Differential binding to and regulation of JAK2 by the SH2 domain and N-terminal region of SH2-bbeta.
Differential binding to and regulation of JAK2 by the SH2 domain and N-terminal region of SH2-bbeta.
复制标题
SH2 结构域和 SH2-bbeta 的 N 末端区域对 JAK2 的差异结合和调节。
DOI：
10.1128/mcb.20.9.3168-3177.2000
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发表时间：
2000
影响因子：
5.3
通讯作者：
Carter-Su,C
中科院分区：
文献类型：
--
作者：
Rui,L;Gunter,DR;Herrington,J;Carter-Su,C
Sh2-Bβ通过其结构域与酪氨酸磷酸化的JAK2结合，并强烈激活JAK2。在这项研究中，我们证明了在SH2-Bβ的N末端区域(氨基酸1到555)中存在与JAK2的额外结合位点(S)，并证明了SH2-B的这一区域抑制JAK2的能力。有四条证据支持这个额外结合位点的存在(S)。在谷胱甘肽转移酶下拉实验中，野生型SH2-Bβ和带有SH2结构域缺陷的SH2-Bβ(R555E)既与生长激素(GH)处理细胞中酪氨酸磷酸化的JAK2结合，又与对照细胞中非酪氨酸磷酸化的JAK2结合，而SH2-Bβ的SH2结构域仅与GH处理细胞中酪氨酸磷酸化的JAK2结合。同样，在没有GH和GH存在的情况下，JAK2存在于αSH2-B免疫沉淀物中，GH显著增加了JAK2与SH2-B的共沉淀。当SH2-Bβ在COS细胞中共表达时，它不仅表达野生型酪氨酸磷酸化的JAK2，而且还表达非酪氨酸磷酸化的野生型JAK2(K882E)，尽管程度较小。ΔC555(SH2-Bβ的1-555位氨基酸)缺乏SH2结构域的大部分，其结合方式类似于野生型JAK2和激酶失活的JAK2(K882E)。使用一系列N-端和C-端截短的SH2-Bβ构建物的实验表明，Pleckstrin同源(PH)结构域(第269到410位氨基酸)和第410到555位氨基酸是SH2-Bβ与失活JAK2最大结合所必需的，但这两个区域本身都不足以实现最大结合。SH2-Bβ的SH2结构域是SH2-Bβ刺激JAK2和JAK2介导的STAT5B酪氨酸磷酸化的充分必要条件。相反，缺少SH2域的ΔC555，以及单独的PH域，在较小程度上抑制JAK2。ΔC555还阻断JAK2介导的STAT5B在COS细胞中的酪氨酸磷酸化和GH刺激的STAT5B在3T3-F442A细胞中的核积聚。这些数据表明，除了SH2结构域，SH2-Bβ在269到555位氨基酸中还与JAK2有一个或多个低亲和力结合位点。通过SH2-B中的这个位点(S)与失活的JAK2相互作用，可能会增加JAK2周围SH2-Bβ的局部浓度，从而促进SH2结构域与配体激活的JAK2结合。这将导致细胞对激活JAK2的激素和细胞因子做出更快速、更强大的反应。失活的JAK2和SH2-B之间的这种相互作用也可能有助于防止JAK2的异常激活。
SH2-Bβ has been shown to bind via its SH2 (Src homology 2) domain to tyrosyl-phosphorylated JAK2 and strongly activate JAK2. In this study, we demonstrate the existence of an additional binding site(s) for JAK2 within the N-terminal region of SH2-Bβ (amino acids 1 to 555) and the ability of this region of SH2-B to inhibit JAK2. Four lines of evidence support the existence of this additional binding site(s). In a glutathioneS-transferase pull-down assay, wild-type SH2-Bβ and SH2-Bβ(R555E) with a defective SH2 domain bind to both tyrosyl-phosphorylated JAK2 from growth hormone (GH)-treated cells and non-tyrosyl-phosphorylated JAK2 from control cells, whereas the SH2 domain of SH2-Bβ binds only to tyrosyl-phosphorylated JAK2 from GH-treated cells. Similarly, JAK2 is present in αSH2-B immunoprecipitates in the absence and presence of GH, with GH substantially increasing the coprecipitation of JAK2 with SH2-B. When coexpressed in COS cells, SH2-Bβ coimmunoprecipitates not only wild-type, tyrosyl-phosphorylated JAK2 but also kinase-inactive, non-tyrosyl-phosphorylated JAK2(K882E), although to a lesser extent. ΔC555 (amino acids 1 to 555 of SH2-Bβ) that lacks most of the SH2 domain binds similarly to wild-type JAK2 and kinase-inactive JAK2(K882E). Experiments using a series of N- and C-terminally truncated SH2-Bβ constructs indicate that the pleckstrin homology (PH) domain (amino acids 269 to 410) and amino acids 410 to 555 are necessary for maximal binding of SH2-Bβ to inactive JAK2, but neither region alone is sufficient for maximal binding. The SH2 domain of SH2-Bβ is necessary and sufficient for the stimulatory effect of SH2-Bβ on JAK2 and JAK2-mediated tyrosyl phosphorylation of Stat5B. In contrast, ΔC555 lacking the SH2 domain, and to a lesser extent the PH domain alone, inhibits JAK2. ΔC555 also blocks JAK2-mediated tyrosyl phosphorylation of Stat5B in COS cells and GH-stimulated nuclear accumulation of Stat5B in 3T3-F442A cells. These data indicate that in addition to the SH2 domain, SH2-Bβ has one or more lower-affinity binding sites for JAK2 within amino acids 269 to 555. The interaction via this site(s) in SH2-B with inactive JAK2 seems likely to increase the local concentration of SH2-Bβ around JAK2, thereby facilitating binding of the SH2 domain to ligand-activated JAK2. This would result in a more rapid and robust cellular response to hormones and cytokines that activate JAK2. This interaction between inactive JAK2 and SH2-B may also help prevent abnormal activation of JAK2.