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Selective loss of substrate recognition induced by the tumour-associated D294G point mutation in protein kinase
C Prévostel1, V Alvaro, A Vallentin
1INSERM U469, 141 rue de la Cardonille, 34094 Montpellier cedex 05, France.
Abstract:
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.
Insights
The D294G mutation in protein kinase Calpha (PKCalpha) impairs its interaction with specific protein substrates, affecting its cellular localization. This suggests a loss of crucial protein-binding interactions for the mutant enzyme.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Protein kinase Calpha (PKCalpha) is crucial for cellular signaling.
- A tumor-associated D294G mutation affects PKCalpha's plasma membrane translocation.
- Wild-type PKCalpha translocates to the plasma membrane upon phorbol 12-myristate 13-acetate (PMA) stimulation.
Purpose of the Study:
- To investigate the functional consequences of the D294G mutation in PKCalpha.
- To determine if the mutation affects catalytic activity or substrate recognition.
- To elucidate the impact of the mutation on PKCalpha-protein interactions and localization.
Main Methods:
- Recombinant wild-type and D294G mutant PKCalpha were used.
- Lipid-dependent catalytic activity was assessed.
- Phosphorylation of histone IIIS, 35F, and 35H substrates was measured.
- Protein binding assays (overlay experiments) were performed.
Main Results:
- The D294G mutation did not alter the intrinsic lipid-dependent catalytic activity of PKCalpha.
- The mutant enzyme showed selective loss of kinase activity towards 35F and 35H substrates.
- Binding affinity of D294G PKCalpha to 35F and 35H proteins was reduced compared to wild-type.
- Histone IIIS phosphorylation efficiency was comparable between wild-type and mutant enzymes.
Conclusions:
- The D294G mutation selectively impairs PKCalpha's recognition and binding to specific protein substrates (35F and 35H).
- This loss of protein interaction may explain the failure of the mutant enzyme to localize to the plasma membrane.
- These findings highlight the importance of protein-protein interactions in regulating PKCalpha function and localization.