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Cloning of a complementary DNA for a protein-tyrosine kinase that specifically phosphorylates a negative regulatory
1Division of Protein Metabolism, Osaka University, Japan.
Abstract:
The protein-tyrosine kinase activity of the proto-oncogene product p60c-src is negatively regulated by the phosphorylation of a tyrosine residue close to the C terminus, tyrosine 527. The phosphorylation might be catalysed by a so-far-unidentified tyrosine kinase, distinct from p60c-src. Recently we purified a protein-tyrosine kinase that specifically phosphorylates tyrosine 527 of p60c-src from neonatal rat brain. We have now confirmed the specificity of this enzyme by using a mutant p60c-src that has a phenylalanine instead of tyrosine 527, and cloned a complementary DNA that encodes the enzyme. The enzyme is similar to kinases of the src family in that it has two conserved regions, Src-homology regions 2 and 3, upstream of a tyrosine kinase domain. The amino-acid identity of each region is no more than 47%, however, and the enzyme lacks phosphorylation sites corresponding to tyrosines 416 and 527 of p60c-src and has no myristylation signal. These results suggest that this protein-tyrosine kinase, which might negatively regulate p60c-src, represents a new type of tyrosine kinase.
Insights
Researchers identified a novel protein-tyrosine kinase that phosphorylates tyrosine 527 of p60c-src, potentially regulating its activity. This enzyme represents a new class of tyrosine kinases.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Proto-oncogene product p60c-src activity is regulated by tyrosine 527 phosphorylation.
- This phosphorylation is catalyzed by an unknown tyrosine kinase distinct from p60c-src.
Purpose of the Study:
- To purify and characterize the tyrosine kinase responsible for phosphorylating tyrosine 527 of p60c-src.
- To confirm the specificity of the purified enzyme and clone its encoding cDNA.
Main Methods:
- Purification of a protein-tyrosine kinase from neonatal rat brain.
- Enzyme specificity confirmation using a mutant p60c-src (Y527F).
- Cloning of the complementary DNA encoding the identified enzyme.
Main Results:
- A novel protein-tyrosine kinase specifically phosphorylating p60c-src tyrosine 527 was purified.
- The enzyme's cDNA was cloned, revealing conserved Src-homology 2 and 3 regions but distinct features.
- The enzyme lacks typical src family phosphorylation sites and myristylation signal.
Conclusions:
- The identified protein-tyrosine kinase represents a new class of tyrosine kinases.
- This enzyme may play a role in the negative regulation of p60c-src activity.
- Further research is needed to elucidate the precise function and regulation of this novel kinase.
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