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Intracellular targeting of pp60src expression: localization of v-src to adhesion plaques is sufficient to transform
1Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Abstract:
To define the effects of pp60v-src activity at different intracellular sites, we have constructed chimeric molecules which target the pp60v-src kinase to specific intracellular locations. pp60v-src was targeted to the nucleus by insertion of the SV40 large T antigen nuclear localization signal. Nuclear pp60v-src was active as a tyrosine kinase and phosphorylated nuclear proteins at tyrosine. However, cells expressing the nuclear pp60v-src were phenotypically normal by a number of criteria, and nuclear src kinase did not induce the expression of an mRNA (CEF-4) whose induction is characteristic of transformation by wild-type v-src. pp60v-src was targeted to perinuclear membranes by fusion to rat growth hormone and vesicular stomatitis G protein sequences. Cells expressing this chimeric molecule were phenotypically normal by most criteria. However the perinuclear src protein did induce elevated levels of CEF-4 mRNA, indicating that the v-src kinase expressed at this site induces partial transformation. The v-src and activated c-src kinases were targeted to adhesion plaques by fusion to the talin-binding sequence of vinculin. Cells expressing these fusion proteins were transformed by morphological, physiological and biochemical criteria, although the foci induced by these viruses were distinct from those induced by wild-type v-src. A chimeric protein which targeted c-src to adhesion plaques was not transforming. Thus targeting pp60src to adhesion plaques, although not sufficient to activate the transforming capacity of c-src, is sufficient to allow transformation by v-src.
Insights
Targeting the pp60src kinase to different cellular locations reveals its role in cell transformation. Localization to adhesion plaques is sufficient for v-src transformation, but not for c-src transformation.
Area of Science:
- Cell Biology
- Molecular Biology
- Oncology
Background:
- The pp60v-src kinase is a proto-oncogene implicated in cell transformation.
- Understanding the intracellular localization and function of pp60v-src is crucial for deciphering its oncogenic potential.
Purpose of the Study:
- To investigate the effects of pp60v-src kinase activity at specific intracellular sites.
- To determine the role of intracellular localization in v-src-mediated cell transformation.
Main Methods:
- Construction of chimeric molecules to target pp60v-src to the nucleus, perinuclear membranes, and adhesion plaques.
- Analysis of cellular phenotype, mRNA expression (CEF-4), and tyrosine kinase activity in cells expressing targeted pp60v-src constructs.
- Comparison with wild-type v-src and activated c-src kinase localization and transforming activity.
Main Results:
- Nuclear pp60v-src exhibited tyrosine kinase activity but did not induce transformation or CEF-4 mRNA expression.
- Perinuclear pp60v-src induced partial transformation, evidenced by elevated CEF-4 mRNA levels.
- Targeting v-src and activated c-src to adhesion plaques resulted in transformation, though distinct from wild-type v-src.
- Targeting c-src to adhesion plaques alone was insufficient for transformation, while v-src targeting to adhesion plaques was sufficient for transformation.
Conclusions:
- The intracellular site of pp60v-src activity significantly influences its transforming potential.
- Adhesion plaques are critical sites for v-src-mediated cell transformation.
- While targeting to adhesion plaques is sufficient for v-src transformation, it is not sufficient for c-src transformation, highlighting differences in their oncogenic mechanisms.