Search

Current anti-epidermal growth factor receptor (EGFR) therapy for oral cancer does not provide satisfactory efficacy due to drug resistance or reduced EGFR level. As an alternative candidate target for therapy, here we identified an oncogene, ROS1, as an important driver for oral squamous cell carcinoma (OSCC) metastasis. Among tumors from 188 oral cancer patients, upregulated ROS1 expression strongly correlated with metastasis to lung and lymph nodes. Mechanistic studies uncover that the activated ROS1 results from highly expressed ROS1 gene instead of gene rearrangement, a phenomenon distinct from other cancers. Our data further reveal a novel mechanism that reduced histone methyltransferase EZH2 leads to a lower trimethylation of histone H3 lysine 27 suppressive modification, relaxes chromatin, and promotes the accessibility of the transcription factor STAT1 to the enhancer and the intron regions of ROS1 target genes, CXCL1 and GLI1, for upregulating their expressions. Down-regulation of ROS1 in highly invasive OSCC cells, nevertheless, reduces cell proliferation and inhibits metastasis to lung in the tail-vein injection and the oral cavity xenograft models. Our findings highlight ROS1 as a candidate biomarker and therapeutic target for OSCC. Finally, we demonstrate that co-targeting of ROS1 and EGFR could potentially offer an effective oral cancer therapy.

Vav3 is a member of the Vav family of guanine nucleotide exchange factors (GEFs) for the Rho family GTPases. Deleting the N-terminal calponin homology (CH) domain to generate Vav3-(5-10) or deleting both the CH and the acidic domain to generate Vav3-(6-10) results in activating the transforming potential of Vav3. Expression of either the full-length Vav3 or its truncation mutants led to activation of phosphatidylinositol 3-kinase (PI3K), mitogen-activated protein kinase (MAPK), focal adhesion kinase (FAK), and Stat3. We investigated the requirement of these signaling molecules for Vav3-induced focus formation and found that PI3K and its downstream signaling molecules, Akt and p70 S6 kinase, are required, albeit to varying degrees. Inhibition of PI3K had a more dramatic effect than inhibition of MAPK on Vav3-(6-10)-induced focus formation. Activated PI3K enhanced the focus-forming activity of Vav3-(6-10). Wild type FAK but not Y397F mutant FAK enhanced Vav3-(6-10)-induced focus formation. Dominant negative (dn) mutant of Stat3 resulted in a 60% inhibition of the focus-forming activity of Vav3-(6-10). Moreover, Rac1, RhoA, and to a lesser extent, Cdc42, are important for Vav3-(6-10)-induced focus formation. Constitutively activated (ca) Rac synergizes with Vav3-(6-10) in focus formation. This synergy requires signaling via Rho-associated kinase (ROK) and p21-activated kinase (PAK), downstream effectors of Rac. Consistently, a ca PAK mutant enhanced, whereas a dn PAK mutant inhibited the focus-forming ability of Vav3-(6-10). Despite having potent focus-forming ability, Vav3-(6-10) has very weak colony-forming ability. This colony-forming ability of Vav3-(6-10) can be enhanced dramatically by co-expressing an activated PI3K and to some extent by co-expressing an activated PAK mutant or c-Myc. Interestingly, inhibition of PI3K and MAPK had no effect on the ability of either wild type or Vav3-(6-10) to induce cytoskeletal changes including formation of lamellipodia and filopodia in NIH 3T3 cells. Over expression of Vav3 or Vav3-(6-10) resulted in an enhancement of cell motility. This enhancement was dependent on PI3K, Rac1, and Cdc42 but not on Rho. Overall, our results show that signaling pathways of PI3K, MAPK, and Rho family GTPases are differentially required for Vav3-induced focus formation, colony formation, morphological changes, and cell motility.

