Pp60src In Cells Research Articles

Autophosphorylation of the focal adhesion kinase, pp125FAK, directs SH2-dependent binding of pp60src.

The phosphorylation of protein tyrosine kinases (PTKs) on tyrosine residues is a critical regulatory event that modulates catalytic activity and triggers the physical association of PTKs with Src homology 2 (SH2)-containing proteins. The integrin-linked focal adhesion kinase, pp125FAK, exhibits extracellular matrix-dependent phosphorylation on tyrosine and physically associates with two nonreceptor PTKs, pp60src and pp59fyn, via their SH2 domains. Herein, we identify Tyr-397 as the major site of tyrosine phosphorylation on pp125FAK both in vivo and in vitro. Tyrosine 397 is located at the juncture of the N-terminal and catalytic domains, a novel site for PTK autophosphorylation. Mutation of Tyr-397 to a nonphosphorylatable residue dramatically impairs the phosphorylation of pp125FAK on tyrosine in vivo and in vitro. The mutation of Tyr-397 to Phe also inhibits the formation of stable complexes with pp60src in cells expressing Src and FAK397F, suggesting that autophosphorylation of pp125FAK may regulate the association of pp125FAK with Src family kinases in vivo. The identification of Tyr-397 as a major site for FAK autophosphorylation provides one of the first examples of a cellular protein containing a high-affinity binding site for a Src family kinase SH2 domain. This finding has implications for models describing the mechanisms of action of pp125FAK, the regulation of the Src family of PTKs, and signal transduction through the integrins.

Autophosphorylation of the focal adhesion kinase, pp125FAK, directs SH2-dependent binding of pp60src.

The phosphorylation of protein tyrosine kinases (PTKs) on tyrosine residues is a critical regulatory event that modulates catalytic activity and triggers the physical association of PTKs with Src homology 2 (SH2)-containing proteins. The integrin-linked focal adhesion kinase, pp125FAK, exhibits extracellular matrix-dependent phosphorylation on tyrosine and physically associates with two nonreceptor PTKs, pp60src and pp59fyn, via their SH2 domains. Herein, we identify Tyr-397 as the major site of tyrosine phosphorylation on pp125FAK both in vivo and in vitro. Tyrosine 397 is located at the juncture of the N-terminal and catalytic domains, a novel site for PTK autophosphorylation. Mutation of Tyr-397 to a nonphosphorylatable residue dramatically impairs the phosphorylation of pp125FAK on tyrosine in vivo and in vitro. The mutation of Tyr-397 to Phe also inhibits the formation of stable complexes with pp60src in cells expressing Src and FAK397F, suggesting that autophosphorylation of pp125FAK may regulate the association of pp125FAK with Src family kinases in vivo. The identification of Tyr-397 as a major site for FAK autophosphorylation provides one of the first examples of a cellular protein containing a high-affinity binding site for a Src family kinase SH2 domain. This finding has implications for models describing the mechanisms of action of pp125FAK, the regulation of the Src family of PTKs, and signal transduction through the integrins.

Novel localization of pp60 src in rous sarcoma virus-transformed rat and goat cells and in chicken cells transformed by viruses rescued from these mammalian cells

We have investigated by indirect immunofluorescence and subcellular fractionation the intracellular location of pp60 src in RSV-transformed mammalian cells and in CEF cells transformed by virus rescued from these cells. Two independently derived cell lines were examined: RR1022 cells isolated from an in vivo sarcoma induced in an Amsterdam rat by infection with SR-RSV-D; and Pcl, cells isolated from a soft agar colony of normal goat skin fibroblasts transformed in vitro by SR-RSV-D. Transforming viruses (RSV-RR and RSV-Pcl) were rescued from RR1022 and Pcl cells by fusion with CEF cells. Immunofluorescence microscopy showed association of pp60 src with the nuclear envelope and the juxtanuclear reticular membranes in the transformed mammalian cells and in CEF cells transformed by the rescued viruses, in contrast to the plasma membrane localization of pp60 src seen in SR-RSV-transformed CEF cells. Results of subcellular fractionation by differential centrifugation and fractionation of particulate fractions by equilibrium centrifugation in discontinuous sucrose gradients were in agreement with the differences in pp60 src distribution observed by immunofluorescence microscopy. Although the mammalian cell lines were independently derived, pp60 srcs isolated from RR1022 and Pcl cells both lacked amino-terminal 21- and 18-kilodalton [ 35S]methionine S. aureus V8 protease peptides found in SR-RSV-D pp60 src. Proteolytic peptides identical to those of pp60 src from the mammalian cells were obtained from pp60 src proteins isolated from rescued virus-transformed CEF cells, suggesting that the alteration in the amino-terminal half of the src protein represents a stable change, and that an alteration in the primary structure of pp60 src is responsible for the altered intracellular membrane localization of pp60 src in these cells.

A cellular protein that associates with the transforming protein of Rous sarcoma virus is also a heat-shock protein.

A single viral protein (pp60src) mediates neoplastic transformation of cells infected with Rous sarcoma virus. Immunoprecipitation of pp60src has revealed two cellular proteins (Mr 50,000 and 89,000) that appear to associate with pp60src in a specific manner. Neither of the cellular proteins has been well characterized, but it is thought that both may participate in the function of pp60src. Treatment of avian cells with unphysiological temperature or certain chemical agents amplifies the production of several proteins in the manner of the "heat shock" response earlier described for Drosophila. We report here that one of these proteins, with a molecular weight of 89,000 is identical to the 89-kilodalton protein found associated with pp60src. The 89-kilodalton protein is a major constituent of both uninfected and infected cells, even in the absence of inducing agents, but only a small fraction of this protein appears to associate with pp60src in cells transformed by Rous sarcoma virus. The complex containing pp60src and the 89-kilodalton protein can be precipitated by an immune reaction involving pp60src alone. The complexed form of the 89-kilodalton protein did not react directly with antibodies but regained its reactivity subsequent to release from the complex. We conclude that the 89-kilodalton protein is bound to pp60src in a relatively stable complex. We suggest that the 89-kilodalton protein may have overlapping roles in viral oncogenesis and the heat shock response, and that evidence on the function of the protein in either setting may illuminate its function in the other. In addition, it may prove profitable to search for other overlaps between the cellular response to heat shock and the neoplastic transformation of cells by pp60src.