Cook Anthony C, Ho Cojen, Kershner Jennifer L, Malinowski Steve A, Moldveen Heath, Stagliano Brigid A, Slater Simon J
Department of Pathology and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Biochemistry. 2006 Dec 5;45(48):14452-65. doi: 10.1021/bi0612420.
Previously, we have shown that protein kinase C (PKC) forms a direct high-affinity, isozyme-specific and membrane lipid-independent interaction with Rho GTPases [Slater, S. J., Seiz, J. L., Stagliano, B. A., and Stubbs, C. D. (2001) Biochemistry 40, 4437-4445]. Since the cellular activation of PKCalpha involves an initial translocation from cytosolic to membrane compartments, the present study investigates the interdependence between the direct protein-protein interaction of PKCalpha with the Rho GTPase, Cdc42, and the protein-lipid interactions of PKCalpha with membranes. It was hypothesized that the interaction of PKCalpha with membrane-bound Cdc42 would contribute to the overall membrane-binding affinity of the kinase by providing an additional anchor. However, it was found that the incorporation of isoprenylated Cdc42 into membranes resulted in an apparent decrease in the membrane-binding affinity of PKCalpha, whereas the association of PKCbetaI, PKCdelta, PKCepsilon, and PKCzeta was each unaffected. The presence of membrane-bound Cdc42 resulted in a rightward shift in both the PS- and Ca2+-concentration response curves for PKCalpha membrane association and for the ensuing activation, whereas the maximal levels of binding and activation attained at saturating PS and Ca2+ concentrations were in each case unaffected. Overall, these findings suggest that PKCalpha undergoes a isozyme-specific interaction with membrane-bound Cdc42 to form a PKCalpha-Cdc42 complex, which possesses a membrane-binding affinity that is reduced relative to that of the individual components due to competition between Cdc42 and PS/Ca2+ for binding to PKCalpha. Consistent with this, it was found that the interaction of PKCalpha with membrane-bound Cdc42 was accompanied by the physical dissociation of the PKCalpha-Cdc42 complex from membranes. Thus, the study provides a novel mechanism by which the membrane association and activation of PKCalpha and Cdc42 may be regulated by competing protein-protein and protein-lipid interactions.
此前，我们已经证明蛋白激酶C（PKC）与Rho GTP酶形成直接的高亲和力、同工酶特异性且不依赖膜脂质的相互作用[斯莱特，S.J.，塞兹，J.L.，斯塔利亚诺，B.A.，和斯塔布斯，C.D.（2001年）《生物化学》40卷，4437 - 4445页]。由于PKCα的细胞激活涉及从胞质溶胶到膜区室的初始转位，本研究调查了PKCα与Rho GTP酶Cdc42的直接蛋白质 - 蛋白质相互作用以及PKCα与膜的蛋白质 - 脂质相互作用之间的相互依赖性。据推测，PKCα与膜结合的Cdc42的相互作用将通过提供额外的锚定作用来促进激酶的整体膜结合亲和力。然而，发现将异戊二烯化的Cdc42掺入膜中导致PKCα的膜结合亲和力明显降低，而PKCβI、PKCδ、PKCε和PKCζ的结合不受影响。膜结合的Cdc42的存在导致PKCα膜结合及随后激活的磷脂酰丝氨酸（PS）和钙离子（Ca2 +）浓度响应曲线向右移动，而在饱和PS和Ca2 +浓度下达到的最大结合和激活水平在每种情况下均不受影响。总体而言，这些发现表明PKCα与膜结合的Cdc42发生同工酶特异性相互作用以形成PKCα - Cdc42复合物，由于Cdc42与PS/Ca2 +竞争结合PKCα，该复合物的膜结合亲和力相对于各个组分有所降低。与此一致的是，发现PKCα与膜结合的Cdc42的相互作用伴随着PKCα - Cdc42复合物从膜上的物理解离。因此，该研究提供了一种新机制，通过这种机制PKCα和Cdc42的膜结合及激活可能受竞争性蛋白质 - 蛋白质和蛋白质 - 脂质相互作用的调节。
