The type II transforming growth factor-beta receptor autophosphorylates not only on serine and threonine but also on tyrosine residues
The type II transforming growth factor-beta receptor autophosphorylates not only on serine and threonine but also on tyrosine residues
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DOI：
10.1074/jbc.272.23.14850
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发表时间：
1997-06-06
影响因子：
4.8
通讯作者：
Derynck, R
中科院分区：
文献类型：
--
作者：
Lawler, S;Feng, XH;Derynck, R
转化生长因子-β（TGF-β）的I型和II型受体是结构上相关的跨膜丝氨酸/苏氨酸激酶，其能够在细胞表面彼此物理相互作用。为了帮助定义TGF-β信号传导中的初始事件，我们表征了II型TGF-β受体的激酶活性。从大肠杆菌和杆状病毒感染的昆虫细胞中纯化了受体的重组胞质结构域。抗磷酸化酪氨酸蛋白质印迹法证实，II型受体激酶可在酪氨酸上自磷酸化。在体外激酶反应后，磷酸化氨基酸分析表明，不仅发生在丝氨酸和苏氨酸，而且也对酪氨酸的胞质结构域和外源性底物的磷酸化的自磷酸化。使用在哺乳动物细胞中表达的免疫沉淀受体也证明了受体的双重激酶特异性，并且体内P-32标记显示丝氨酸和酪氨酸上的受体磷酸化。此外，酪氨酸激酶抑制剂tyrphostin可抑制胞质结构域的激酶活性。胰蛋白酶定位和氨基酸测序的体外自磷酸化的II型受体胞质结构域允许的酪氨酸磷酸化位点的位置259，336和424。替换所有三个酪氨酸与苯丙氨酸强烈抑制受体的激酶活性，表明酪氨酸自磷酸化可能发挥autoregulatory作用，这种受体的激酶活性。这些结果表明，II型TGF-β受体可以作为一种双重特异性激酶发挥作用，并表明酪氨酸自磷酸化在TGF-β受体信号传导中的作用。
The type I and type II receptors for transforming growth factor-beta (TGF-beta) are structurally related transmembrane serine/threonine kinases, which are able to physically interact with each other at the cell surface. To help define the initial events in TGF-beta signaling, we characterized the kinase activity of the type II TGF-beta receptor. A recombinant cytoplasmic domain of the receptor was purified from Escherichia coli and baculovirus-infected insect cells. Anti-phosphotyrosine Western blotting demonstrated that the type II receptor kinase can autophosphorylate on tyrosine. Following an in vitro kinase reaction, the autophosphorylation of the cytoplasmic domain and phosphorylation of exogenous substrate was shown by phosphoamino acid analysis to occur not only on serine and threonine but also on tyrosine. The dual kinase specificity of the receptor was also demonstrated using immunoprecipitated receptors expressed in mammalian cells and in vivo P-32 labeling showed phosphorylation of the receptor on serine and tyrosine. In addition, the kinase activity of the cytoplasmic domain was inhibited by the tyrosine kinase inhibitor tyrphostin. Tryptic mapping and amino acid sequencing of in vitro autophosphorylated type II receptor cytoplasmic domain allowed the localization of the sites of tyrosine phosphorylation to positions 259, 336, and 424. Replacement of all three tyrosines with phenylalanines strongly inhibited the kinase activity of the receptor, suggesting that tyrosine autophosphorylation may play an autoregulatory role for the kinase activity of this receptor. These results demonstrate that the type II TGF-beta receptor can function as a dual specificity kinase and suggest a role for tyrosine autophosphorylation in TGF-beta receptor signaling.