Host range mutants of v-src: alterations in kinase activity and substrate interactions

Area of Science:

• Molecular Biology
• Oncology
• Virology

Background:

• Schmidt-Ruppin v-src is a viral oncogene known to transform cells.
• Previous studies generated host range mutants of v-src using linker insertion-deletion mutagenesis.
• These mutants exhibit differential transformation capabilities in chicken embryo fibroblasts (CEF) and Rat-2 cells.

Purpose of the Study:

• To investigate the mechanisms underlying the host-dependent transformation phenotype of Schmidt-Ruppin v-src mutants.
• To determine the role of kinase activity and substrate specificity in v-src-mediated cell transformation.
• To elucidate the influence of host cell factors on v-src kinase function.

Main Methods:

• Generation of host range mutants of Schmidt-Ruppin v-src via linker insertion-deletion mutagenesis.
• Analysis of kinase activity in vitro and in vivo in different cell types (CEF and Rat-2).
• Assessment of protein phosphorylation patterns, including specific substrates like p36, 120-kDa, and 62-kDa proteins, and tyrosine phosphorylation of GAP and p62.

Main Results:

• Mutant SRX5, with Tyr-416 substitution, showed reduced p36 phosphorylation, indicating altered substrate recognition.
• Mutants SPX1, SHX13, and XD6 (SH2 region alterations) had reduced kinase activity, insufficient to explain transformation failure.
• Mutant XD4 (lacking SH2/SH3 domains) was active in CEF but inactive in Rat-2 cells, highlighting host factor influence on tyrosine phosphorylation and activity.

Conclusions:

• The host-dependent transformation phenotype of v-src is attributed to alterations in both kinase activity and substrate specificity.
• Host cell factors play a critical role in regulating v-src tyrosine phosphorylation and kinase activity, even in the absence of SH2 and SH3 domains.
• Specific substrate phosphorylation defects, such as for GAP and p62, may contribute to the transformation defect in Rat-2 cells.