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Rapid and long-term effects on protein kinase C on receptor tyrosine kinase phosphorylation and degradation
K Seedorf1, M Shearman, A Ullrich
1Department of Molecular Biology, Max-Planck-Institut für Biochemie, Martinsried, Germany.
Abstract:
Rapid and long term effects of protein kinase C alpha activation on receptor tyrosine kinase signaling parameters were investigated in human 293 embryonic fibroblasts and mouse NIH 3T3 cells. Within minutes of phorbol 12-myristate 13-acetate treatment, epidermal growth factor receptor and HER2 tyrosine phosphorylation was decreased, while platelet-derived growth factor receptor and insulin receptor autophosphorylation was upregulated. These effects are not mediated by protein kinase C-dependent receptor tyrosine kinase phosphorylation but apparently by activation or inactivation of receptor tyrosine kinase-specific phosphatases, as indicated by neutralization of these phenomena upon treatment of cells with sodium orthovanadate. In contrast to these short term effects, sustained activation of protein kinase C alpha by phorbol 12-myristate 13-acetate results in translocation of protein kinase C from the cytosol to the membrane fraction where it forms stable complexes with all receptor tyrosine kinases investigated. Ligand-induced receptor tyrosine kinase/protein kinase C association in NIH 3T3 fibroblasts is accompanied by a mobility shift of the receptor, indicating phosphorylation by activated protein kinase C. This phenomenon correlates with the disappearance of receptor tyrosine kinases from the cell surface, implying that this interaction plays a role in the process of receptor internalization and degradation. Interestingly, ligand-stimulated receptor down-regulation is also enhanced by overexpression of phospholipase C gamma, which strongly indicates a role for this common receptor tyrosine kinase substrate in negative regulation of growth factor signals.
Insights
Protein kinase C alpha activation rapidly alters receptor tyrosine kinase (RTK) signaling by modulating phosphatases. Sustained activation leads to RTK complex formation, phosphorylation, and degradation, impacting growth factor signaling.
Area of Science:
- Cellular Biology
- Molecular Signaling
- Biochemistry
Background:
- Receptor tyrosine kinases (RTKs) are crucial for cell growth and differentiation.
- Protein kinase C (PKC) is a key signaling enzyme involved in various cellular processes.
- The interplay between PKC and RTK signaling is complex and requires further elucidation.
Purpose of the Study:
- To investigate the rapid and long-term effects of Protein Kinase C alpha (PKCα) activation on RTK signaling.
- To determine the mechanisms underlying PKC-mediated modulation of RTK activity and stability.
Main Methods:
- Utilized human embryonic 293 and mouse NIH 3T3 fibroblasts.
- Employed phorbol 12-myristate 13-acetate (PMA) to activate PKCα.
- Assessed RTK phosphorylation, phosphatase activity (using sodium orthovanadate), protein complex formation, and receptor localization via cell surface assays.
Main Results:
- Short-term PKCα activation rapidly altered RTK autophosphorylation, decreasing epidermal growth factor receptor (EGFR) and HER2, while upregulating platelet-derived growth factor receptor (PDGFR) and insulin receptor.
- These rapid effects were mediated by RTK-specific phosphatases, not direct PKC phosphorylation of RTKs.
- Sustained PKCα activation resulted in PKC translocation, stable complex formation with RTKs, RTK phosphorylation by PKC, and subsequent receptor internalization and degradation.
- Overexpression of phospholipase C gamma (PLCγ) enhanced ligand-stimulated RTK down-regulation, highlighting PLCγ's role in negative growth factor signal regulation.
Conclusions:
- PKCα activation has differential and time-dependent effects on RTK signaling parameters.
- PKCα influences RTK stability and degradation through direct interaction and phosphorylation.
- The findings reveal a novel regulatory pathway involving PKCα, phosphatases, and PLCγ in controlling RTK-mediated cellular responses.