c-Ab1 has high intrinsic tyrosine kinase activity that is stimulated by mutation of the Src homology 3 domain and by autophosphorylation at two distinct regulatory tyrosines
c-Ab1 has high intrinsic tyrosine kinase activity that is stimulated by mutation of the Src homology 3 domain and by autophosphorylation at two distinct regulatory tyrosines
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DOI：
10.1074/jbc.m005401200
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发表时间：
2000-11-10
影响因子：
4.8
通讯作者：
Van Etten, RA
中科院分区：
文献类型：
--
作者：
Brasher, BB;Van Etten, RA
使用特异性 Abl 酪氨酸激酶抑制剂 STI 571，我们纯化了未磷酸化的鼠 IV 型 c-Abl，并使用基于肽的测定法测量了溶液中 c-Abl 酪氨酸激酶活性的动力学参数。未磷酸化的c-Abl 表现出显着的肽激酶活性，K-m 为204 muM，V-max 为33 pmol min(-1)。与之前使用免疫复合物激酶测定的观察结果相反，我们发现具有 Src 同源 3 结构域点突变 (P131L) 的转化 c-Abl 突变体比野生型 c-Abl 具有显着（约 6 倍）的内在激酶活性（K-m = 91 muM，V-max = 112 pmol min(-1)）。自磷酸化刺激野生型 c-Abl 的活性约 18 倍，c-Abl P131L 约 3.6 倍，产生具有相似催化速率的高活性激酶，自磷酸化速率取决于与分子间反应一致的 Abl 蛋白浓度，与 c-Src 催化结构域自磷酸化位点同源的 c-Abl 残基处的酪氨酸至苯丙氨酸突变 (Y412F)使野生型 c-Abl 的激活减弱 90%，但仅使 c-Abl P131L 的激活降低 45%。 Src 同源性 2 和 Abl 家族中保守的催化结构域之间的连接区中酪氨酸 (Tyr-245) 的突变抑制了野生型 c-Abl 的自磷酸化诱导的激活 50%，而 c-Abl Y245F/Y412F 双突变体通过自磷酸化被最小程度地激活。这些结果支持了这样的模型，其中 c-Abl 通过分子内 Src 同源性 3 连接体相互作用，并通过 Tyr-412 和 Tyr-245 处的连续磷酸化激发完全催化活性。
Using the specific Abl tyrosine kinase inhibitor STI 571, we purified unphosphorylated murine type IV c-Abl and measured the kinetic parameters of c-Abl tyrosine kinase activity in a solution with a peptide-based assay. Unphosphorylated c-Abl exhibited substantial peptide kinase activity with K-m of 204 muM and V-max of 33 pmol min(-1). Contrary to previous observations using immune complex kinase assays, we found that a transforming c-Abl mutant with a Src homology 3 domain point mutation (P131L) had significantly (about 6-fold) higher intrinsic kinase activity than wild-type c-Abl (K-m = 91 muM, V-max = 112 pmol min(-1)). Autophosphorylation stimulated the activity of wild-type c-Abl about 18-fold and c-Abl P131L about 3.6-fold, resulting in highly active kinases with similar catalytic rates, The autophosphorylation rate was dependent on Abl protein concentration consistent with an intermolecular reaction, A tyrosine to phenylalanine mutation (Y412F) at the c-Abl residue homologous to the c-Src catalytic domain autophosphorylation site impaired the activation of wildtype c-Abl by 90% but reduced activation of c-Abl P131L by only 45%. Mutation of a tyrosine (Tyr-245) in the linker region between the Src homology 2 and catalytic domains that is conserved among the Abl family inhibited the autophosphorylation-induced activation of wild-type c-Abl by 50%, whereas the c-Abl Y245F/Y412F double mutant was minimally activated by autophosphorylation, These results support a model where c-Abl is inhibited in part through an intramolecular Src homology 3-linker interaction and stimulated to full catalytic activity by sequential phosphorylation at Tyr-412 and Tyr-245.