title: The deubiquitinating enzyme UBPy/USP8 interacts with TrkA and inhibits neuronal differentiation in PC12 cells.
abstract: The tropomyosin-related kinase (Trk) family of receptor tyrosine kinases controls synaptic function, plasticity and sustains differentiation, morphology, and neuronal cell survival. Understanding Trk receptors down-regulation and recycling is a crucial step to point out sympathetic and sensory neuron function and survival. PC12 cells derived from pheochromocytoma of the rat adrenal medulla have been widely used as a model system for studies of neuronal differentiation as they respond to nerve growth factor (NGF) with a dramatic change in phenotype and acquire a number of properties characteristic of sympathetic neurons. In this study we demonstrated that in PC12 cells the TrkA receptor interacts with the deubiquitinating enzyme USP8/UBPy in a NGF-dependent manner and that it is deubiquitinated in vivo and in vitro by USP8. USP8 overexpression blocked NGF-induced neurites outgrowth while the overexpression of the catalytically inactive mutant USP8/UBPy(C748A) caused a marked increase of cell differentiation. Localization and biochemical experiments have point out that USP8 and TrkA partially co-localize in endosomes after NGF stimulation. Finally we have studied the role played by USP8 on TrkA turnover; using specific siRNA for USP8 we found that USP8 knockdown increases TrkA half-life, suggesting that the deubiquitinating activity of USP8 promotes TrkA degradation.
authors: Department of Biotechnology and Biosciences, University of Milano-Bicocca, Piazza della Scienza 2, 20126 Milan, Italy. Electronic address: michela.ceriani@unimib.it.
authors: Michela
authors: M
authors: Ceriani
authors: Department of Biotechnology and Biosciences, University of Milano-Bicocca, Piazza della Scienza 2, 20126 Milan, Italy. Electronic address: loredana.amigoni1@unimib.it.
authors: Loredana
authors: L
authors: Amigoni
authors: Department of Biotechnology and Biosciences, University of Milano-Bicocca, Piazza della Scienza 2, 20126 Milan, Italy. Electronic address: a.daloia1@campus.unimib.it.
authors: Alessia
authors: A
authors: D'Aloia
authors: Department of Biosciences, University of Milan, Via Celoria 26, 20133 Milan, Italy. Electronic address: giovanna.berruti@unimi.it.
authors: Giovanna
authors: G
authors: Berruti
authors: Department of Biotechnology and Biosciences, University of Milano-Bicocca, Piazza della Scienza 2, 20126 Milan, Italy. Electronic address: enzo.martegani@unimib.it.
authors: Enzo
authors: E
authors: Martegani
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D011992:Endosomes / Q000201:enzymology; D057809:HEK293 Cells; D006801:Humans; D020932:Nerve Growth Factor / Q000502:physiology; D009474:Neurons / Q000502:physiology*; D016716:PC12 Cells; D021381:Protein Transport; D059748:Proteolysis; D051381:Rats; D020917:Receptor, trkA / Q000378:metabolism*; D064570:Ubiquitin-Specific Proteases / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D020932:Nerve Growth Factor; D020917:Receptor, trkA; C000598578:USP8 protein, rat; D064570:Ubiquitin-Specific Proteases
keywords: Deubiquitination; Differentiation; PC12 cells; Signaling endosomes; TrkA; USP8/UBPy