title: Protein Kinase Cα (PKCα) Is Resistant to Long Term Desensitization/Down-regulation by Prolonged Diacylglycerol Stimulation.
abstract: Sustained activation of PKCα is required for long term physiological responses, such as growth arrest and differentiation. However, studies with pharmacological agonists (e.g. phorbol 12-myristate 13-acetate (PMA)) indicate that prolonged stimulation leads to PKCα desensitization via dephosphorylation and/or degradation. The current study analyzed effects of chronic stimulation with the physiological agonist diacylglycerol. Repeated addition of 1,2-dioctanoyl-sn-glycerol (DiC8) resulted in sustained plasma membrane association of PKCα in a pattern comparable with that induced by PMA. However, although PMA potently down-regulated PKCα, prolonged activation by DiC8 failed to engage known desensitization mechanisms, with the enzyme remaining membrane-associated and able to support sustained downstream signaling. DiC8-activated PKCα did not undergo dephosphorylation, ubiquitination, or internalization, early events in PKCα desensitization. Although DiC8 efficiently down-regulated novel PKCs PKCδ and PKCϵ, differences in Ca(2+) sensitivity and diacylglycerol affinity were excluded as mediators of the selective resistance of PKCα. Roles for Hsp/Hsc70 and Hsp90 were also excluded. PMA, but not DiC8, targeted PKCα to detergent-resistant membranes, and disruption of these domains with cholesterol-binding agents demonstrated a role for differential membrane compartmentalization in selective agonist-induced degradation. Chronic DiC8 treatment failed to desensitize PKCα in several cell types and did not affect PKCβI; thus, conventional PKCs appear generally insensitive to desensitization by sustained diacylglycerol stimulation. Consistent with this conclusion, prolonged (several-day) membrane association/activation of PKCα is seen in self-renewing epithelium of the intestine, cervix, and skin. PKCα deficiency affects gene expression, differentiation, and tumorigenesis in these tissues, highlighting the importance of mechanisms that protect PKCα from desensitization in vivo.
authors.affiliation: From the Eppley Institute for Research in Cancer and Allied Diseases and the Fred and Pamela Buffet Cancer Center, University of Nebraska Medical Center, Omaha, Nebraska 68198-5950 and the Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, New York 14263.
authors.forename: Michelle A
authors.initials: MA
authors.lastname: Lum
authors.affiliation: From the Eppley Institute for Research in Cancer and Allied Diseases and the Fred and Pamela Buffet Cancer Center, University of Nebraska Medical Center, Omaha, Nebraska 68198-5950 and.
authors.forename: Carter J
authors.initials: CJ
authors.lastname: Barger
authors.affiliation: From the Eppley Institute for Research in Cancer and Allied Diseases and the Fred and Pamela Buffet Cancer Center, University of Nebraska Medical Center, Omaha, Nebraska 68198-5950 and.
authors.forename: Alice H
authors.initials: AH
authors.lastname: Hsu
authors.affiliation: the Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, New York 14263.
authors.forename: Olga V
authors.initials: OV
authors.lastname: Leontieva
authors.affiliation: From the Eppley Institute for Research in Cancer and Allied Diseases and the Fred and Pamela Buffet Cancer Center, University of Nebraska Medical Center, Omaha, Nebraska 68198-5950 and the Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, New York 14263.
authors.forename: Adrian R
authors.initials: AR
authors.lastname: Black
authors.affiliation: From the Eppley Institute for Research in Cancer and Allied Diseases and the Fred and Pamela Buffet Cancer Center, University of Nebraska Medical Center, Omaha, Nebraska 68198-5950 and the Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, New York 14263 jennifer.black@unmc.edu.
authors.forename: Jennifer D
authors.initials: JD
authors.lastname: Black
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D004075:Diglycerides / Q000494:pharmacology*; D015536:Down-Regulation; D004789:Enzyme Activation; D006801:Humans; D007413:Intestinal Mucosa / Q000201:enzymology; D021962:Membrane Microdomains / Q000201:enzymology; D051571:Protein Kinase C-alpha / Q000378:metabolism*; D021381:Protein Transport; D059748:Proteolysis; D051381:Rats; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D004075:Diglycerides; C044295:1,2-dioctanoylglycerol; C496628:PRKCA protein, human; D051571:Protein Kinase C-alpha; D013755:Tetradecanoylphorbol Acetate
keywords: 70-kilodalton heat shock protein (Hsp70); PKCα, desensitization; diacylglycerol; heat shock protein 90 (Hsp90); lipid raft; protein degradation; protein kinase C (PKC); protein phosphorylation; signal transduction