Identification of the conserved serine/threonine residues important for gibberellin-sensitivity of Arabidopsis RGL2 protein
Identification of the conserved serine/threonine residues important for gibberellin-sensitivity of Arabidopsis RGL2 protein
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DOI：
10.1111/j.1365-313x.2005.02512.x
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发表时间：
2005-10-01
期刊：
影响因子：
7.2
通讯作者：
Peng, JR
中科院分区：
文献类型：
--
作者：
Hussain, A;Cao, DN;Peng, JR
拟南芥中的DELLA蛋白GAI、RGA、RGL 1和RGL 2是植物生长抑制因子，抑制多种发育过程。研究表明，赤霉素（GA）通过蛋白酶体途径触发DELLA蛋白的降解，从而减弱DELLA蛋白的抑制功能。然而，目前尚不清楚GA诱导的蛋白降解是否是调节DELLA蛋白生物活性的唯一途径。我们在这里表明，烟草BY2细胞代表了一个合适的系统，用于研究GA信号。RGL 2以磷酸化形式存在于BY2细胞中。RGL 2经历GA诱导的降解，并且该过程被蛋白酶体抑制剂和丝氨酸/苏氨酸磷酸酶抑制剂阻断;然而，丝氨酸/苏氨酸激酶抑制剂没有可检测的作用，表明丝氨酸/苏氨酸的去磷酸化可能是RGL 2通过蛋白酶体途径降解的先决条件。所有17个保守的丝氨酸和苏氨酸残基的定点取代表明，模拟组成性磷酸化状态的6个突变体（RGL 2（S441 D）、RGL 2（S542 D）、RGL 2（T271 E）、RGL 2（T319 E）、RGL 2（T411 E）和RGL 2（T535 E））对GA诱导的降解具有抗性。这表明这些位点是潜在的磷酸化位点。基于GA 20-氧化酶表达的功能测定揭示RGL 2（T271 E）可能是无效突变体，RGL 2（S441 D）、RGL 2（S542 D）、RGL 2（T319 E）和RGL 2（T411 E）仅保留野生型RGL 2的活性的约4-17%，而RGL 2（T535 E）保留野生型RGL 2的活性的约66%。然而，在表达这些突变蛋白的BY2细胞中，GA 20-氧化酶的表达仍然对GA有响应，这表明RGL 2蛋白的稳定化不是调节其生物活性的唯一途径。
The DELLA proteins GAI, RGA, RGL1 and RGL2 in Arabidopsis are plant growth repressors, repressing diverse developmental processes. Studies have shown that gibberellin (GA) attenuates the repressive function of DELLA proteins by triggering their degradation via the proteasome pathway. However, it is not known if GA-induced protein degradation is the only pathway for regulating the bioactivity of DELLA proteins. We show here that tobacco BY2 cells represent a suitable system for studying GA signaling. RGL2 exists in a phosphorylated form in BY2 cells. RGL2 undergoes GA-induced degradation, and this process is blocked by proteasome inhibitors and serine/threonine phosphatase inhibitors; however, serine/threonine kinase inhibitors had no detectable effect, suggesting that dephosphorylation of serine/threonine is probably a prerequisite for degradation of RGL2 via the proteasome pathway. Site-directed substitution of all 17 conserved serine and threonine residues showed that six mutants (RGL2(S441D), RGL2(S542D), RGL2(T271E), RGL2(T319E), RGL2(T411E) and RGL2(T535E)) mimicking the status of constitutive phosphorylation are resistant to GA-induced degradation. This suggests that these sites are potential phosphorylation sites. A functional assay based on the expression of GA 20-oxidase revealed that RGL2(T271E) is probably a null mutant, RGL2(S441D), RGL2(S542D), RGL2(T319E) and RGL2(T411E) only retained about 4-17% of the activity of the wild type RGL2, whereas RGL2(T535E) retained about 66% of the activity of the wild type RGL2. However, expression of GA 20-oxidase in BY2 cells expressing these mutant proteins is still responsive to GA, suggesting that the stabilization of RGL2 protein is not the only pathway for regulating its bioactivity.