Molecular events leading to fusiform morphological transformation by partial src deletion mutant of rous sarcoma virus

Iwashita, Shintaro; Kitamura, Naomi; Yoshida, Mitsuaki

doi:10.1016/0042-6822(83)90213-1

Cited by 31 publications

(13 citation statements)

References 20 publications

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“…The requirement for low-affinity P-Tyr interactions for transformation of rat cells stands in remarkable contrast to chicken cells, which are transformed (albeit to a fusiform morphology) even in the complete absence of an SH2 domain (e.g., d15; Iswashita et al, 1983). In fact, most host-dependent alleles induce fusiform morphological transformation in chicken cells (DeClue and Martin, 1989;Verderame et al, 1989;Hirai and Varmus, 1990b;Verderame and Varmus, 1994).…”

Section: Discussionmentioning

confidence: 99%

pp60v-src transformation of rat cells but not chicken cells strongly correlates with low-affinity phosphopeptide binding by the SH2 domain.

Verderame

1997

MBoC

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Substrates critical for transformation by pp6Ov-src remain unknown, as does the precise role of the src homology 2 (SH2) domain in this process. To continue exploring the role of the SH2 domain in pp60vsrc-mediated transformation, site-directed mutagenesis was used to create mutant v-src alleles predicted to encode proteins with overall structural integrity intact but with reduced ability to bind phosphotyrosine-containing peptides. Arginine-175, which makes critical contacts in the phosphotyrosine-binding pocket, was mutated to lysine or alanine. Unexpectedly, both mutations created v-src alleles that transform chicken cells with wild-type (wt) efficiency and are reduced for transformation of rat cells; these alleles are host dependent for transformation. Additionally, these alleles resulted in a round morphological transformation of chicken cells, unlike 12 of the 13 known host-dependent src SH2 mutations that result in a fusiform morphology. Analysis of phosphopeptide binding by the mutant SH2 domains reveal that the in vitro ability to bind phosphopeptides known to have a high affinity for wt src SH2 correlates with wt (round) morphological transformation in chicken cells and the in vitro ability to bind phosphopeptides known to have a low affinity for wt src SH2 correlates with rat cell transformation. These results suggest that the search for critical substrates in rat cells should be among proteins that interact with pp6ov-src with low affinity.

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Section: Discussionmentioning

confidence: 99%

pp60v-src transformation of rat cells but not chicken cells strongly correlates with low-affinity phosphopeptide binding by the SH2 domain.

Verderame

1997

MBoC

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“…However, this activity has not been linked unequivocally to transformation, and the physiological action of these proteins remains in doubt. We now have found that immunoprecipitated p68v-s also is associated with phosphatidylinositol (PtdIns) kinase (22,23), tyrosine kinase activity shows little substrate specificity in vitro, and the phosphorylation stoichiometries observed are often too low to be of obvious regulatory significance (21). The possibility that the physiological substrate of the oncogene kinases might not be tyrosine was raised by recent observations that p6fV-src can phosphorylate glycerol (24,25).…”

mentioning

confidence: 99%

“…It has been widely assumed that this kinase activity is directly related to transformation, and a great deal of effort has been expended to identify the substrates of the oncogene kinases (19)(20)(21). However, expression of the transformed phenotype does not correlate well with phosphorylation of putative substrates (22,23), tyrosine kinase activity shows little substrate specificity in vitro, and the phosphorylation stoichiometries observed are often too low to be of obvious regulatory significance (21). The possibility that the physiological substrate of the oncogene kinases might not be tyrosine was raised by recent observations that p6fV-src can phosphorylate glycerol (24,25).…”

mentioning

confidence: 99%

Transforming protein of avian sarcoma virus UR2 is associated with phosphatidylinositol kinase activity: possible role in tumorigenesis.

Macara¹,

Marinetti²,

Balduzzi³

1984

Proc. Natl. Acad. Sci. U.S.A.

244

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The transforming protein of avian sarcoma virus UR2, p68v-s, has an associated tyrosine-specific protein kinase activity similar to that of p6O-SrC and several other oncogene products. However, this activity has not been linked unequivocally to transformation, and the physiological action of these proteins remains in doubt. We now have found that immunoprecipitated p68v-s also is associated with phosphatidylinositol (PtdIns) kinase (22,23), tyrosine kinase activity shows little substrate specificity in vitro, and the phosphorylation stoichiometries observed are often too low to be of obvious regulatory significance (21). The possibility that the physiological substrate of the oncogene kinases might not be tyrosine was raised by recent observations that p6fV-src can phosphorylate glycerol (24,25). Because of (i) the similarities between the activity of oncogene kinases and that of growth factor receptors (16-18) and (ii) the implication of P-inositide turnover in cell proliferation, it occurred to us to test the phosphorylating activity of one of these kinases, p68vr's, towards phosphatidylinositol (PtdIns). The protein p68v-ros is the oncogene product of avian sarcoma virus UR2 (26, 27); although unrelated to the p60vsrc of Rous sarcoma virus (28), it will phosphorylate itself and exogenous protein substrates on tyrosine residues (27). We found that immunoprecipitated p68v-ros is also capable of phosphorylating PtdIns to Ptdlns 4-phosphate (PtdIns4P). Viruses and Antisera. The origins of UR2 and some of the properties of its transforming protein have been described (26,27). A temperature-sensitive (ts) mutant, UR2-R0200, has been isolated by one of us (P.B.). This mutant produces full expression of the transformed phenotype at 37°C but only partial expression at 42°C. Anti-gag antiserum was obtained from E. J. Smith.Cell Cultures and Conditions. Chicken embryo fibroblast cultures were prepared and maintained as described (26). Infected cells were passaged 2 or 3 times until extensive morphological conversion was apparent.Immunoprecipitation of p68vros. Cell lysates were prepared with either RIPA buffer or a buffer containing 1% NP-40 (or Triton X-100), 1% aprotinin, 10 mM Tris, 100 mM NaCl, and 1 mM EDTA (pH 7.4). Plates (10 cm) containing about 107 cells were washed three times in Tris/glucose, and

