PMID- 26769967 OWN - NLM STAT- MEDLINE DCOM- 20160808 LR - 20210205 IS - 1083-351X (Electronic) IS - 0021-9258 (Print) IS - 0021-9258 (Linking) VI - 291 IP - 12 DP - 2016 Mar 18 TI - Protein Kinase Calpha (PKCalpha) Is Resistant to Long Term Desensitization/Down-regulation by Prolonged Diacylglycerol Stimulation. PG - 6331-46 LID - 10.1074/jbc.M115.696211 [doi] AB - Sustained activation of PKCalpha 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 PKCalpha 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 PKCalpha in a pattern comparable with that induced by PMA. However, although PMA potently down-regulated PKCalpha, 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 PKCalpha did not undergo dephosphorylation, ubiquitination, or internalization, early events in PKCalpha desensitization. Although DiC8 efficiently down-regulated novel PKCs PKCdelta and PKCϵ, differences in Ca(2+) sensitivity and diacylglycerol affinity were excluded as mediators of the selective resistance of PKCalpha. Roles for Hsp/Hsc70 and Hsp90 were also excluded. PMA, but not DiC8, targeted PKCalpha 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 PKCalpha in several cell types and did not affect PKCbetaI; thus, conventional PKCs appear generally insensitive to desensitization by sustained diacylglycerol stimulation. Consistent with this conclusion, prolonged (several-day) membrane association/activation of PKCalpha is seen in self-renewing epithelium of the intestine, cervix, and skin. PKCalpha deficiency affects gene expression, differentiation, and tumorigenesis in these tissues, highlighting the importance of mechanisms that protect PKCalpha from desensitization in vivo. CI - (c) 2016 by The American Society for Biochemistry and Molecular Biology, Inc. FAU - Lum, Michelle A AU - Lum MA AD - 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. FAU - Barger, Carter J AU - Barger CJ AD - 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. FAU - Hsu, Alice H AU - Hsu AH AD - 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. FAU - Leontieva, Olga V AU - Leontieva OV AD - the Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, New York 14263. FAU - Black, Adrian R AU - Black AR AD - 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. FAU - Black, Jennifer D AU - Black JD AD - 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. LA - eng GR - DK060632/DK/NIDDK NIH HHS/United States GR - R21 CA191894/CA/NCI NIH HHS/United States GR - P30 CA016056/CA/NCI NIH HHS/United States GR - R01 DK060632/DK/NIDDK NIH HHS/United States GR - CA036727/CA/NCI NIH HHS/United States GR - P30 CA036727/CA/NCI NIH HHS/United States GR - CA191894/CA/NCI NIH HHS/United States GR - R01 DK054909/DK/NIDDK NIH HHS/United States GR - R56 DK060632/DK/NIDDK NIH HHS/United States GR - DK054909/DK/NIDDK NIH HHS/United States GR - CA016056/CA/NCI NIH HHS/United States GR - T32 CA009476/CA/NCI NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural DEP - 20160114 PL - United States TA - J Biol Chem JT - The Journal of biological chemistry JID - 2985121R RN - 0 (Diglycerides) RN - 1069-87-0 (1,2-dioctanoylglycerol) RN - EC 2.7.11.13 (PRKCA protein, human) RN - EC 2.7.11.13 (Protein Kinase C-alpha) RN - NI40JAQ945 (Tetradecanoylphorbol Acetate) SB - IM MH - Animals MH - Cell Line, Tumor MH - Diglycerides/*pharmacology MH - Down-Regulation MH - Enzyme Activation MH - Humans MH - Intestinal Mucosa/enzymology MH - Membrane Microdomains/enzymology MH - Protein Kinase C-alpha/*metabolism MH - Protein Transport MH - Proteolysis MH - Rats MH - Signal Transduction MH - Tetradecanoylphorbol Acetate/pharmacology PMC - PMC4813581 OTO - NOTNLM OT - 70-kilodalton heat shock protein (Hsp70) OT - PKCalpha, desensitization OT - diacylglycerol OT - heat shock protein 90 (Hsp90) OT - lipid raft OT - protein degradation OT - protein kinase C (PKC) OT - protein phosphorylation OT - signal transduction EDAT- 2016/01/16 06:00 MHDA- 2016/08/09 06:00 PMCR- 2017/03/18 CRDT- 2016/01/16 06:00 PHST- 2015/10/01 00:00 [received] PHST- 2016/01/16 06:00 [entrez] PHST- 2016/01/16 06:00 [pubmed] PHST- 2016/08/09 06:00 [medline] PHST- 2017/03/18 00:00 [pmc-release] AID - S0021-9258(20)44327-3 [pii] AID - M115.696211 [pii] AID - 10.1074/jbc.M115.696211 [doi] PST - ppublish SO - J Biol Chem. 2016 Mar 18;291(12):6331-46. doi: 10.1074/jbc.M115.696211. Epub 2016 Jan 14.