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Characterization of two activated mutants of human pp60c-src that escape c-Src kinase regulation by distinct
J D Bjorge1, C Bellagamba, H C Cheng
1Department of Medical Biochemistry, University of Calgary, Canada.
Abstract:
Two activated transforming mutants of human pp60c-src were found to possess single point mutations within the regulatory carboxyl terminus (E527K in CY CST201) and the kinase domain (E381G in WO CST1), respectively, that do not directly interfere with either the regulatory c-Src kinase (CSK) phosphorylation site (Tyr530) or the SH2/3 domains. In vivo, both mutant proteins are hypophosphorylated on their carboxyl-terminal regulatory tyrosines and are hyperactive. In an in vitro Src kinase inactivation assay, both mutant Src proteins exhibited resistance to inactivation by CSK relative to wild-type Src. Under these in vitro conditions, E381G c-Src was found to be phosphorylated by CSK to wild-type levels, while E527K c-Src was not detectably phosphorylated. The ability of CSK to phosphorylate a carboxyl-terminal peptide modelled against E527K c-Src was also impaired, suggesting that CSK is unable to recognize E527K c-Src as an efficient substrate. In the case of E381G c-Src, examination of whether its SH2/3 domains were accessible to the carboxyl-terminal regulatory phosphotyrosine revealed a highly reduced ability of autophosphorylated E381G c-Src to bind to a synthetic phosphopeptide modelled from the SH2-binding region of polyoma middle-T antigen which binds to Src SH2 with high affinity. This suggests that the E381G c-Src mutation results in an altered or reduced accessibility of the SH2 domain of the autophosphorylated form of E381G c-Src and may represent a previously undescribed mode of Src activation. Further study of these and other Src mutants may offer additional new insights into the regulation of "Src family" kinases.
Insights
Two human pp60c-src mutants with single point mutations show increased activity. These mutants resist inactivation by c-Src kinase (CSK) and display altered substrate recognition or SH2 domain accessibility, revealing new insights into Src kinase regulation.
Area of Science:
- Molecular Biology
- Cellular Signaling
- Oncogenesis
Background:
- The proto-oncogene pp60c-src (c-Src) is a non-receptor tyrosine kinase regulating various cellular processes.
- Dysregulation of c-Src activity is implicated in cancer development.
- Understanding c-Src regulation is crucial for targeted cancer therapies.
Purpose of the Study:
- To investigate the functional consequences of specific point mutations in human pp60c-src.
- To elucidate the mechanisms underlying the hyperactivation of these c-Src mutants.
- To explore novel modes of c-Src kinase activation and regulation.
Main Methods:
- Site-directed mutagenesis to generate E527K and E381G c-Src mutants.
- In vivo analysis of protein phosphorylation status.
- In vitro kinase inactivation assays using c-Src kinase (CSK).
- Peptide phosphorylation assays and phosphopeptide binding assays to assess SH2 domain accessibility.
Main Results:
- Both E527K and E381G mutants exhibited hyperactivation and resistance to CSK-mediated inactivation in vivo.
- E527K c-Src showed impaired phosphorylation by CSK, suggesting altered substrate recognition.
- E381G c-Src displayed reduced SH2 domain accessibility in its autophosphorylated state, indicating a novel activation mechanism.
Conclusions:
- Single point mutations can lead to activated transforming pp60c-src variants.
- Mutations can confer resistance to regulatory phosphorylation by CSK.
- Altered SH2 domain accessibility represents a potential new mechanism for Src kinase activation, offering therapeutic targets.