Molecular Characterization of Functional Domains in the Protein Kinase SOS2 That Is Required for Plant Salt Tolerance
Molecular Characterization of Functional Domains in the Protein Kinase SOS2 That Is Required for Plant Salt Tolerance
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DOI：
10.1105/tpc.010021
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发表时间：
2001-06
期刊：
影响因子：
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通讯作者：
Yan Guo;U. Halfter;M. Ishitani;Jian‐Kang Zhu
中科院分区：
文献类型：
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作者：
Yan Guo;U. Halfter;M. Ishitani;Jian‐Kang Zhu
SOS 3（for SALT OVERLY SENSITIVE 3）钙结合蛋白和SOS 2蛋白激酶是拟南芥钠、钾离子稳态和耐盐性所必需的。我们之前已经证明，SOS 3与SOS 2蛋白激酶相互作用并激活SOS 2蛋白激酶。我们在这里报告的SOS 2，也作为激酶自抑制域的SOS 3结合基序的鉴定。酵母双杂交试验以及体外结合试验揭示了在SOS 2的调节结构域中的21个氨基酸基序，其对于与SOS 3的相互作用是必要且足够的。数据库搜索发现了一个大家族的SOS 2样蛋白激酶含有这样的SOS 3结合基序。使用酵母双杂交系统，我们表明，这些SOS 2样激酶与钙结合蛋白的SOS 3家族的成员相互作用。双杂交试验还揭示了N-末端激酶结构域和C-末端调节结构域之间的相互作用，表明调节结构域可以通过阻断底物进入催化位点来抑制激酶活性。去除包括SOS 3结合基序在内的SOS 2的调节结构域，导致蛋白激酶的组成性激活，表明SOS 3结合基序可以作为激酶自抑制结构域。组成型活性的SOS 2是SOS 3独立的，也产生了改变Thr 168到Asp的SOS 2激酶结构域的激活环。将Thr 168-to-Asp突变与自抑制结构域缺失相结合，产生了超活性的SOS 2激酶。这些结果提供了对SOS 2和SOS 2蛋白激酶家族的激酶活性调节的见解。
The SOS3 (for SALT OVERLY SENSITIVE3) calcium binding protein and SOS2 protein kinase are required for sodium and potassium ion homeostasis and salt tolerance in Arabidopsis. We have shown previously that SOS3 interacts with and activates the SOS2 protein kinase. We report here the identification of a SOS3 binding motif in SOS2 that also serves as the kinase autoinhibitory domain. Yeast two-hybrid assays as well as in vitro binding assays revealed a 21–amino acid motif in the regulatory domain of SOS2 that is necessary and sufficient for interaction with SOS3. Database searches revealed a large family of SOS2-like protein kinases containing such a SOS3 binding motif. Using a yeast two-hybrid system, we show that these SOS2-like kinases interact with members of the SOS3 family of calcium binding proteins. Two-hybrid assays also revealed interaction between the N-terminal kinase domain and the C-terminal regulatory domain within SOS2, suggesting that the regulatory domain may inhibit kinase activity by blocking substrate access to the catalytic site. Removal of the regulatory domain of SOS2, including the SOS3 binding motif, resulted in constitutive activation of the protein kinase, indicating that the SOS3 binding motif can serve as a kinase autoinhibitory domain. Constitutively active SOS2 that is SOS3 independent also was produced by changing Thr168 to Asp in the activation loop of the SOS2 kinase domain. Combining the Thr168-to-Asp mutation with the autoinhibitory domain deletion created a superactive SOS2 kinase. These results provide insights into regulation of the kinase activities of SOS2 and the SOS2 family of protein kinases.