Entwicklungsbiologie und Physiologie der Pflanzen, Universität Kiel, Am Botanischen Garten 5, Kiel, 24118, Germany.
Plant J. 2014 Apr;78(2):192-202. doi: 10.1111/tpj.12460. Epub 2014 Mar 5.
The cell growth-promoting peptide phytosulfokine (PSK) is perceived by leucine-rich repeat (LRR) receptor kinases. To elucidate PSK receptor function we analyzed PSKR1 kinase activity and binding to Ca(2+) sensors and evaluated the contribution of these activities to growth control in planta. Ectopically expressed PSKR1 was capable of auto- and transphosphorylation. Replacement of a conserved lysine within the ATP-binding region by a glutamate resulted in the inhibition of auto- and transphosphorylation kinase activities. Expression of the kinase-inactive PSKR1(K762E) receptor in the pskr null background did not restore root or shoot growth. Instead, the mutant phenotype was enhanced suggesting that the inactive receptor protein exerts growth-inhibitory activity. Bioinformatic analysis predicted a putative calmodulin (CaM)-binding site within PSKR1 kinase subdomain VIa. Bimolecular fluorescence complementation analysis demonstrated that PSKR1 binds to all isoforms of CaM, more weakly to the CaM-like protein CML8 but apparently not to CML9. Mutation of a conserved tryptophan (W831S) within the predicted CaM-binding site strongly reduced CaM binding. Expression of PSKR1(W831S) in the pskr null background resulted in growth inhibition that was similar to that of the kinase-inactive receptor. We conclude that PSK signaling requires Ca(2+) /CaM binding and kinase activity of PSKR1 in planta. We further propose that the inactivated kinase interferes with other growth-promoting signaling pathway(s).
植物生长促进肽 PSK（phytosulfokine）被富含亮氨酸重复（LRR）受体激酶感知。为了阐明 PSK 受体的功能，我们分析了 PSKR1 激酶活性及其与 Ca2+传感器的结合，并评估了这些活性对植物中生长调控的贡献。异位表达的 PSKR1 能够自动和转磷酸化。在 ATP 结合区域内保守的赖氨酸被谷氨酸取代，导致自动和转磷酸化激酶活性的抑制。在 pskr 缺失背景下表达激酶失活的 PSKR1（K762E）受体不能恢复根或茎的生长。相反，突变表型增强，表明失活的受体蛋白具有生长抑制活性。生物信息学分析预测 PSKR1 激酶亚结构域 VIa 内存在一个假定的钙调蛋白（CaM）结合位点。双分子荧光互补分析表明 PSKR1 与所有 CaM 同工型结合，与 CaM 样蛋白 CML8 结合较弱，但显然不与 CML9 结合。预测的 CaM 结合位点内保守色氨酸（W831S）的突变强烈降低了 CaM 的结合。在 pskr 缺失背景下表达 PSKR1（W831S）导致的生长抑制与激酶失活受体相似。我们得出结论，PSK 信号需要 Ca2+/CaM 结合和 PSKR1 的激酶活性在植物中。我们进一步提出失活的激酶干扰了其他促进生长的信号通路。
