title: Agonist-induced down-regulation of endogenous protein kinase c α through an endolysosomal mechanism.
abstract: Protein kinase C (PKC) isozymes undergo down-regulation upon sustained stimulation. Previous studies have pointed to the existence of both proteasome-dependent and -independent pathways of PKCα processing. Here we demonstrate that these down-regulation pathways are engaged in different subcellular compartments; proteasomal degradation occurs mainly at the plasma membrane, whereas non-proteasomal processing occurs in the perinuclear region. Using cholesterol depletion, pharmacological inhibitors, RNA interference, and dominant-negative mutants, we define the mechanisms involved in perinuclear accumulation of PKCα and identify the non-proteasomal mechanism mediating its degradation. We show that intracellular accumulation of PKCα involves at least two clathrin-independent, cholesterol/lipid raft-mediated pathways that do not require ubiquitination of the protein; one is dynamin-dependent and likely involves caveolae, whereas the other is dynamin- and small GTPase-independent. Internalized PKCα traffics through endosomes and is delivered to the lysosome for degradation. Supportive evidence includes (a) detection of the enzyme in EEA1-positive early endosomes, Rab7-positive late endosomes/multivesicular bodies, and LAMP1-positive lysosomes and (b) inhibition of its down-regulation by lysosome-disrupting agents and leupeptin. Only limited dephosphorylation of PKCα occurs during trafficking, with fully mature enzyme being the main target for lysosomal degradation. These studies define a novel and widespread mechanism of desensitization of PKCα signaling that involves endocytic trafficking and lysosome-mediated degradation of the mature, fully phosphorylated protein.
authors.affiliation: The Eppley Institute, University of Nebraska Medical Center, Omaha, Nebraska 68198-5950, USA.
authors.forename: Michelle A
authors.initials: MA
authors.lastname: Lum
authors.forename: Krista E
authors.initials: KE
authors.lastname: Pundt
authors.forename: Benjamin E
authors.initials: BE
authors.lastname: Paluch
authors.forename: Adrian R
authors.initials: AR
authors.lastname: Black
authors.forename: Jennifer D
authors.initials: JD
authors.lastname: Black
mesh_terms: D000818:Animals; D015536:Down-Regulation; D011992:Endosomes / Q000201:enzymology* / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D006367:HeLa Cells; D006801:Humans; D008247:Lysosomes / Q000201:enzymology* / Q000235:genetics; D021962:Membrane Microdomains / Q000201:enzymology / Q000235:genetics; D010766:Phosphorylation / Q000235:genetics; D051571:Protein Kinase C-alpha / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport; D059748:Proteolysis; D051381:Rats; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D000090802:rab7 GTP-Binding Proteins
chemical_list: D033921:Vesicular Transport Proteins; C093846:early endosome antigen 1; D000090802:rab7 GTP-Binding Proteins; C000716463:rab7 GTP-binding proteins, human; C000716466:rab7 GTP-binding proteins, rat; C496628:PRKCA protein, human; D051571:Protein Kinase C-alpha; D020691:rab GTP-Binding Proteins
keywords: Caveolae; Endocytosis; Lipid Raft; Lysosomal; Lysosomes; PKCα Degradation; Protein Degradation; Protein Kinase C (PKC)