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“…However, other workers observed no correlation between the morphology of transformed cells and phosphorylation in vinculin (23,30 tion in the amounts of both vinculin and fibronectin than does the wild-type RSV. In this study, we found no direct correlation between vinculin content and cellular morphology (data not shown).…”

mentioning

confidence: 91%

Increased phosphorylation of tyrosine in vinculin does not occur upon transformation by some avian sarcoma viruses.

Antler¹,

Greenberg²,

Edelman³

et al. 1985

Mol. Cell. Biol.

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The level of phosphotyrosine in vinculin was determined in chicken embryo fibroblasts transformed by various strains of avian sarcoma virus. As previously reported (Sefton et al., Cell 24:165-174, 1981), vinculin was phosphorylated at tyrosine residues in most cultures examined, but the level varied greatly and no detectable change was found in cultures infected with Fujinami sarcoma virus or UR2 sarcoma virus.Regardless of the level of vinculin phosphorylation, the number of organized microfilament bundles was found to be decreased in all transformed cells. These results strongly suggest that tyrosine phosphorylation of vinculin is not an obligatory step in cell transformation by this class of oncogenes, nor is it correlated with the associated cytoskeletal disarray.The transforming protein of Rous sarcoma virus (RSV), pp6Osrc, is a phosphoprotein of Mr 60,000 with a protein kinase activity that phosphorylates tyrosine (12). A similar protein kinase activity associated with the oncogene products of other avian retroviruses has been found, and there is evidence that tyrosine phosphorylation plays an important role in the process of transformation by these viruses (3). Primary substrates of the transforming proteins have been sought on the basis of enhanced phosphotyrosine content. Vinculin is a 130,000-dalton cytoskeletal protein which has been shown to contain a significantly greater amount of phosphotyrosine in cells transformed by RSV, Y73 sarcoma virus, or Abelson leukemia virus (32). The increase in phosphotyrosine content in vinculin is thermolabile in chick cells infected with an RSV mutant which is temperature sensitive in transformation (33). Vinculin has therefore been considered a possible substrate of the transforming proteins of these viruses in vivo.Vinculin is associated with focal adhesion plaques, which are regions of close contact and anchorage between cell and substratum and are the sites of actin-membrane interaction at the ends of microfilament bundles (5, 18). In RSV-transformed cells, focal contacts are fewer and smaller, the cytoskeleton becomes disordered, and cells round up and are generally less adhesive (7). pp6Osrc has been shown to colocalize with vinculin in transformed cells (27,29 Fujinami sarcoma virus (FSV); Schmidt Ruppin RSV, subgroup A (SR-A); Bryan high-titer strain of RSV (BH-RSV); and a fusiform morphological mutant of BH-RSV (BH-RSVf) isolated in this laboratory. These viruses are described in Table 1, which lists their transforming genes, their transforming proteins, and the transformed morphology which they induce. Criteria for transformation included morphology, refractility, and loss of anchorage dependence. Each plate containing log-phase cultures was labeled with 1.5 mCi of carrier-free 32p, (Amersham Corp., Arlington Heights, Ill.) per ml. Incubation was continued for 18 h, after which cell extracts were made as previously described (13), except for the use of phosphate-buffered RIPA (13) as lysis buffer. In some experiments, duplicate cultures were lysed in 1 ml ...

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Molecular events leading to fusiform morphological transformation by partial src deletion mutant of rous sarcoma virus

Cited by 31 publications

References 20 publications

pp60v-src transformation of rat cells but not chicken cells strongly correlates with low-affinity phosphopeptide binding by the SH2 domain.

pp60v-src transformation of rat cells but not chicken cells strongly correlates with low-affinity phosphopeptide binding by the SH2 domain.

Transforming protein of avian sarcoma virus UR2 is associated with phosphatidylinositol kinase activity: possible role in tumorigenesis.

Increased phosphorylation of tyrosine in vinculin does not occur upon transformation by some avian sarcoma viruses.

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