SELECTIVE LOSS OF SUBSTRATE RECOGNITION INDUCED BY THE TUMOR-ASSOCIATED D294G POINT MUTATION IN PROTEIN-KINASE-C-ALPHA

The tumour-associated D294G mutant of protein kinase C alpha (PKC alph a) was recently shown not to be translocated to the plasma membrane on stimulation with PMA, in contrast with the wildtype enzyme. Using rec ombinant wild-type and mutant PKC alpha, we establish here that, altho ugh the PKC alpha intrinsic lipid-dependent catalytic activity remains unaltered by the D294G mutation, the mutant enzyme exhibits a selecti ve loss of substrate recognition. Indeed, whereas the mutant enzyme is still able to phosphorylate histone IIIS with comparable efficiency t o 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 PKC alpha to the 35F a nd 35H proteins compared with that of the wild-type enzyme. Because th e 35H and 35F proteins are predicted to be PKC alpha-anchoring protein s, these findings suggest a selective loss of PKC alpha-protein intera ctions that might fail to stabilize the location of the PKC alpha muta nt at the plasma membrane.