Sasaki M
Department of Neurosurgery, Kobe University School of Medicine.
Kobe J Med Sci. 1990 Dec;36(5-6):137-51.
The products of the viral & cellular src gene, p60v-src and p60c-src, appear to be composed of multiple functional domains. Highly conserved regions called src homology 2 and 3 (SH2 and SH3), comprising amino acid residues 88 to 250, are believed to modulate the protein-tyrosine kinase activity present in the carboxy-terminal halves of the src proteins. To explore the functions of these regions more fully, we have introduced deletion mutations within the amino-terminal half of p56lyn, a member of the src-related family of the protein tyrosine kinases. These mutant alleles were expressed under the control of the Moloney murine leukemia virus long terminal repeat in the vector plasmid pZip-neo-SV(X) to examine the biochemical and biological properties of the mutant proteins after introduction into NIH3T3 cells and WEHI-231(immature B-cell line). No mutant proteins induced transformed foci in NIH3T3 transfectants. Deletional mutant affecting residues 163 to 234 (lyn-dC) impaired kinase activity severely and residues 35 to 60 (lyn-dV), to the lesser extent. In WEHI-231 transfectants, we could also found the inhibition of kinase activity with deletions of residues 163 to 234 (lyn-dC) and 35 to 60 (lyn-dV). WEHI-231 transfectant with lyn-dC, named WE-C was induced lesser growth arrest by crosslinking membrane IgM compared with wild type (wt) WEHI-231 cells. Down Regulation of the mutant protein stimulated with anti-IgM antibodies was detected in wt WEHI-231, but not in WEHI-231 transfectants, WE-V and WE-C. We show here the effect of the mutations in the regulatory domain on the kinase activity and the biological function of the lyn gene product, p56lyn.
病毒src基因和细胞src基因的产物p60v-src和p60c-src似乎由多个功能域组成。被称为src同源2和3（SH2和SH3）的高度保守区域，包含88至250位氨基酸残基，被认为可调节src蛋白羧基末端部分存在的蛋白酪氨酸激酶活性。为了更全面地探索这些区域的功能，我们在蛋白酪氨酸激酶src相关家族的成员p56lyn的氨基末端部分引入了缺失突变。这些突变等位基因在载体质粒pZip-neo-SV(X)中的莫洛尼鼠白血病病毒长末端重复序列的控制下表达，以检测将突变蛋白引入NIH3T3细胞和WEHI-231（未成熟B细胞系）后其生化和生物学特性。没有突变蛋白在NIH3T3转染子中诱导转化灶。影响163至234位残基的缺失突变体（lyn-dC）严重损害激酶活性，而35至60位残基的缺失突变体（lyn-dV）对激酶活性的损害程度较小。在WEHI-231转染子中，我们也发现163至234位残基（lyn-dC）和35至60位残基（lyn-dV）缺失时激酶活性受到抑制。与野生型（wt）WEHI-231细胞相比，携带lyn-dC的WEHI-231转染子（命名为WE-C）通过交联膜IgM诱导的生长停滞较少。在wt WEHI-231中检测到抗IgM抗体刺激后突变蛋白的下调，但在WEHI-231转染子WE-V和WE-C中未检测到。我们在此展示了调节域中的突变对lyn基因产物p56lyn的激酶活性和生物学功能的影响。
