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Regulation of c-SRC activity and function by the adapter protein CAS
M R Burnham1, P J Bruce-Staskal, M T Harte
1Department of Microbiology and Cancer Center, Health Sciences Center, University of Virginia, Charlottesville, Virginia 22908, Trinity College, Dublin 2, Ireland. ahb8y@virginia.edu
Abstract:
SRC family kinases play essential roles in a variety of cellular functions, including proliferation, survival, differentiation, and apoptosis. The activities of these kinases are regulated by intramolecular interactions and by heterologous binding partners that modulate the transition between active and inactive structural conformations. p130(CAS) (CAS) binds directly to both the SH2 and SH3 domains of c-SRC and therefore has the potential to structurally alter and activate this kinase. In this report, we demonstrate that overexpression of full-length CAS in COS-1 cells induces c-SRC-dependent tyrosine phosphorylation of multiple endogenous cellular proteins. A carboxy-terminal fragment of CAS (CAS-CT), which contains the c-SRC binding site, was sufficient to induce c-SRC-dependent protein tyrosine kinase activity, as measured by tyrosine phosphorylation of cortactin, paxillin, and, to a lesser extent, focal adhesion kinase. A single amino acid substitution located in the binding site for the SRC SH3 domain of CAS-CT disrupted CAS-CT's interaction with c-SRC and inhibited its ability to induce tyrosine phosphorylation of cortactin and paxillin. Murine C3H10T1/2 fibroblasts that expressed elevated levels of tyrosine phosphorylated CAS and c-SRC-CAS complexes exhibited an enhanced ability to form colonies in soft agar and to proliferate in the absence of serum or growth factors. CAS-CT fully substituted for CAS in mediating growth in soft agar but was less effective in promoting serum-independent growth. These data suggest that CAS plays an important role in regulating specific signaling pathways governing cell growth and/or survival, in part through its ability to interact with and modulate the activity of c-SRC.
Insights
p130(CAS) protein activates SRC kinase, promoting cell growth and survival. This interaction is crucial for cellular functions like proliferation and colony formation, highlighting CAS as a key regulator of SRC signaling pathways.
Area of Science:
- Cellular Biology
- Molecular Biology
- Biochemistry
Background:
- SRC family kinases are crucial regulators of cellular processes such as proliferation, survival, differentiation, and apoptosis.
- Kinase activity is modulated by intramolecular interactions and binding partners, influencing active and inactive structural states.
- p130(CAS) (CAS) directly binds to c-SRC's SH2 and SH3 domains, suggesting a role in structural alteration and activation.
Purpose of the Study:
- To investigate the role of p130(CAS) in modulating c-SRC activity and its downstream effects on cellular functions.
- To determine the specific domains of CAS involved in c-SRC interaction and kinase activation.
- To assess the impact of CAS-mediated SRC activation on cell growth and survival characteristics.
Main Methods:
- Overexpression of full-length CAS and a carboxy-terminal fragment (CAS-CT) in COS-1 cells.
- Assessing c-SRC-dependent tyrosine phosphorylation of cellular proteins (cortactin, paxillin, focal adhesion kinase).
- Utilizing site-directed mutagenesis to disrupt the CAS-CT/c-SRC interaction.
- Evaluating colony formation in soft agar and serum-independent proliferation of murine fibroblasts.
Main Results:
- Overexpression of CAS induced c-SRC-dependent tyrosine phosphorylation of multiple proteins.
- CAS-CT alone was sufficient to induce c-SRC-dependent kinase activity, evidenced by cortactin and paxillin phosphorylation.
- A mutation disrupting CAS-CT binding to c-SRC's SH3 domain inhibited its ability to activate cortactin and paxillin.
- Fibroblasts with elevated tyrosine phosphorylated CAS and c-SRC-CAS complexes showed enhanced soft agar colony formation and serum-independent proliferation.
- CAS-CT mediated soft agar growth but was less effective in promoting serum-independent growth compared to full-length CAS.
Conclusions:
- CAS plays a significant role in regulating cell growth and survival signaling pathways.
- CAS modulates c-SRC activity through direct interaction, influencing key cellular processes.
- The interaction between CAS and c-SRC is critical for promoting anchorage-independent growth and survival under growth factor-limiting conditions.
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