Stahelin Robert V, Subramanian Preeti, Vora Mohsin, Cho Wonhwa, Chalfant Charles E
Department of Biochemistry, Medical College of Virginia Campus, Virginia Commonwealth University, 1101 East Marshall Street, Richmond, VA 23298-0614, USA.
J Biol Chem. 2007 Jul 13;282(28):20467-74. doi: 10.1074/jbc.M701396200. Epub 2007 Apr 30.
Previously, ceramide-1-phosphate (C1P) was demonstrated to be a potent and specific activator of group IV cytosolic phospholipase A(2)alpha (cPLA(2)alpha) via interaction with the C2 domain. In this study, we hypothesized that the specific interaction site for C1P was localized to the cationic beta-groove (Arg(57), Lys(58), Arg(59)) of the C2 domain of cPLA(2)alpha. In this regard, mutants of this region of cPLA(2)alpha were generated (R57A/K58A/R59A, R57A/R59A, K58A/R59A, R57A/K58A, R57A, K58A, and R59A) and examined for C1P affinity by surface plasmon resonance. The triple mutants (R57A/K58A/R59A), the double mutants (R57A/R59A, K58A/R59A, and R57A/K58A), and the single mutant (R59A) demonstrated significantly reduced affinity for C1P-containing vesicles as compared with wild-type cPLA(2)alpha. Examining these mutants for enzymatic activity demonstrated that these five mutants of cPLA(2)alpha also showed a significant reduction in the ability of C1P to: 1) increase the V(max) of the reaction; and 2) significantly decrease the dissociation constant (K (A)(s)) of the reaction as compared with the wild-type enzyme. The mutational effect was specific for C1P as all of the cationic mutants of cPLA(2)alpha demonstrated normal basal activity as well as normal affinities for phosphatidylcholine and phosphatidylinositol-4,5-bisphosphate as compared with wild-type cPLA(2)alpha. This study, for the first time, demonstrates a novel C1P interaction site mapped to the cationic beta-groove of the C2 domain of cPLA(2)alpha.
此前，已证明神经酰胺 -1- 磷酸（C1P）通过与 C2 结构域相互作用，是 IV 型胞质磷脂酶 A(2)α（cPLA(2)α）的一种强效且特异性激活剂。在本研究中，我们假设 C1P 的特异性相互作用位点定位于 cPLA(2)α 的 C2 结构域的阳离子β-凹槽（Arg(57)、Lys(58)、Arg(59)）。就此而言，构建了 cPLA(2)α 该区域的突变体（R57A/K58A/R59A、R57A/R59A、K58A/R59A、R57A/K58A、R57A、K58A 和 R59A），并通过表面等离子体共振检测其对 C1P 的亲和力。与野生型 cPLA(2)α 相比，三突变体（R57A/K58A/R59A）、双突变体（R57A/R59A、K58A/R59A 和 R57A/K58A）以及单突变体（R59A）对含 C1P 的囊泡的亲和力显著降低。检测这些突变体的酶活性表明，cPLA(2)α 的这五个突变体在以下方面也显示出 C1P 能力的显著降低：1）增加反应的 V(max)；2）与野生型酶相比，显著降低反应的解离常数（K (A)(s)）。突变效应对 C1P 具有特异性，因为与野生型 cPLA(2)α 相比，cPLA(2)α 的所有阳离子突变体均表现出正常的基础活性以及对磷脂酰胆碱和磷脂酰肌醇 -4,5- 二磷酸的正常亲和力。本研究首次证明了一个新的 C1P 相互作用位点定位于 cPLA(2)α 的 C2 结构域的阳离子β-凹槽。
