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Mutants affected in the putative diacylglycerol binding site of yeast protein kinase C
J J Jacoby1, H P Schmitz, J J Heinisch
1Institut für Mikrobiologie, Heinrich-Heine-Universität Düsseldorf, Germany.
Abstract:
In an attempt to study the functional similarities between protein kinase C from the yeast Saccharomyces cerevisiae and its human homologues we have started in vitro mutagenesis to alter specific domains. Here we report on the exchange of four cysteine residues by serines in yeast Pkc1p that have been shown to be essential for diacylglycerol (DAG) binding and activation by this compound in humans. The mutant yeast protein leads to sensitivity to caffeine and low concentrations of SDS when expressed in a pkc1 deletion strain. However, sensitivity to staurosporine was not affected. Our data indicate that the conserved DAG binding domain serves an important function in yeast Pkc1p.
Insights
Mutating key sites in yeast protein kinase C (PKC) revealed its diacylglycerol (DAG) binding domain is crucial for function. This study highlights conserved functions between yeast and human PKC proteins.
Area of Science:
- Molecular Biology
- Biochemistry
- Yeast Genetics
Background:
- Protein kinase C (PKC) is a family of enzymes involved in cell signaling.
- Human and yeast homologues of PKC share some functional similarities.
- The diacylglycerol (DAG) binding domain is critical for human PKC activation.
Purpose of the Study:
- To investigate the functional role of the DAG binding domain in yeast Protein Kinase C 1 (Pkc1p).
- To compare the function of yeast Pkc1p with its human homologues using in vitro mutagenesis.
Main Methods:
- In vitro mutagenesis was used to exchange four cysteine residues with serines in yeast Pkc1p.
- The mutant yeast protein was expressed in a pkc1 deletion strain.
- Sensitivity to caffeine, SDS, and staurosporine was assessed.
Main Results:
- The mutant yeast Pkc1p exhibited sensitivity to caffeine and low concentrations of SDS.
- Sensitivity to staurosporine was not altered in the mutant.
- The conserved DAG binding domain was shown to be important for yeast Pkc1p function.
Conclusions:
- The diacylglycerol (DAG) binding domain plays a significant role in the function of yeast Pkc1p.
- This finding supports the conservation of function for this domain between yeast and human PKC proteins.