GH is required for normal postnatal growth and metabolism. GH stimulates postnatal growth through induction of IGF-I gene expression. Although the liver is the major site of GH-regulated IGF-I, recent evidence indicates that GH-regulated IGF-I expression in nonhepatic tissues is sufficient for normal postnatal growth. One potentially important nonhepatic site of GH-stimulated IGF-I expression is skeletal muscle, as injection of GH into animals leads to increased IGF-I mRNA in this tissue. Nevertheless, direct effects of GH in skeletal muscle cells in culture have not been reported. We therefore tested the C2C12 myogenic cell line for its response to GH and demonstrate that C2C12 skeletal muscle cells rapidly respond to physiological levels of GH with increased tyrosine phosphorylation of the GH receptor, Janus kinase 2, signal transducer and activator of transcription-5a and -5b, insulin receptor substrate-1, and activation of MAPKs/ERKs and protein kinase B/Akt. In these cells, GH stimulates the expression of IGF-I and two members of the suppressors of cytokine signaling family, cytokine-inducible SH2-containing protein and suppressor of cytokine signaling-2. Treatment of C2C12 myoblasts with either the MAPK kinase inhibitor PD98059 or the PI3K inhibitor wortmannin results in higher levels of GH-induced IGF-I and suppressor of cytokine signaling-2 mRNA expression, suggesting that activation of MAPK and PI3K pathways has an inhibitory role in IGF-I and suppressor of cytokine signaling-2 gene regulation. Therefore, C2C12 cells provide the first in vitro model system to study various aspects of GH action in skeletal muscle.

The precise regulation of both the magnitude and the duration of Janus kinase (JAK) catalytic activity is essential for the cytokine orchestration of many biological processes, and the dysregulation of JAK activity has pathological implications. Immunosuppressive disease states, such as X-linked severe combined immunodeficiency, arise from inappropriate JAK inhibition. In contrast, a limited number of cancers, primarily leukemias, result from constitutive or enhanced activation of JAK activity. JAKs are no longer implicated only in classic cytokine receptor-mediated signaling pathways, but are now also known to integrate indirectly into other receptor-mediated signal transduction processes. Therefore, an increasing number of therapeutic applications exist for biological-response modifiers that can restore aberrant JAK activity to normal levels. Exciting breakthroughs in both physiological and pharmacological methods of selective inhibition of cytokine-JAK-signal transducers and activators of transcription pathways have recently emerged in the form of suppressors of cytokine signaling (also known as cytokine-inducible SH2 protein, JAK-binding protein, or STAT-induced STAT inhibitor) proteins and novel dimethoxyquinazoline derivatives, respectively. The basis of these and other mechanisms of negative regulation of JAK activity, including the suppression of jak expression levels caused by tumor- or pathogen-derived agents, the complex interactions of JAKs with phosphatases, and the redox regulation of JAK catalytic activity, is the focus of this review.

A recently reported new member of the Vav family proteins, Vav3 has been identified as a Ros receptor protein tyrosine kinase (RPTK) interacting protein by yeast two-hybrid screening. Northern analysis shows that Vav3 has a broad tissue expression profile that is distinct from those of Vav and Vav2. Two species of Vav3 transcripts, 3.4 and 5.4 kb, were detected with a differential expression pattern in various tissues. Transient expression of Vav in 293T and NIH 3T3 cells demonstrated that ligand stimulation of several RPTKs (epidermal growth factor receptor [EGFR], Ros, insulin receptor [IR], and insulin-like growth factor I receptor [IGFR]) led to tyrosine phosphorylation of Vav3 and its association with the receptors as well as their downstream signaling molecules, including Shc, Grb2, phospholipase C (PLC-gamma), and phosphatidylinositol 3 kinase. In vitro binding assays using glutathione S-transferase-fusion polypeptides containing the GTPase-binding domains of Rok-alpha, Pak, or Ack revealed that overexpression of Vav3 in NIH 3T3 cells resulted in the activation of Rac-1 and Cdc42 whereas a deletion mutant lacking the N-terminal calponin homology and acidic region domains activated RhoA and Rac-1 but lost the ability to activate Cdc42. Vav3 induced marked membrane ruffles and microspikes in NIH 3T3 cells, while the N-terminal truncation mutants of Vav3 significantly enhanced membrane ruffle formation but had a reduced ability to induce microspikes. Activation of IR further enhanced the ability of Vav3 to induce membrane ruffles, but IGFR activation specifically promoted Vav3-mediated microspike formation. N-terminal truncation of Vav3 activated its transforming potential, as measured by focus-formation assays. We conclude that Vav3 mediates RPTK signaling and regulates GTPase activity, its native and mutant forms are able to modulate cell morphology, and it has the potential to induce cell transformation.

