Selective loss of substrate recognition induced by the tumour-associated D294G point mutation in protein kinase Calpha.
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.
INSERM U469, 141 rue de la Cardonille, 34094 Montpellier cedex 05, France.
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Prévostel
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Alvaro
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Vallentin
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Martin
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Jaken
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Joubert
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D006657:Histones / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009369:Neoplasms / Q000201:enzymology / Q000235:genetics; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D017354:Point Mutation; D011493:Protein Kinase C / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera; D013379:Substrate Specificity; D014162:Transfection
D006657:Histones; D007527:Isoenzymes; D010446:Peptide Fragments; D011994:Recombinant Proteins; C496628:PRKCA protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha