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A novel antiproliferative PKCα-Ras-ERK signaling axis in intestinal epithelial cells
Navneet Kaur1, Michelle A Lum1, Robert E Lewis1
1Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska, USA.
Abstract:
We have previously shown that the serine/threonine kinase PKCα triggers MAPK/ERK kinase (MEK)-dependent G1→S cell cycle arrest in intestinal epithelial cells, characterized by downregulation of cyclin D1 and inhibitor of DNA-binding protein 1 (Id1) and upregulation of the cyclin-dependent kinase inhibitor p21Cip1. Here, we use pharmacological inhibitors, genetic approaches, siRNA-mediated knockdown, and immunoprecipitation to further characterize antiproliferative ERK signaling in intestinal cells. We show that PKCα signaling intersects the Ras-Raf-MEK-ERK kinase cascade at the level of Ras small GTPases and that antiproliferative effects of PKCα require active Ras, Raf, MEK, and ERK, core ERK pathway components that are also essential for pro-proliferative ERK signaling induced by epidermal growth factor (EGF). However, PKCα-induced antiproliferative signaling differs from EGF signaling in that it is independent of the Ras guanine nucleotide exchange factors (Ras-GEFs), SOS1/2, and involves prolonged rather than transient ERK activation. PKCα forms complexes with A-Raf, B-Raf, and C-Raf that dissociate upon pathway activation, and all three Raf isoforms can mediate PKCα-induced antiproliferative effects. At least two PKCα-ERK pathways that collaborate to promote growth arrest were identified: one pathway requiring the Ras-GEF, RasGRP3, and H-Ras, leads to p21Cip1 upregulation, while additional pathway(s) mediate PKCα-induced cyclin D1 and Id1 downregulation. PKCα also induces ERK-dependent SOS1 phosphorylation, indicating possible negative crosstalk between antiproliferative and growth-promoting ERK signaling. Importantly, the spatiotemporal activation of PKCα and ERK in the intestinal epithelium in vivo supports the physiological relevance of these pathways and highlights the importance of antiproliferative ERK signaling to tissue homeostasis in the intestine.
Insights
Protein kinase C alpha (PKCα) activates anti-proliferative ERK signaling in intestinal cells by interacting with Ras GTPases. This pathway, distinct from EGF signaling, involves prolonged ERK activation and promotes cell cycle arrest.
Area of Science:
- Molecular Biology
- Cell Signaling
- Gastrointestinal Physiology
Background:
- Previously established that serine/threonine kinase PKCα induces G1→S cell cycle arrest in intestinal epithelial cells.
- This arrest is characterized by specific changes in cell cycle regulators: downregulation of cyclin D1 and Id1, and upregulation of p21Cip1.
- The precise mechanisms by which PKCα signaling intersects with the MAPK/ERK pathway to mediate antiproliferative effects remained to be fully elucidated.
Purpose of the Study:
- To further characterize the antiproliferative ERK signaling initiated by PKCα in intestinal cells.
- To identify the specific components and pathways involved in PKCα-mediated cell cycle arrest.
- To investigate the crosstalk between PKCα-induced antiproliferative signaling and growth-promoting signaling pathways.
Main Methods:
- Utilized pharmacological inhibitors and genetic approaches to dissect the signaling cascade.
- Employed siRNA-mediated knockdown to specifically target key signaling molecules.
- Used immunoprecipitation to identify protein complexes and interactions.
- Investigated Ras-GEF independence and ERK activation kinetics.
Main Results:
- PKCα signaling intersects the Ras-Raf-MEK-ERK cascade at Ras small GTPases, requiring active Ras, Raf, MEK, and ERK for antiproliferative effects.
- PKCα-induced signaling is distinct from EGF signaling, being independent of SOS1/2 Ras-GEFs and characterized by prolonged ERK activation.
- PKCα forms complexes with A-Raf, B-Raf, and C-Raf, with all isoforms mediating antiproliferative effects.
- Two distinct PKCα-ERK pathways were identified: one involving RasGRP3 and H-Ras leading to p21Cip1 upregulation, and others mediating cyclin D1 and Id1 downregulation.
- PKCα induces ERK-dependent SOS1 phosphorylation, suggesting negative crosstalk with pro-proliferative pathways.
Conclusions:
- PKCα orchestrates antiproliferative ERK signaling in intestinal cells through distinct pathways involving Ras GTPases and Raf isoforms.
- The prolonged ERK activation and Ras-GEF independence differentiate PKCα signaling from growth-promoting EGF signaling.
- Spatiotemporal activation of PKCα and ERK in vivo confirms the physiological relevance of these findings for intestinal homeostasis.
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