Recent evidence indicates that STAT proteins can be activated by a variety of receptor and non-receptor protein-tyrosine kinases. Unlike cytokine-induced activation of STATs, where JAKs are known to play a pivotal role in phosphorylating STATs, the mechanism for receptor protein-tyrosine kinase-mediated activation of STATs remains elusive. In this study, we investigated the activation of STAT proteins by the insulin-like growth factor I receptor (IGF-IR) in vitro and in vivo and assessed the role of JAKs in the process of activation. We found that STAT3, but not STAT5, was activated in response to IGF-I in 293T cells cotransfected with IGF-IR and STAT expression vectors. Moreover, tyrosine phosphorylation of STAT3, JAK1, and JAK2 was increased upon IGF-I stimulation of endogenous IGF-IR in 293T cells transfected with the respective STAT or JAK expression vector. Supporting the observation in 293T cells, endogenous STAT3 was tyrosine-phosphorylated upon IGF-I stimulation in the muscle cell line C2C12 as well as in various embryonic and adult mouse organs during different stages of development. Dominant-negative JAK1 or JAK2 was able to block the IGF-IR-mediated tyrosine phosphorylation of STAT3 in 293T cells. A newly identified family of proteins called SOCS (suppressor of cytokine signaling), including SOCS1, SOCS2, SOCS3 and CIS, was able to inhibit the IGF-I-induced STAT3 activation as well with varying degrees of potency, in which SOCS1 and SOCS3 appeared to have the higher inhibitory ability. Inhibition of STAT3 activation by SOCS could be overcome by overexpression of native JAK1 and JAK2. We conclude that IGF-I/IGF-IR is able to mediate activation of STAT3 in vitro and in vivo and that JAKs are essential for the process of activation.

Interleukin 6 (IL-6) is a cytokine that regulates not only immune and inflammatory responses but also the growth of some tumors, including prostate carcinomas. IL-6 signals through Janus kinase, signal transducer and activator of transcription 3 (STAT3), and mitogen-activated protein kinase and is also able to induce androgen receptor (AR)-mediated gene activation in prostate cancer, which is an important process in prostate cancer androgen-independent progression. We now show that IL-6-induced AR-mediated gene activation requires the activation of STAT3 by IL-6 in LNCaP prostate cancer cells. In particular, STAT3 associates with AR in an androgen-independent but IL-6-dependent manner. Inhibition of STAT3 rather than mitogen-activated protein kinase results in inhibition of AR-mediated gene activation in response to IL-6. These findings not only identify STAT3 as an important signaling molecule required for IL-6-signaling to induce AR-mediated gene activation in prostate carcinoma cells but also reveal the importance of activated STAT3 in human tumor development and progression.

In an effort to determine the role of protein kinase C-delta (PKC-delta) in cellular transformation mediated by the sis proto-oncogene, we cotransfected expression vectors containing cDNAs that encode for c-sis with an ATP binding mutant of PKC-delta (PKC-delta K376R) or wild type PKC-delta (PKC-delta WT) into NIH3T3 cells. Our results showed that expression of PKC-delta K376R severely impaired Sis-induced focus formation, whereas cotransfection of PKC-delta WT cDNA had no effect on Sis-mediated transformation. Consistent with this result, PKC-delta K376R expression also inhibited PDGF-BB-mediated anchorage-independent colony formation. While cotransfection of a vector containing a dominant negative mutant of ras (N17 ras) cDNA potently inhibited Sis-induced transformation, the expression of PKC-delta K376R did not block transformation mediated by v-H-Ras or v-Raf. In addition, PDGF-BB-induced Raf and mitogen-activated protein kinase activation, which are known to be downstream molecules in the Ras cascade, were not affected by the expression of PKC-delta K376R, indicating that PKC-delta and Ras are segregated in mediating Sis-induced transformation. Interestingly, expression of PKC-delta K376R strongly reduced TPA responsive element (TRE) transactivation induced by PDGF stimulation, suggesting that activation of TRE-containing genes, which may be involved in Sis-mediated transformation, are negatively regulated by expression of PKC-delta K376R.

