Prévostel C, Alvaro V, Vallentin A, Martin A, Jaken S, Joubert D
INSERM U469, 141 rue de la Cardonille, 34094 Montpellier cedex 05, France.
Biochem J. 1998 Sep 1;334 ( Pt 2)(Pt 2):393-7. doi: 10.1042/bj3340393.
The tumour-associated D294G mutant of protein kinase Calpha (PKCalpha) was recently shown not to be translocated to the plasma membrane on stimulation with PMA, in contrast with the wild-type enzyme. Using recombinant wild-type and mutant PKCalpha, we establish here that, although the PKCalpha intrinsic lipid-dependent catalytic activity remains unaltered by the D294G mutation, the mutant enzyme exhibits a selective loss of substrate recognition. Indeed, whereas the mutant enzyme is still able to phosphorylate histone IIIS with comparable efficiency to that of the wild-type enzyme, it exhibits a lack of kinase activity towards the previously cloned 35F and 35H substrates for PKC. Overlay experiments demonstrate that this selective loss of kinase activity is correlated with a decrease in binding of D294G PKCalpha to the 35F and 35H proteins compared with that of the wild-type enzyme. Because the 35H and 35F proteins are predicted to be PKCalpha-anchoring proteins, these findings suggest a selective loss of PKCalpha-protein interactions that might fail to stabilize the location of the PKCalpha mutant at the plasma membrane.
蛋白激酶Cα（PKCα）的肿瘤相关D294G突变体最近被发现，与野生型酶不同，在用佛波酯（PMA）刺激时它不会转位到质膜上。利用重组野生型和突变型PKCα，我们在此证实，尽管D294G突变未改变PKCα固有的脂质依赖性催化活性，但突变酶表现出底物识别的选择性丧失。实际上，虽然突变酶仍能以与野生型酶相当的效率磷酸化组蛋白IIIS，但它对先前克隆的PKC的35F和35H底物缺乏激酶活性。覆盖实验表明，与野生型酶相比，激酶活性的这种选择性丧失与D294G PKCα与35F和35H蛋白结合的减少相关。由于预测35H和35F蛋白是PKCα锚定蛋白，这些发现表明PKCα-蛋白相互作用的选择性丧失，这可能无法稳定PKCα突变体在质膜上的定位。
