Tyrosines 868, 966, and 972 in the Kinase Domain of JAK2 Are Autophosphorylated and Required for Maximal JAK2 Kinase Activity
Tyrosines 868, 966, and 972 in the Kinase Domain of JAK2 Are Autophosphorylated and Required for Maximal JAK2 Kinase Activity
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DOI：
10.1210/me.2009-0355
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发表时间：
2010-05-01
影响因子：
--
通讯作者：
Carter-Su, Christin
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文献类型：
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作者：
Argetsinger, Lawrence S.;Stuckey, Jeanne A.;Carter-Su, Christin
Janus kinase2(JAK2)被大多数细胞因子家族受体激活，包括生长激素受体、瘦素受体和促红细胞生成素受体。为了确定新的JAK2调节和/或结合位点，我们开始确定JAK2激活域中的自动磷酸化位点。体外自磷酸化JAK2的二维磷酸肽图确定酪氨酸868、966和972是自磷酸化的位点。通过对促红细胞生成素结合的嵌合促红细胞生成素受体/瘦素受体激活的JAK2的质谱分析，也鉴定了磷酸化酪氨酸868和972。磷酸特异性抗体表明，所有三种酪氨酸的磷酸化都会随着生长激素的反应而增加。与野生型JAK2相比，野生型JAK2在过表达时具有结构性活性，缺乏酪氨酸868、966或972的JAK2活性显著降低。通过GH受体和蛋白酪氨酸磷酸酶1B的共同表达，我们可以研究这些突变的JAK2在人胚胎肾脏293T细胞中的GH依赖激活。所有三个突变的JAK2都被生长激素激活，尽管程度低于野生型JAK2。这3个突变的JAK2还介导信号转导和转录激活因子3(STAT3)、信号转导和转录激活因子5b(Stat5b)和ERK1的GH激活，但水平降低。与Src同源2B1β(SH2B1β)共表达，与GH结合GH受体共表达一样，部分恢复了所有三个JAK2突变体的活性。根据这些结果和JAK2激动域的晶体结构，我们假设JAK2酪氨酸868、966和972周围区域的构象发生微小变化，例如由于磷酸化、与配体结合的细胞因子受体和/或与Src同源2B1的结合，可能是JAK2具有最大活性构象所必需的。(分子内分泌学24：1062-1076,2010)
Janus kinase 2 (JAK2) is activated by a majority of cytokine family receptors including receptors for GH, leptin, and erythropoietin. To identify novel JAK2-regulatory and/or -binding sites, we set out to identify autophosphorylation sites in the kinase domain of JAK2. Two-dimensional phosphopeptide mapping of in vitro autophosphorylated JAK2 identified tyrosines 868, 966, and 972 as sites of autophosphorylation. Phosphorylated tyrosines 868 and 972 were also identified by mass spectrometry analysis of JAK2 activated by an erythropoietin-bound chimeric erythropoietin receptor/leptin receptor. Phosphospecific antibodies suggest that the phosphorylation of all three tyrosines increases in response to GH. Compared with wild-type JAK2, which is constitutively active when overexpressed, JAK2 lacking tyrosine 868, 966, or 972 has substantially reduced activity. Coexpression with GH receptor and protein tyrosine phosphatase1B allowed us to investigate GH-dependent activation of these mutated JAK2s in human embryonic kidney 293T cells. All three mutated JAK2s are activated by GH, although to a lesser extent than wild-type JAK2. The three mutated JAK2s also mediate GH activation of signal transducer and activator of transcription 3 (Stat3), signal transducer and activator of transcription 5b (Stat5b) and ERK1, but at reduced levels. Coexpression with Src-homology 2B1 beta (SH2B1 beta), like coexpression with GH-bound GH receptor, partially restores the activity of all three JAK2 mutants. Based on these results and the crystal structure of the JAK2 kinase domain, we hypothesize that small changes in the conformation of the regions of JAK2 surrounding tyrosines 868, 966, and 972 due to e. g. phosphorylation, binding to a ligand-bound cytokine receptor, and/or binding to Src-homology 2B1, may be essential for JAK2 to assume a maximally active conformation. (Molecular Endocrinology 24: 1062-1076, 2010)