Two chimeric receptors, ER1 and ER2, were constructed. ER1 contains the extracellular and transmembrane (TM) domains derived from epidermal growth factor receptor and the cytoplasmic domain from c-Ros; ER2 is identical to ER1 except that its TM domain is derived from c-Ros. Both chimeras can be activated by epidermal growth factor and are capable of activating or phosphorylating an array of cellular signaling proteins. Both chimeras promote colony formation in soft agar with about equal efficiency. Surprisingly, ER1 inhibits while ER2 stimulates cell growth on monolayer culture. Cell cycle analysis revealed that all phases, in particular the S and G2/M phases, of the cell cycle in ER1 cells were elongated whereas G1 phase of ER2 cells was shortened threefold. Comparison of signaling pathways mediated by the two chimeras revealed several differences. Several early signaling proteins are activated or phosphorylated to a higher extent in ER1 than in ER2 cells in response to epidermal growth factor. ER1 is less efficiently internalized and remains tyrosine phosphorylated for a longer time than ER2. However, phosphorylation of the 66-kDa She protein, activation of mitogen activated protein kinase, and induction of c-fos and c-jun occur either to a lesser extent or for a shorter time in ER1 cells. Cellular protein phosphorylation patterns are also different in ER1 and ER2 cells. In particular, a 190-kDa Shc-associated protein is tyrosine phosphorylated in ER2 but not in ER1 cells. Our results indicate that the TM domains have a profound effect on the signal transduction and biological activity of those chimeric receptors. The results also imply that sustained stimulation of ER1 due to its retarded internalization apparently triggers an inhibitory response that dominantly counteracts the receptor-mediated mitogenic signals. These two chimeras, expressed at similar levels in the same cell type but having opposite effects on cell growth, provide an ideal system to study the mechanism by which a protein tyrosine kinase inhibits cell growth.

Our previous study has shown that chicken c-ros is specifically expressed in certain epithelial cells of kidney, intestine, lung, bursa, thymus, and testis, and the expression is regulated temporally and spatially. To explore the molecular basis for the regulation of c-ros expression, we have cloned and characterized the chicken c-ros promoter. The most 5' c-ros cDNA was isolated and sequenced. Using the 5' cDNA as a probe, three genomic DNA clones containing the 5' c-ros cDNA sequence were isolated. Primer extension and RNase protection analysis were used to map the transcription initiation site for the c-ros mRNA in kidney and intestine. The sequence of the 1.3-kb region upstream of the initiation site contains TATA and CAAT boxes at 26 and 54 nucleotides, respectively, upstream of the initiation site. In addition, transcription factor binding sites for AP1, AP2, and Oct1 and several direct and inverted repeats are present within 1 kb upstream of the initiation site. The 1.3-kb DNA, when placed upstream of the chloramphenicol acetyltransferase gene, was shown to be functionally active. Serial deletions of this putative c-ros promoter allowed us to define a minimum c-ros promoter and to identify positive and negative regulatory regions. Using two oligonucleotides corresponding to a positive regulatory and potential factor binding region, we have demonstrated, by gel mobility shift experiments, their specific binding to nuclear extracts from kidney, intestine, and thymus. The binding pattern corresponds to the tissue specificity and temporal control of c-ros mRNA expression.

There is a 3-aa insertion in the transmembrane (TM) domain of the p68gag-ros protein-tyrosine kinase encoded by avian sarcoma virus UR2 v-ros as compared with that of the protooncogene c-ros. The effect of this insertion on biological function and biochemical properties of v-Ros protein was investigated by deleting these 3 aa to generate the mutant TM1. This mutant has greatly reduced transforming, mitogenic, and tumorigenic activities despite the fact that the protein-tyrosine kinase activity and cell-surface localization of TM1 protein are unaffected. However, unlike UR2 protein, mutant TM1 protein becomes glycosylated, is differentially phosphorylated, and fails to induce tyrosine phosphorylation of a 88-kDa protein and a major substrate of insulin receptor, insulin receptor substrate 1. The TM1 protein is unable to associate with phosphatidylinositol 3-kinase and fails to promote association of insulin receptor substrate 1 with phosphatidylinositol 3-kinase. By contrast, tyrosine phosphorylation of Shc protein and phospholipase C gamma as well as interaction of Grb2 protein with Shc and SOS protein signaling components are unaltered in the TM1 infected cells. Our results show that the TM-domain sequence of p68gag-ros profoundly affects its function and substrate interaction. The mutant defines a signaling pathway including phosphatidylinositol 3-kinase, insulin receptor substrate 1, and possibly an 88-kDa protein that does not overlap the Ras pathway and is important for full transforming and mitogenic potency of v-ros protein-tyrosine kinase.

