Three-dimensional structures of complexes of the SH2 domain of the v-src oncogene product with two phosphotyrosyl peptides have been determined by X-ray crystallography at resolutions of 1.5 and 2.0 A, respectively. A central antiparallel beta-sheet in the structure is flanked by two alpha-helices, with peptide binding mediated by the sheet, intervening loops and one of the helices. The specific recognition of phosphotyrosine involves amino-aromatic interactions between lysine and arginine side chains and the ring system in addition to hydrogen-bonding interactions with the phosphate.

Amino Acid Sequence↗
PubMed1992

Tyrosine-phosphorylated epidermal growth factor receptor and cellular p130 provide high affinity binding substrates to analyze Crk-phosphotyrosine-dependent interactions in vitro.

The genome of CT10 avian sarcoma virus encodes a 47-kDa fusion protein that consists of viral gag sequences fused to a cell-derived sequence containing SH2 and SH3 domains (v-crk). Genetic and biochemical evidence suggests that v-Crk can induce transformation of chicken embryo fibroblasts by influencing the activity of cellular proteins involved in growth regulation. In this report, we have developed an in vitro microtiter assay to study the binding of bacterially expressed glutathione S-transferase-fusion proteins of v-Crk and its cellular homolog, c-Crk, to the phosphorylated epidermal growth factor receptor (EGFR). Competitive binding data are presented that compare the abilities of heterologous glutathione S-transferase-fusion proteins containing GAPSH2[N], AblSH2, SrcSH2, and PLC-gamma SH2[N] sequences to inhibit Crk binding. Results indicate that both full-length Crk and GAPSH2[N] bind the phosphorylated EGFR with high affinity and can quantitatively compete the binding of each other by competitive enzyme-linked immunosorbent assay. Binding of full-length Crk or the isolated SH2 domains of GAP or Abl resulted in a significant protection of phosphorylated EGFR against dephosphorylation by cellular phosphatase activity, but did not appear to stimulate the intrinsic tyrosine kinase activity of the EGFR. To extend these findings to p130, the major phosphotyrosine-containing protein in CT10-transformed cells, we utilized a nitrocellulose filter binding assay. Results demonstrate high affinity binding of Crk toward denatured p130 and, as is the case for phosphorylated EGFR, Crk binding can partially protect p130 from phosphatase activity. However, no apparent competition of Crk binding was noted with heterologous SH2-containing proteins including GAPSH2[N], suggesting a possible specificity of Crk-p130 binding. These data are consistent with a direct role of SH2 in the modulation of cellular phosphotyrosine status in vivo.

Animals↗
PubMed1992

Characterization of the murine BEK fibroblast growth factor (FGF) receptor: activation by three members of the FGF family and requirement for heparin.

The bek gene encodes a member of the high-affinity fibroblast growth factor receptor family. The BEK/FGFR-2 receptor is a membrane-spanning tyrosine kinase with the typical features of FGF receptors. We have cloned a murine bek cDNA and expressed it in receptor-negative Chinese hamster ovary cells and in 32D myeloid cells. The BEK receptor expressed in Chinese hamster ovary cells binds acidic FGF, basic FGF, and Kaposi FGF equally well but does not bind keratinocyte growth factor or FGF-5 appreciably. Upon treatment with basic FGF or Kaposi FGF, the BEK receptor is phosphorylated and a mitogenic response is achieved. Heparan sulfate proteoglycans have been shown to play an obligate role in basic FGF binding to the high-affinity FLG receptor. Unlike the BEK-expressing Chinese hamster ovary cells, 32D cells expressing the BEK receptor require the addition of exogenous heparin in order to grow in the presence of basic FGF or Kaposi FGF. We show that the addition of heparin greatly enhances the binding of radio-labeled basic FGF to the receptor. Thus the BEK receptor, like FLG, also requires an interaction with heparan sulfate proteoglycans to facilitate binding to its ligands.

Amino Acid Sequence↗
PubMed1992

Molecular cloning and expression of chicken C-terminal Src kinase: lack of stable association with c-Src protein.

Cloning and sequencing of chicken C-terminal Src kinase (CSK), a tyrosine kinase that phosphorylates the regulatory C-terminal tyrosine residue present on cytoplasmic tyrosine kinases of the Src family, demonstrated a high degree of interspecies conservation as well as src homology 2 and 3 domains N-terminal to the kinase domain. The lack of autophosphorylation sites distinguishes CSK from other tyrosine kinases. CSK is unique and does not belong to a gene family, suggesting that it may phosphorylate other members of the Src family of tyrosine kinases in addition to c-Src. Since complex formation between c-Src and CSK seemed a likely regulatory step in the control of c-Src kinase activity, such an association was investigated by immunoprecipitation and Western blotting as well as intracellular localization studies. Although some portions of CSK were found in a membrane fraction, no complex formation between CSK and c-Src was observed, suggesting that the src homology 2 domain of CSK does not play a role in the direct interaction of c-Src.

