Agonist-induced down-regulation of endogenous protein kinase C α through an endolysosomal mechanism

Michelle A. Lum, Krista E. Pundt, Benjamin E. Paluch, Adrian R Black, Jennifer D Black

Research output: Contribution to journalArticle

11 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)13093-13109
Number of pages17
JournalJournal of Biological Chemistry
Volume288
Issue number18
DOIs
StatePublished - May 3 2013

Fingerprint

Protein Kinase C
Down-Regulation
Lysosomes
Endosomes
Degradation
Dynamins
Cholesterol
Multivesicular Bodies
Caveolae
Clathrin
Monomeric GTP-Binding Proteins
Ubiquitination
Proteasome Endopeptidase Complex
Enzymes
Cell membranes
Processing
RNA Interference
Isoenzymes
Proteins
Cell Membrane

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Agonist-induced down-regulation of endogenous protein kinase C α through an endolysosomal mechanism. / Lum, Michelle A.; Pundt, Krista E.; Paluch, Benjamin E.; Black, Adrian R; Black, Jennifer D.

In: Journal of Biological Chemistry, Vol. 288, No. 18, 03.05.2013, p. 13093-13109.

Research output: Contribution to journalArticle

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