v-Src

Abstract: The recently described focal adhesion kinase (FAK) has been implicated in signal transduction pathways initiated by cell adhesion receptor integrins and by neuropeptide growth factors To examine the mechanisms by which FAK relays signals from the membrane to the cell interior, we carried out a series of experiments to detect potential FAK interactions with proteins containing Src homology 2 (SH2) domains that are important intracellular signaling molecules Using v-Src-transformed NIH3T3 cells, we showed that FAK was present in the immune-complex precipitated by anti-Src antibody, suggesting potential interaction of FAK with v-Src in vivo We also showed potentially direct interaction of FAK with v-Src in vivo using the yeast two-hybrid system Using recombinant FAK expressed in insect cells and bacterial fusion proteins containing Src SH2 domains, we showed direct binding of FAK to the Src SH2 domain but not to the SH3 domain in vitro A kinase-defective mutant of FAK, which is not autophosphorylated, did not interact with the Src SH2 domain under the same conditions, suggesting the involvement of the FAK autophosphorylation sites Treatment of FAK with a protein-tyrosine phosphatase decreased its binding to the Src SH2 domain, whereas autophosphorylation in vitro increased its binding These results confirm the importance of FAK autophosphorylation sites in its interaction with SH2 domain-containing proteins Taken together, these results suggest that FAK may mediate signal transduction events initiated on the cell surface by kinase activation and autophosphorylation that result in its binding to other key intracellular signaling molecules

Abstract: Chicken embryo fibroblasts (CEF) infected with a temperature-sensitive Rous sarcoma virus (RSV) mutant, tsNY72-4, express a set of pp60v-src-induced RNAs soon after shift to the permissive temperature. By subtractive and differential screening, we have cloned 12 of these sequences, 2 of which were c-fos and krox-24. Serum induced all the v-src-inducible genes tested, suggesting that these genes serve roles in normal cell division and are not specific to transformation per se. Significantly, however, v-src produced prolonged, and in some cases kinetically complex, patterns of induction compared to serum. For most of the clones, phorbol 12-tetradecanoate 13-acetate (TPA) induced mRNAs with kinetics similar to that of serum. However, one clone (CEF-4) was expressed in a biphasic manner. Another (CEF-10) was repressed by TPA at 1 hr, after which this mRNA was permanently induced. The pattern of repression-induction of CEF-10 mRNA is the inverse of protein kinase C (PKC) activity in the cell, suggesting that PKC actively represses this gene. In vivo expression of CEF-10 mRNA is restricted predominantly to the lung. A full-length CEF-10 cDNA encodes a 41-kDa protein that has an amino-terminal signal peptide for secretion, contains a markedly high number of cysteine residues, and shows no sequence similarity to known proteins.

Abstract: PC 12 rat phaeochromocytoma cells1,2 are a model system that can be used to study both neuronal differentiation and the mechanism of action of nerve growth factor (NGF)3. PC12 cells respond to NGF protein by shifting from a chromaffin-cell-like phenotype to a neurite-bearing sympathetic neurone-like phenotype2,4,5. Here we present data on the effect of infection of PC 12 cells with retroviruses carrying the src oncogene of Rous sarcoma virus6. Previous studies have demonstrated that the expression of src severely affects the synthesis and accumulation of differentiated cell products in a variety of cell types7–9. We show that in the PC 12 cell system, expression of v-src appears to have an inductive effect on differentiation that resembles the action of a ‘physiological’ growth factor.

Abstract: Chicken embryo fibroblasts (CEF) infected with a temperature-sensitive Rous sarcoma virus (RSV) mu- tant, tsNY72-4, express a set of pp6vWc-induced RNAs soon after shift to the permissive temperature. By subtractive and differential screening, we have cloned 12 of these sequences, 2 of which were c-fos and krox-24. Serum induced all the v-src-inducible genes tested, suggesting that these genes serve roles in normal cell division and are not specific to transfor- mation per se. Significantly, however, v-src produced pro- longed, and in some cases kinetically complex, patterns of Induction compared to serum. For most of the clones, phorbol 12-tetradecanoate 13-acetate (TPA) induced mRNAs with ki- netics similar to that of serum. However, one clone (CEF-4) was expressed in a biphasic manner. Another (CEF-10) was re- pressed by TPA at 1 hr, after which this mRNA was perma- nently induced. The pattern of repression-induction of CEF-10 mRNA is the inverse of protein kinase C (PKC) activity in the cell, suggesting that PKC actively represses this gene. In vivo expression of CEF-10 mRNA is restricted predominantly to the lung. A full-length CEF-10 cDNA encodes a 41-kDa protein that has an amino-terminal signal peptide for secretion, con- tains a markedly high number of cysteine residues, and shows no sequence similarity to known proteins.