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The kinase activity of the v-fms encoded protein has a low pH optimum
1Fred Hutchinson Cancer Research Center, Seattle, Washington 98104.
Abstract:
The protein encoded by v-fms, the oncogene of the Susan McDonough strain of feline sarcoma virus, is a member of the protein tyrosine kinase family. The kinase activity of the v-fms encoded protein has been reported to be low compared to other members of this enzyme family. We found that the optimal pH in vitro for the autophosphorylation of the immunoprecipitated v-fms encoded protein kinase activity was about pH 5.0; the activity at this pH was 15-fold higher than at the pH (7.4) used in standard kinase assays. The low pH optimum of the kinase activity of the v-fms encoded protein was observed when this protein was immunoprecipitated with each of four independent polyclonal antisera. v-fms proteins from transfected rat, mink or hamster cells all showed the same pH optimum for the kinase activity, as did the protein encoded by the feline c-fms gene. Autophosphorylation of v-fms in vitro at pH 5.0 occurred exclusively on tyrosine residues. Enolase was a substrate for the v-fms encoded protein kinase, and the pH profile for phosphorylation of this substrate in vitro paralleled that seen for the autophosphorylation of v-fms encoded proteins. The discovery of the low pH optimum of the kinase activity exhibited by v-fms proteins may be useful for further characterization of this activity in vitro, as well as for phenotypic classification of other members of the protein tyrosine kinase family.
Insights
The feline sarcoma virus v-fms oncogene protein tyrosine kinase exhibits optimal activity at pH 5.0, 15-fold higher than standard assays. This low pH optimum aids in characterizing kinase activity and classifying related enzymes.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- The v-fms protein, an oncogene from feline sarcoma virus, belongs to the protein tyrosine kinase family.
- Previous studies indicated low kinase activity for v-fms compared to other tyrosine kinases.
Purpose of the Study:
- To investigate the optimal conditions for v-fms protein kinase activity.
- To characterize the pH dependency of v-fms kinase activity in vitro.
- To explore the implications for classifying protein tyrosine kinases.
Main Methods:
- Immunoprecipitation of v-fms protein from various cell lines.
- In vitro kinase assays measuring autophosphorylation and substrate phosphorylation (enolase).
- Assessment of kinase activity across a range of pH values.
Main Results:
- The optimal pH for v-fms protein kinase autophosphorylation was found to be approximately pH 5.0.
- Kinase activity at pH 5.0 was 15-fold higher than at pH 7.4.
- This low pH optimum was consistent across different antisera and host cell types, and also observed for the feline c-fms gene product.
- Phosphorylation occurred exclusively on tyrosine residues during autophosphorylation at pH 5.0.
- The phosphorylation of enolase by v-fms kinase showed a similar pH profile.
Conclusions:
- The v-fms protein kinase exhibits a distinct low pH optimum for its activity.
- This finding provides a valuable tool for in vitro characterization of v-fms kinase.
- The unique pH profile may aid in the phenotypic classification of other protein tyrosine kinases.