Phosphorylation of p125FAK and paxillin focal adhesion proteins in src-transformed cells with different metastatic capacity

AbstractHamster fibroblasts transformed by Rous sarcoma virus (RSV) display different metastatic potentials that are associated with specific structural features of the v-src oncoprotein. This diverse metastatic activity could be due to various tyrosine phosphorylation levels of specific src protein substrates. To check this hypothesis, phosphorylation of the FAK and paxillin proteins, involved in signal transduction pathways and known as src protein substrates, was tested. It was shown that FAK and paxillin are hyperphosphorylated in the high metastatic cell lines as compared with the phosphotyrosine level of these proteins found in the low metastatic cell lines. In addition, our data confirm that v-src protein plays a direct role in paxillin phosphorylation

Two Novel Variants of the v-srcOncogene Isolated from Low and High Metastatic RSV-Transformed Hamster Cells

AbstractFour different transformed cell lines were isolated as a result of independent infection of primary hamster fibroblasts by Rous sarcoma virus (RSV SR-D stocks). These lines differ by the level of their spontaneous metastatic activity: HET-SR-1, HET-SR-8, and HET-SR-10 cell lines induced 70–200 metastatic nodules in the lung and/or lymph nodes of inoculated animals (high metastatic lines, HM). Metastatic activity was not identified after injection of HET-SR cells (low metastatic line, LM). All cell lines contained one copy of integrated and expressed intact RSV provirus. The difference in the amount of v-srcprotein in cell lines was not correlated with their metastatic potentialin vivo.Complete v-srcHM and v-srcLM genes were cloned from corresponding gene libraries and sequenced. In the unique region of both v-srcisoforms a GC-rich insert of 60 nucleotides (20 a.a.) was found. The presence of this insert explains the unusual apparent molecular weight of protein encoded by v-srcHM and v-srcLM: 62 kDa. Both genes had 10 identical amino acid changes when compared to the known RSV SR-D v-srcsequence. v-srcHM and v-srcLM differ by several amino acid changes. Most of them are localized in the unique domain and the extreme carboxy-terminal region of the oncoprotein. Both v-srcvariants and chimeric v-srcwith mutually substituted parts were subcloned in a retroviral vector and introduced into avian neuroretina cells. Significant differences in the morphology of transformed neuroretina cells were associated with the mutations in the carboxy-terminal region of the v-srconcogene. Low metastatic HET-SR cells transfected with v-srcHM and the chimeric gene v-src-LH remarkably increased their metastatic potential. In contrast, this effect was not observed when the same cells were transfected with v-srcLM and the chimeric v-srcHL gene. Specific changes in the distribution of fibronectin matrix typical for high metastatic cells were found in the lines transfected with v-srcHM