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Updated: Aug 30, 2026
In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability
Published on: October 19, 2018
Identification of two distinct pathways of protein kinase Calpha down-regulation in intestinal epithelial cells
Olga V Leontieva1, Jennifer D Black
1Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, New York 14263, USA.
Abstract:
Signal transduction pathways are controlled by desensitization mechanisms, which can affect receptors and/or downstream signal transducers. It has long been recognized that members of the protein kinase C (PKC) family of signal transduction molecules undergo down-regulation in response to activation. Previous reports have indicated that key steps in PKCalpha desensitization include caveolar internalization, priming site dephosphorylation, ubiquitination of the dephosphorylated protein, and degradation by the proteasome. In the current study, comparative analysis of PKCalpha processing induced by the PKC agonists phorbol 12-myristate 13-acetate and bryostatin 1 in IEC-18 rat intestinal epithelial cells demonstrates that: (a) at least two pathways of PKCalpha down-regulation can co-exist within cells, and (b) a single PKC agonist can activate both pathways at the same time. Using a combined biochemical and morphological approach, we identify a novel pathway of PKCalpha desensitization that involves ubiquitination of mature, fully phosphorylated activated enzyme at the plasma membrane and subsequent down-regulation by the proteasome. The phosphatase inhibitors okadaic acid and calyculin A accelerated PKCalpha down-regulation and inhibitors of vesicular trafficking did not prevent degradation of the protein, indicating that neither internalization nor priming site dephosphorylation are requisite intermediate steps in this ubiquitin/proteasome dependent pathway of PKCalpha down-regulation. Instead, caveolar trafficking and dephosphorylation are involved in a second, proteasome-independent mechanism of PKCalpha desensitization in this system. Our findings highlight subcellular distribution and phosphorylation state as critical determinants of PKCalpha desensitization pathways.
Insights
Protein kinase C alpha (PKCalpha) desensitization involves multiple pathways. This study identifies a novel ubiquitin/proteasome dependent pathway for PKCalpha down-regulation, distinct from internalization and dephosphorylation mechanisms.
Area of Science:
- Cellular signaling
- Molecular biology
- Signal transduction
Background:
- Signal transduction pathways rely on desensitization mechanisms affecting receptors and transducers.
- Protein kinase C (PKC) family members, including PKCalpha, are known to down-regulate upon activation.
- Prior research suggested PKCalpha desensitization involves caveolar internalization, dephosphorylation, ubiquitination, and proteasomal degradation.
Purpose of the Study:
- To comparatively analyze PKCalpha processing induced by phorbol 12-myristate 13-acetate and bryostatin 1 in IEC-18 cells.
- To identify and characterize distinct pathways of PKCalpha down-regulation.
- To elucidate the role of ubiquitination, phosphorylation, and cellular trafficking in PKCalpha desensitization.
Main Methods:
- Comparative analysis of PKCalpha processing using PKC agonists (phorbol 12-myristate 13-acetate, bryostatin 1).
- Biochemical and morphological approaches in IEC-18 rat intestinal epithelial cells.
- Utilized phosphatase inhibitors (okadaic acid, calyculin A) and inhibitors of vesicular trafficking.
Main Results:
- Identified at least two co-existing pathways for PKCalpha down-regulation within cells.
- Demonstrated that a single PKC agonist can activate both pathways simultaneously.
- Characterized a novel ubiquitin/proteasome dependent pathway involving ubiquitination of activated PKCalpha at the plasma membrane.
- Showed that this novel pathway does not require internalization or priming site dephosphorylation.
- Indicated that caveolar trafficking and dephosphorylation are involved in a separate, proteasome-independent desensitization mechanism.
Conclusions:
- PKCalpha desensitization can occur through at least two distinct pathways.
- A novel pathway for PKCalpha down-regulation involves direct ubiquitination and proteasomal degradation of the activated enzyme at the plasma membrane.
- Caveolar trafficking and dephosphorylation constitute a separate, proteasome-independent desensitization route.
- Subcellular localization and phosphorylation status critically determine the PKCalpha desensitization pathway utilized.
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