Proto-oncogene c-ros codes for a receptor-type protein tyrosine kinase (PTK) sharing high homology with the Drosophila sevenless protein. Recent studies of c-ros expression in mouse by in situ hybridization showed that c-ros was expressed specifically and transiently in the epithelial cells of late embryonic kidney collecting duct and intestine villi. Those investigators suggested that c-ros may play a role in the development of those organelles. In the present study, we have examined the expression profile of c-ros in chicken by RNAase protection and in situ hybridization with riboprobes. Our results showed that in addition to kidney and intestine, low levels of c-ros mRNA could also be detected in lung, testis, thymus and bursa. Expression of c-ros commences during middle to late embryonic stages in those organs and persists into the adult life. In situ hybridization revealed that expression of c-ros was restricted to the epithelial cells of all the tissues examined including kidney, intestine, bursa, thymus and testis. In kidney c-ros was detected in all the epithelial cells of the collecting ducts, in intestine it was detected in the epithelial cells of villi and the underneath crypts. Our finding of c-ros expression in chicken differs from those in mouse in (1) instead of transient expression during the embryonic stage, expression of c-ros in chicken kidney and intestine persists into the adult life and (2) expression of c-ros in renal collecting ducts is not restricted to its growing tips, instead it is expressed in the entire epithelial layer of the ducts. Our results suggest that c-ros may play a role not only in the initial induction events in the organogenesis, but also in the mature function of those organs.

The transforming gene of avian sarcoma virus UR2, v-ros, encodes a receptor-like protein tyrosine kinase and differs from its proto-oncogene, c-ros, in its 5' truncation and fusion to viral gag, a three-amino-acid (aa) insertion in the transmembrane (TM) domain, and changes in the carboxyl region. To explore the basis for activation of the c-ros transforming potential, various c-ros retroviral vectors containing those changes were constructed and studied for their biological and biochemical properties. Ufcros codes for the full-length c-ros protein of 2,311 aa, Uppcros has 1,661-aa internal deletion in the extracellular domain, CCros contains the 3' c-ros cDNA fused 150 aa upstream of the TM domain to the UR2 gag, CVros is the same as CCros except that the 3' region is replaced by that of v-ros, and VCros is the same as CCros except that the 5' region is replaced by that of v-ros. The Ufcros, Uppcros, CCros, and CVros are inactive in transforming chicken embryo fibroblasts, whereas VCros is as potent as UR2 in cell-transforming and tumorigenic activities. Upon passages of CCros and CVros viruses, the additional extracellular sequence in comparison with that of v-ros was delected; concurrently, both viruses (named CC5d and CV5d, respectively) attained moderate transforming activity, albeit significantly lower than that of UR2 or VCros. The native c-ros protein has a very low protein tyrosine kinase activity, whereas the ppcros protein is constitutively activated in kinase activity. The inability of CCros and CVros to transform chicken embryo fibroblasts is consistent with the inefficient membrane association, instability, and low kinase activity of their encoded proteins. The CC5d and CV5d proteins are indistinguishable in kinase activity, membrane association, and stability from the v-ros protein. The reduced transforming potency of CC5d and CV5d proteins can be attributed only to their differential substrate interaction, notably the failure to phosphorylate a 88-kDa protein. We conclude that the 5' rather than the 3' modification of c-ros is essential for its oncogenic activation; the sequence upstream of the TM domain has a negative effect on the transforming activity of CCros and CVros and needs to be deleted to activate their biological activity.

Our earlier study predicted that proto-oncogene c-ros codes for a receptor-like tyrosine protein kinase (TPK). To further understand its protein structure and physiological function, we have analysed its expression in various tissues of chicken and have isolated and sequenced cDNA clones containing the entire coding region of the gene. Confirming our earlier study, we found that kidney is the organ that expresses the highest level of c-ros mRNA, in addition, we found a lower level of expression in gonad, thymus, bursa and brain. A distinctive 8.3 kb c-ros mRNA is present in kidney. No detectable amount of c-ros mRNA was found in the rest of tissues examined. Nucleotide sequence of the c-ros cDNA predicts that it codes for a transmembrane (TM) TPK molecule of 2311 amino acids (aa). The extracellular domain consists of 1873 amino acids which share 20 to 43% homology with that of the Drosophila sevenless protein and TPK domains of the two genes have 58 to 74% homology. The extracellular domain containing 37 potential N-linked glycosylation sites is preceded by a 5' hydrophobic sequence resembling a typical signal peptide. An internal hydrophobic domain of 26 amino acids, the presumed transmembrane domain, is followed by a spacer sequence of 58 amino acids, a TPK domain of 270 amino acids and a carboxyl tail of 84 amino acids. Overall, our result indicates that c-ros codes for a glycosylated transmembrane TPK molecule which shares a remarkable sequence and structural homology with that of Drosophila sevenless protein.

