Interaction Partners of the Postsynaptic Protein Sharpin : Involvement in Protein Degradation in Rattus norvegicus and a Human Cell Line

Abstract

Sharpin is a protein which has been initially described as an interacting protein for the post-synaptic protein Shank. Shank functions as a scaffold protein at the glutamatergic synapse in neurons of the central nervous system. However, as Sharpin is widely expressed, and even in neurons is not exclusively localized to synaptic sites, it is likely that the cellular function of Sharpin is not limited to a role at the synapse. This work set out to identify and characterize novel protein-protein interactions of Sharpin in order to shed some light on its potential cellular functions. Sharpin contains two domains that in the context of other proteins have previously been described to play a role in the ubiquitin proteasome system (UPS): a Npl4 zinc finger (NZF) domain and a type 2 ubiquitin-like domain (Ubl). The NZF domain of Sharpin was shown here to bind to ubiquitin in a yeast two-hybrid system. The interaction was demonstrated for monoubiquitin as well as for Lys48- and Lys63-linked polyubiquitin chains. Interestingly, in a yeast two-hybrid screen with the Ubl domain of Sharpin (aa 171-304) as bait, OS-9, a protein upregulated in osteosarcoma, and the E3 ubiquitin ligase EDD1 were identified here as novel interaction partners of Sharpin. In human cells, all these interactions of Sharpin (including the interaction with Shank) were confirmed but required previous inhibition of the proteasome by MG132. Subcellular fractionation revealed that Sharpin/OS-9 complexes were predominantly present in the fractions with the highest amount of proteasome subunits again linking this interaction to UPS function. Sharpin also interacted with coexpressed Rpt1, a 19S proteasome subunit. MG132 treatment of cultured cortical neurons resulted in a substantially increased amount of endogenous Sharpin present in postsynaptic density preparations. Under these conditions, also the subcellular localization of ectopically expressed Sharpin changed significantly. A reduction in the nuclear localization of Sharpin was accompanied by its accumulation in the cytoplasm, where it was found colocalized with Shank1 in aggregate-like structures. Taken together the results presented in this work suggest a possible shuttling role for Sharpin in the UPS. Via its interaction with OS-9, Sharpin could be involved in regulating the ubiquitination and degradation of components of the hypoxia response pathway. Based on the recent identification of a mouse line which suffers from severe immunological deficits due to a spontaneous mutation in the Sharpin gene, it can also be speculated that Sharpin similarly plays a role as a ubiquitin shuttling protein for components of the non-canonical NF-kappaB signaling pathway.