Huang J Z, Hardin S C, Huber S C
Department of Biological Sciences, Zhejiang University, Huajiachi Campus, Hangzhou, Zhejiang, 310029, China.
Arch Biochem Biophys. 2001 Sep 1;393(1):61-6. doi: 10.1006/abbi.2001.2476.
The Ca(2+)-dependent protein kinases (CDPKs) are members of a large subfamily of protein kinases in plants that have been implicated in the control of numerous aspects of plant growth and development. One known substrate of the CDPKs is the ER-located ACA2 calcium pump, which is regulated by phosphorylation of Ser(45). In the present study, a synthetic peptide based on the known regulatory phosphorylation site (RRFRFTANLS(45)KRYEA) was efficiently phosphorylated in vitro by CDPKs but not a plant SNF1-related protein kinase. Phosphorylation of the Ser(45)-ACA2 peptide was surprising because the sequence lacks basic residues at P-3/P-4 (relative to the phosphorylated Ser at position P) that are considered to be essential recognition elements for CDPKs. We demonstrate that phosphorylation of the Ser(45)-ACA2 peptide is dependent on the cluster of basic residues found N-terminal (P-6 to P-9) as well as C-terminal (P + 1/P + 2) to the phosphorylated Ser. The results establish a new general phosphorylation motif for CDPKs: [Basic-Basic-X-Basic]-phi-X(4)-S/T-X-Basic (where phi is a hydrophobic residue). The motif predicts a number of new phosphorylation sites in plant proteins. Evidence is presented that the novel motif may explain the phosphorylation by CDPKs of Ser271 in the aquaporin PM28A.
钙依赖蛋白激酶（CDPKs）是植物中蛋白激酶大家族的成员，参与调控植物生长和发育的诸多方面。CDPKs的一个已知底物是内质网定位的ACA2钙泵，它通过Ser(45)的磷酸化进行调控。在本研究中，基于已知调控磷酸化位点（RRFRFTANLS(45)KRYEA）的合成肽在体外能被CDPKs有效磷酸化，但不能被植物SNF1相关蛋白激酶磷酸化。Ser(45)-ACA2肽的磷酸化令人惊讶，因为该序列在P-3/P-4（相对于磷酸化的Ser在P位）缺乏碱性残基，而这些残基被认为是CDPKs的关键识别元件。我们证明Ser(45)-ACA2肽的磷酸化依赖于在磷酸化Ser残基N端（P-6至P-9）以及C端（P + 1/P + 2）发现的碱性残基簇。这些结果为CDPKs建立了一个新的通用磷酸化基序：[碱性-碱性-X-碱性]-φ-X(4)-S/T-X-碱性（其中φ是一个疏水残基）。该基序预测了植物蛋白中的一些新磷酸化位点。有证据表明，这个新基序可能解释了水通道蛋白PM28A中Ser271被CDPKs磷酸化的现象。
