calyculin-a and bryostatin-1

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.

Topics: Animals; Antineoplastic Agents; Blotting, Western; Bryostatins; Calpain; Cell Line, Tumor; Cell Membrane; Cells, Cultured; Cysteine Endopeptidases; Dose-Response Relationship, Drug; Down-Regulation; Enzyme Inhibitors; Epithelial Cells; Intestinal Mucosa; Lactones; Macrolides; Marine Toxins; Microscopy, Fluorescence; Models, Biological; Multienzyme Complexes; Okadaic Acid; Oxazoles; Phosphorylation; Precipitin Tests; Proteasome Endopeptidase Complex; Protein Kinase C; Protein Kinase C-alpha; Rats; Signal Transduction; Subcellular Fractions; Tetradecanoylphorbol Acetate; Time Factors; Ubiquitin