Mutations in the parkin gene result in an autosomal recessive juvenile-onset form of Parkinson's disease. As an E3 ubiquitin-ligase, parkin promotes the attachment of ubiquitin onto specific substrate proteins. Defects in the ubiquitination of parkin substrates are therefore believed to lead to neurodegeneration in Parkinson's disease. Here, we identify the PSD-95/Discs-large/Zona Occludens-1 (PDZ) protein PICK1 as a novel parkin substrate. We find that parkin binds PICK1 via a PDZ-mediated interaction, which predominantly promotes PICK1 monoubiquitination rather than polyubiquitination. Consistent with monoubiquitination and recent work implicating parkin in proteasome-independent pathways, parkin does not promote PICK1 degradation. However, parkin regulates the effects of PICK1 on one of its other PDZ partners, the acid-sensing ion channel (ASIC). Overexpression of wild-type, but not PDZ binding-or E3 ubiquitinligase-defective parkin abolishes the previously described, protein kinase C-induced, PICK1-dependent potentiation of ASIC2a currents in non-neuronal cells. Conversely, the loss of parkin in hippocampal neurons from parkin knockout mice unmasks prominent potentiation of native ASIC currents, which is normally suppressed by endogenous parkin in wild-type neurons. Given that ASIC channels contribute to excitotoxicity, our work provides a mechanism explaining how defects in parkin-mediated PICK1 monoubiquitination could enhance ASIC activity and thereby promote neurodegeneration in Parkinson's disease. INTRODUCTIONParkinson's disease (PD) is characterized by the selective and progressive loss of midbrain dopamine neurons resulting in motor dysfunction and disability. Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism, which accounts for a large proportion of genetically linked PD cases (Kitada et al., 1998). Parkin encodes a 465-amino acid protein (ϳ52 kDa) that is expressed in multiple tissues and functions in the ubiquitin (Ub) system as an E3 Ub-ligase (Shimura et al., 2000). Ubiquitination of substrate proteins is a tightly regulated process, requiring the combined activity of three enzymes: an E1 Ub-activating enzyme, E2 Ub-conjugating enzymes, and E3 Ub-ligases (Hershko and Ciechanover, 1998). E3 Ub-ligases bind substrate proteins and therefore regulate and confer specificity to the ubiquitination reaction. Typically, ubiquitination leads to the assembly of a K48-linked polyubiquitin chain on the substrate, which targets it for degradation by the 26S proteasome, a large multimeric proteolytic complex (Voges et al., 1999). Accordingly, defects in parkin-mediated ubiquitination have been proposed to result in the failure to target parkin substrates to the proteasome for degradation (Kahle et al., 2000;Feany and Pallanck, 2003;Giasson and Lee, 2003). The ensuing accumulation of parkin substrates is believed, in turn, to induce the cellular toxicity and dopamine neuron loss seen in PD. Although numerous parkin substrates have been identified (Zhang et al., 2000;Chung et al., 2001...

A monoclonal antibody raised against adenovirus E1A-associated cellular proteins recognized Nek9, a NimA-related protein kinase. Subcellular fractionation and immunofluorescence indicated that Nek9 was primarily cytoplasmic with a small portion located in the nucleus whereas E1A was primarily nuclear. Although co-immunoprecipitation experiments indicated that nuclear Nek9 interacted, directly or indirectly, with E1A, the major effect of E1A was to diminish the amount of Nek9 in the nucleus suggesting that E1A alters the subcellular distribution of Nek9 and that the interaction is transient. A Nek9 deletion mutant lacking a central RCC1-like domain interacted stably with E1A and accumulated in the nucleus in the presence of E1A, possibly representing an intermediate stage of the normally transient Nek9/E1A interaction. The interaction of Nek9 with E1A was dependent on the N-terminal sequences of E1A. Attempts to stably overexpress either Nek9 or the kinase-inactive mutant in various cell lines were unsuccessful; however, the presence of E1A allowed stable overexpression of both proteins. These results suggest that E1A disrupts a nuclear function of Nek9.

Patients who survive injuries experience residual impairments in FC and QOL for as long as 1 year after injury. Changes to the long-term management of these patients should be considered.