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Tyrosine phosphorylation of protein kinase C-delta in response to its activation
1Laboratory of Cellular and Molecular Biology, National Cancer Institute, Bethesda, Maryland 20892.
Abstract:
Retroviral vectors containing five different protein kinase C (PKC) isoenzymes (alpha, delta, epsilon, eta, zeta) were expressed in 32D hematopoietic cells and NIH-3T3 fibroblasts. In an effort to investigate signaling events regulated by PKC activation, we analyzed whether tyrosine phosphorylation of cellular proteins would occur after 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment of the various transfectants. While no detectable tyrosine-specific phosphorylation was observed after treatment of the majority of the transfectants, pronounced TPA-dependent tyrosine phosphorylation of an 82-kDa protein was detected in the 32D/PKC-delta and NIH-3T3/PKC-delta lines. Interestingly, the 82-kDa substrate proved to be PKC-delta itself. Tyrosine phosphorylation of purified PKC-delta by src family or receptor tyrosine kinases in vitro enhanced PKC-delta activity, suggesting that tyrosine phosphorylation of PKC-delta may positively affect its function.
Insights
Protein kinase C delta (PKC-delta) undergoes tyrosine phosphorylation upon 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulation. This phosphorylation enhances PKC-delta activity, suggesting a regulatory role in cellular signaling pathways.
Area of Science:
- Cellular signaling
- Molecular biology
- Biochemistry
Background:
- Protein kinase C (PKC) is a family of enzymes involved in various cellular processes.
- Investigating the specific roles of different PKC isoenzymes is crucial for understanding cellular regulation.
- Tyrosine phosphorylation is a key post-translational modification regulating protein function.
Purpose of the Study:
- To investigate the signaling events regulated by protein kinase C (PKC) activation.
- To determine if tyrosine phosphorylation of cellular proteins occurs following 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment in cells expressing different PKC isoenzymes.
- To identify the specific PKC isoenzyme, if any, that undergoes tyrosine phosphorylation.
Main Methods:
- Retroviral vectors were used to express five different PKC isoenzymes (alpha, delta, epsilon, eta, zeta) in 32D hematopoietic cells and NIH-3T3 fibroblasts.
- Cells were treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) to activate PKC.
- Tyrosine phosphorylation of cellular proteins was analyzed using Western blotting or similar techniques.
- Purified PKC-delta was subjected to in vitro phosphorylation by src family or receptor tyrosine kinases.
Main Results:
- TPA treatment induced pronounced, time-dependent tyrosine phosphorylation of an 82-kDa protein specifically in cells expressing PKC-delta.
- The 82-kDa protein undergoing tyrosine phosphorylation was identified as PKC-delta itself.
- Tyrosine phosphorylation of purified PKC-delta by src family or receptor tyrosine kinases in vitro enhanced its kinase activity.
Conclusions:
- PKC-delta is a substrate for tyrosine phosphorylation in response to TPA stimulation.
- Tyrosine phosphorylation of PKC-delta may serve as a positive regulatory mechanism, enhancing its enzymatic activity.
- This finding reveals a novel regulatory mechanism for PKC-delta function in cellular signaling pathways.