Autophosphorylation is required for high kinase activity and efficient transformation ability of proteins encoded by host range alleles of v-src.
Autophosphorylation is required for high kinase activity and efficient transformation ability of proteins encoded by host range alleles of v-src.
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v-src 宿主范围等位基因编码的蛋白质的高激酶活性和高效转化能力需要自磷酸化。
DOI：
10.1128/jvi.68.11.7267-7274.1994
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发表时间：
1994
影响因子：
5.4
通讯作者：
Verderame,MF
中科院分区：
文献类型：
--
作者：
Woods,KM;Verderame,MF
Pp60v-src是一种非受体蛋白酪氨酸激酶，可以转化鸡和啮齿动物的成纤维细胞。该蛋白的src同源性2 （SH2）结构域在蛋白酪氨酸激酶活性的调控中起关键作用。宿主范围蛋白pp60v-src-L（包含SH2结构域高度保守残基（Phe-172）的缺失）和pp60v-src-PPP（包含SH2结构域186个氨基酸从Leu变为Phe）转化鸡细胞，而不是大鼠细胞，并且在体外测量的激酶活性略有降低。这里的数据表明，这些改变的蛋白质需要在Tyr-416上进行自磷酸化才能获得高激酶活性和转化能力。在没有自磷酸化的情况下，相对于磷酸化的宿主亲本蛋白，体外激酶活性进一步降低至少三倍，没有形态转化，锚定独立生长减少，肌动蛋白细胞骨架没有破坏。此外，这些SH2突变破坏了SH2结构域结合与pp60src自磷酸化位点相对应的磷酸化肽的能力。因此，与c-src编码转化能力蛋白的突变等位基因一样，与v-src不同，pp60v-src-F172 delta和pp60v-src-L186F的转化依赖于Y-416的磷酸化，以获得高激酶活性和转化能力。转化对磷酸酪氨酸的依赖并不能反映自磷酸化位点和SH2结构域之间的分子内相互作用，因为纯化的SH2结构域在体外不能结合磷酸化的自磷酸化位点肽。
pp60v-src is a nonreceptor protein tyrosine kinase that can transform both chicken and rodent fibroblasts. The src homology 2 (SH2) domain of this protein serves a critical role in the regulation of protein tyrosine kinase activity. The host range proteins pp60v-src-L, which contains a deletion of a highly conserved residue (Phe-172) in the SH2 domain, and pp60v-src-PPP, which contains a change from a Leu to a Phe at amino acid 186 in the SH2 domain, transform chicken but not rat cells and have slightly reduced kinase activity measured in vitro. The data presented here show that these altered proteins require autophosphorylation on Tyr-416 for high kinase activity and transforming ability. In the absence of autophosphorylation, there is a further decrease of at least threefold in in vitro kinase activity relative to the phosphorylated host range parental protein, no morphological transformation, a reduction in anchorage independent growth, and no disruption of the actin cytoskeleton. In addition, these SH2 mutations abolish the ability of the SH2 domain to bind a phosphorylated peptide that corresponds to the autophosphorylation site of pp60src. Thus, like mutant alleles of c-src encoding transformation competent proteins, and unlike v-src, transformation by pp60v-src-F172 delta and pp60v-src-L186F is dependent on phosphorylation of Y-416 for high kinase activity and transformation ability. The dependence of transformation on phosphotyrosine is not a reflection of an intramolecular interaction between the autophosphorylation site and the SH2 domains since purified SH2 domains are incapable of binding phosphorylated autophosphorylation site peptides in vitro.