Hirai H, Varmus H E
Department of Microbiology, University of California, San Francisco 94143-0502.
Mol Cell Biol. 1990 Apr;10(4):1307-18. doi: 10.1128/mcb.10.4.1307-1318.1990.
The products of the viral and cellular src genes, 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 made 34 site-directed mutations in a transformation-competent c-src gene encoding phenylalanine in place of tyrosine 527 (Y527F c-src). Twenty of the new mutations change only one or two amino acids, and the remainder delete small or large portions of the SH2-SH3 region. These mutant alleles have been incorporated into a replication-competent Rous sarcoma virus vector to examine the biochemical and biological properties of the mutant proteins after infection of chicken embryo fibroblasts. Four classes of mutant proteins were observed: class 1, mutants with only slight differences from the parental gene products; class 2, mutant proteins with diminished transforming and specific kinase activities; class 3, mutant proteins with normal or enhanced specific kinase activity but impaired biological activity, often as a consequence of instability; and class 4, mutant proteins with augmented biological and catalytic activities. In general, there was a strong correlation between total kinase activity (or amounts of intracellular phosphotyrosine-containing proteins) and transforming activity. Deletion mutations and some point mutations affecting residues 109 to 156 inhibited kinase and transforming functions, whereas deletions affecting residues 187 to 226 generally had positive effects on one or both of those functions, confirming that SH2-SH3 has complex regulatory properties. Five mutations that augmented the transforming and kinase activities of Y527F c-src [F172P, R175L, delta(198-205), delta(206-226), and delta(176-226)] conferred transformation competence on an otherwise normal c-src gene, indicating that mutations in SH2 (like previously described lesions in SH3, the kinase domain, and a carboxy-terminal inhibitory domain) can activate c-src.
病毒src基因和细胞src基因的产物，即p60v-src和p60c-src，似乎由多个功能结构域组成。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，我们在一个具有转化能力的c-src基因中进行了34个定点突变，该基因编码苯丙氨酸以取代酪氨酸527（Y527F c-src）。其中20个新突变仅改变一两个氨基酸，其余突变则删除了SH2-SH3区域的小部分或大部分。这些突变等位基因已被整合到一个具有复制能力的劳氏肉瘤病毒载体中，以检测感染鸡胚成纤维细胞后突变蛋白的生化和生物学特性。观察到四类突变蛋白：第1类，与亲本基因产物仅有轻微差异的突变体；第2类，转化和特异性激酶活性降低的突变蛋白；第3类，特异性激酶活性正常或增强但生物学活性受损的突变蛋白，通常是由于不稳定性导致；第4类，生物学和催化活性增强的突变蛋白。一般来说，总激酶活性（或细胞内含磷酸酪氨酸蛋白的量）与转化活性之间存在很强的相关性。影响109至156位残基的缺失突变和一些点突变抑制了激酶和转化功能，而影响187至226位残基的缺失通常对这些功能中的一项或两项有积极影响，证实SH2-SH3具有复杂的调节特性。五个增强Y527F c-src转化和激酶活性的突变 [F172P、R175L、delta(198 - 205)、delta(206 - 226) 和 delta(176 - 226)] 赋予了一个原本正常的c-src基因转化能力，表明SH2中的突变（如先前描述的SH3、激酶结构域和羧基末端抑制结构域中的损伤）可以激活c-src。