Amino Acid Sequence↗
PubMed1992

Identification and evaluation of new primer sets for the detection of lentivirus proviral DNA.

We have developed sets of degenerate oligonucleotides designed to detect pol gene sequences from any member of the lentivirus subfamily when used as primers in amplification techniques such as the polymerase chain reaction (PCR). This pan-lentivirus-specific primer set (PLSPS) consists of primers, LV1, LV2, and LV3, based on conserved regions common to lentiviruses only. Our protocol is based on primary amplification with LV1 and LV2 followed by secondary amplification with a nested primer set based on the YM/VDD motif found in all reverse transcriptases (or "DDMY," in the opposite direction), and LV3, a block of lentivirus homology nested just downstream of LV1. PLSPS-PCR analysis of DNA from cells infected with HIV-1, HIV-2, SIVmac239, BIV, visna, EIAV, CAEV, OPPV, or FIV resulted in the amplification of appropriately sized products. Sequence analysis of the LV1/2 products, cloned into pBluescript (pBS), indicated that at least 20% (most often, > 80%) contained the predicted lentivirus pol sequence. Greater than 95% of the LV3/DDMY products contained the expected lentiviral sequences. Using the PLSPS, lentivirus pol sequences could typically be detected at levels of one copy in 2 x 10(6) cells after secondary amplification. No specific lentiviral PCR products were detected in DNA from uninfected human or mouse monocytes, feline or bovine leukocytes, mouse, rat or human fibroblast cell lines, chicken embryo fibroblasts, Tahr lung cells, or cell lines infected with the following retroviruses which are not lentiviruses: Rous sarcoma virus, Moloney leukemia virus or Kirsten sarcoma virus, mouse mammary tumor virus, human T-cell lymphotropic virus I, and feline leukemia virus.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

Biological and biochemical activity of v-Crk chimeras containing the SH2/SH3 regions of phosphatidylinositol-specific phospholipase C-gamma and Src.

The chicken CT10 virus oncogene product, P47gag-crk, contains SH2/SH3 domains that have been identified as conserved domains among proteins involved in signal transduction. We studied the functional similarity of the SH2/SH3 domains by replacing those of v-Crk with those of phosphatidylinositol-specific phospholipase C-gamma, v-Src, or c-Src. The transforming activity of v-Crk was partially retained in a mutant with a v-Src SH3 domain but not in the other mutants with heterologous SH2/SH3 domains. Mutant viruses with Crk-SH2/SH2' domains induced tyrosine phosphorylation of cellular proteins, but mutants with phosphatidylinositol-specific phospholipase C-gamma or Src SH2/SH2' domains did not. However, the mutant proteins with heterologous SH2/SH2' regions were able to weakly associate with some phosphotyrosine-containing proteins in vitro. These results indicate that in the context of the P47gag-crk structure, the requirement of Crk-SH2/SH3 is more stringent for its activity to induce cell transformation than to cause phosphorylation of cellular proteins. The substitution with heterologous sequences least perturbs the capacity to bind phosphotyrosine-containing proteins. In each case, the SH3 domain is more flexible to substitution than is the SH2 domain.

Animals↗
PubMed1992

A novel oncogene, v-ryk, encoding a truncated receptor tyrosine kinase is transduced into the RPL30 virus without loss of viral sequences.

The RPL viruses are acute oncogenic avian retroviruses isolated from chicken tumors. We carried out a genetic analysis of three of the viruses, RPL25, RPL28, and RPL30. While RPL25 and RPL28 were shown to contain the erbB oncogene, RPL30 appeared to contain a novel protein tyrosine kinase oncogene. This gene, v-ryk, was cloned and sequenced. The v-ryk oncogene contains a 1.39-kb nonretroviral sequence that includes a tyrosine kinase domain which was inserted into the viral envelope protein gp37-coding region and fused in frame with upstream gp37 to generate a P69gp37-ryk fusion oncoprotein. Unlike that of other acutely transforming retroviruses, transduction of the v-ryk gene into RPL30 did not result in deletion of viral sequences. Sequence analysis suggested that v-Ryk is more homologous to receptor-type tyrosine kinases than to nonreceptor-type kinases. By reconstitution of a virus from its cDNA, the v-ryk oncogene has been shown to be fully responsible for the transforming activity of the RPL30 virus. Antibodies specific to v-Ryk immunoprecipitated the v-Ryk oncoprotein from cells transformed by the RPL30 virus. The v-Ryk protein was shown to be first synthesized as a 150-kDa precursor and then cleaved into the mature 69-kDa gp37-Ryk fusion protein, both parts of which were found to be localized to the membrane fraction. As expected from the sequence of v-Ryk, immunoprecipitates of v-Ryk from RPL30-transformed cells were found to display a protein tyrosine kinase activity in vitro, and the levels of tyrosine-phosphorylated proteins are elevated in v-ryk-transformed cells.

Amino Acid Sequence↗