Charpenteau Martine, Jaworski Krzysztof, Ramirez Bertha C, Tretyn Andrzej, Ranjeva Raoul, Ranty Benoît
UMR 5546 CNRS/Université Paul Sabatier, Pôle de Biotechnologie Végétale, BP 17 Auzeville, 31326 Castanet-Tolosan cedex, France.
Biochem J. 2004 May 1;379(Pt 3):841-8. doi: 10.1042/BJ20031045.
Screening a cDNA expression library with a radiolabelled calmodulin (CaM) probe led to the isolation of AtCaMRLK, a receptor-like kinase (RLK) of Arabidopsis thaliana. AtCaMRLK polypeptide sequence shows a modular organization consisting of the four distinctive domains characteristic of receptor kinases: an amino terminal signal sequence, a domain containing seven leucine-rich repeats, a single putative membrane-spanning segment and a protein kinase domain. Using truncated versions of the protein and a synthetic peptide, we demonstrated that a region of 23 amino acids, located near the kinase domain of AtCaMRLK, binds CaM in a calcium-dependent manner. Real-time binding experiments showed that AtCaMRLK interacted in vitro with AtCaM1, a canonical CaM, but not with AtCaM8, a divergent isoform of the Ca2+ sensor. The bacterially expressed kinase domain of the protein was able to autophosphorylate and to phosphorylate the myelin basic protein, using Mn2+ preferentially to Mg2+ as an ion activator. Site-directed mutagenesis of the conserved lysine residue (Lys423) to alanine, in the kinase subdomain II, resulted in a complete loss of kinase activity. CaM had no influence on the autophosphorylation activity of AtCaMRLK. AtCaMRLK was expressed in reproductive and vegetative tissues of A. thaliana, except in leaves. Disruption in the AtCaMRLK coding sequence by insertion of a DsG transposable element in an Arabidopsis mutant did not generate a discernible phenotype. The CaM-binding motif of AtCaMRLK was found to be conserved in several other members of the plant RLK family, suggesting a role for Ca2+/CaM in the regulation of RLK-mediated pathways.
用放射性标记的钙调蛋白（CaM）探针筛选cDNA表达文库，分离出了拟南芥的一种类受体激酶（RLK）——AtCaMRLK。AtCaMRLK多肽序列呈现出模块化结构，由受体激酶特有的四个不同结构域组成：一个氨基末端信号序列、一个包含七个富含亮氨酸重复序列的结构域、一个单一的假定跨膜片段和一个蛋白激酶结构域。利用该蛋白的截短版本和合成肽，我们证明了位于AtCaMRLK激酶结构域附近的一个23个氨基酸的区域以钙依赖的方式结合CaM。实时结合实验表明，AtCaMRLK在体外与典型的CaM——AtCaM1相互作用，但不与Ca2+传感器的一种不同亚型AtCaM8相互作用。该蛋白的细菌表达激酶结构域能够自磷酸化，并能磷酸化髓鞘碱性蛋白，优先使用Mn2+而非Mg2+作为离子激活剂。将激酶亚结构域II中保守的赖氨酸残基（Lys423）定点突变为丙氨酸，导致激酶活性完全丧失。CaM对AtCaMRLK的自磷酸化活性没有影响。AtCaMRLK在拟南芥的生殖和营养组织中表达，但在叶片中不表达。在拟南芥突变体中插入DsG转座元件破坏AtCaMRLK编码序列，未产生明显的表型。发现AtCaMRLK的CaM结合基序在植物RLK家族的其他几个成员中保守，表明Ca2+/CaM在RLK介导的信号通路调控中起作用。
