Bittova L, Stahelin R V, Cho W
Department of Chemistry (M/C 111), University of Illinois at Chicago, Chicago, Illinois 60607-7061, USA.
J Biol Chem. 2001 Feb 9;276(6):4218-26. doi: 10.1074/jbc.M008491200. Epub 2000 Oct 11.
On the basis of extensive structure-function studies of protein kinase C-alpha (PKC-alpha), we have proposed an activation mechanism for conventional PKCs in which the C2 domain and the C1 domain interact sequentially with membranes (Medkova, M., and Cho, W. (1999) J. Biol. Chem. 274, 19852-19861). To further elucidate the interactions between the C1 and C2 domains during PKC activation and the origin of phosphatidylserine specificity, we mutated several charged residues in two C1 domains (C1a and C1b) of PKC-alpha. We then measured the membrane binding affinities, activities, and monolayer penetration of these mutants. Results indicate that cationic residues of the C1a domain, most notably Arg(77), interact nonspecifically with anionic phospholipids prior to the membrane penetration of hydrophobic residues. The mutation of a single aspartate (Asp(55)) in the C1a domain to Ala or Lys resulted in dramatically reduced phosphatidylserine specificity in vesicle binding, activity, and monolayer penetration. In particular, D55A showed much higher vesicle affinity, activity, and monolayer penetration power than wild type under nonactivating conditions, i.e. with phosphatidylglycerol and in the absence of Ca(2+), indicating that Asp(55) is involved in the tethering of the C1a domain to another part of PKC-alpha, which keeps it in an inactive conformation at the resting state. Based on these results, we propose a refined model for the activation of conventional PKC, in which phosphatidylserine specifically disrupts the C1a domain tethering by competing with Asp(55), which then leads to membrane penetration and diacylglycerol binding of the C1a domain and PKC activation.
基于对蛋白激酶C-α（PKC-α）广泛的结构-功能研究，我们提出了一种传统蛋白激酶C的激活机制，即C2结构域和C1结构域依次与膜相互作用（Medkova，M.，和Cho，W.（1999年）《生物化学杂志》274，19852 - 19861）。为了进一步阐明PKC激活过程中C1和C2结构域之间的相互作用以及磷脂酰丝氨酸特异性的起源，我们对PKC-α的两个C1结构域（C1a和C1b）中的几个带电荷残基进行了突变。然后我们测量了这些突变体的膜结合亲和力、活性和单层穿透能力。结果表明，在疏水残基穿透膜之前，C1a结构域的阳离子残基，最显著的是精氨酸（Arg(77)），与阴离子磷脂非特异性相互作用。C1a结构域中单个天冬氨酸（Asp(55)）突变为丙氨酸或赖氨酸会导致在囊泡结合、活性和单层穿透方面磷脂酰丝氨酸特异性显著降低。特别是，在非激活条件下，即存在磷脂酰甘油且不存在Ca(2+)时，D55A显示出比野生型高得多的囊泡亲和力、活性和单层穿透能力，这表明Asp(55)参与将C1a结构域拴系到PKC-α的另一部分，使其在静息状态下保持无活性构象。基于这些结果，我们提出了一个传统PKC激活的改进模型，其中磷脂酰丝氨酸通过与Asp(55)竞争特异性地破坏C1a结构域的拴系，进而导致C1a结构域的膜穿透和二酰基甘油结合以及PKC激活。