Chicken c-src sequences have been transduced by avian leukosis viruses (ALV) and by partial src-deletion (td) mutants of Rous sarcoma virus in several independent events. Analyses of the recombination junctions in the genomes of src-containing viruses and the c-src DNA have shed light on the mechanism of transduction, which involves at least two steps of recombination. The initial recombination between a viral genome and the 5' region of c-src appears to occur at the DNA level. This step does not require extensive homology and can be mediated by stretches of sequences with only partial homology. The 5' recombination junction can also be formed by splicing between viral and c-src sequences. The second recombination is presumed to occur between the transducing ALV or td viral RNA and the viral-c-src hybrid RNA molecule generated from the initial recombination. This step involving recombination at the 3' ends of those molecules restores the 3' viral sequences essential for replication to the viral-c-src hybrid molecule. High frequency of c-src transduction by partial td mutants suggests that the second recombination is greatly enhanced when there is sequence homology between the transducing virus and the 3' region of c-src. Incorporation of the c-src sequences into an ALV genome results in greatly elevated expression of the gene. However, increased expression of c-src alone is insufficient to activate its transforming potential. Structural changes in c-src are necessary to convert it into a transforming gene. The changes can be as small as single nucleotide changes resulting in single amino aid substitutions at certain positions. Mutations can occur rapidly during viral replication after c-src is incorporated into the viral genome. Therefore, it is most likely that transduction of c-src by ALV is followed by subsequent mutation and selection for the sarcomagenic virus. In the case of transduction by td viruses that retain certain src sequences, joining of these sequences with the transduced c-src apparently is sufficient to activate its transforming potential.

Expression of the c-src locus in skeletal muscle of the chicken was found to be different from other tissues. A 4 kilobases (kb) cellular src (c-src) mRNA, which most likely codes for pp60c-src, is expressed in various chicken tissues examined, and most of its approximately 2.2 kb non-coding sequences are present at the 3' end of the RNA molecule. Whereas the 4 kb RNA is also expressed in embryonic chicken muscle, it disappears shortly before hatching and is replaced by a smaller-sized class of c-src mRNAs of 2.8 to 3.3 kb. The transition of expression from the 4 kb to the 2.8-3.3 kb RNA in skeletal muscle occurs shortly before hatching of the chicken and persists into adulthood. The two classes of the c-src mRNA share sequences both within the 5' coding and the 3' non-coding regions of pp60c-src, however, the 2.8 to 3.3 kb RNA lacks most of the kinase domain, but it contains upstream c-src sequences missing in the 4 kb RNA. The two classes of c-src mRNA most likely are generated by alternative splicing, and the smaller c-src RNA most likely codes for a product other than tyrosine protein kinase.

A cellular src (c-src) cDNA clone was isolated from a chicken embryonic brain cDNA library and characterized by DNA sequence analysis. Comparison with the published sequence of a chicken genomic c-src clone indicated that the brain cDNA clone contained an 18-base-pair insertion located between exons 3 and 4 of the c-src gene. The six amino acids encoded by the insertion caused an alteration in the electrophoretic mobility of the c-src gene product similar to that of the structurally distinct form of the src protein detected in neuronal cultures.

We first describe the characterization of proto-oncogene of v-ros in chicken and human genomes. v-ros sequence of UR2 avian sarcoma virus carries a hydrophobic short stretch upstream of the kinase domain, suggesting that its proto-oncogene encodes for a receptor molecule. Using v-ros DNA as a probe we isolated chicken and human c-ros proto-oncogenes. These c-ros DNAs contained a tyrosine kinase domain, transmembrane domain and a part of an extracellular domain carrying an N glycosylation site which was not acquired by UR2 sarcoma virus. These results strongly suggest that proto-oncogene c-ros encodes for a receptor of cell growth or differentiation factor(s) and that the v-ros sequence is a truncated form of this receptor molecule. Structural alteration and overexpression under the control of viral promoter may be crucial for transforming activity of v-ros gene. We then report another example where a receptor-type oncogene is qualitatively and quantitatively activated. By screening with various onc probes we detected two human glioblastomas which have amplification of structurally altered c-erbB1 (epidermal growth factor (EGF) receptor) gene. c-erbB1 gene in these tumors bears a small deletion within the extracellular domain, and the gene product 140 kd protein shorter than the normal 170 kd EGF receptor was heavily phosphorylated on tyrosine residue even without ligand in in vitro phosphorylation reaction. Thus, these mutated EGF receptors seem to be fixed as a "switch-on" form in signal transduction for cell growth and might be involved in the transformation of glial cells.