Ubiquitination is emerging as a key regulatory mechanism in controlling the fate and function of neuronal proteins (28
). However, not much is known about the particular substrates or ubiquitin enzymes involved in this process. In this article, we present findings that Mib2 is an E3 ubiquitin ligase that associates with and ubiquitinates the NR2B subunit of the NMDAR. We found that the interaction between Mib2 and the NR2B subunit is facilitated by Fyn kinase, suggesting that the association occurs in a phosphorylation-dependent manner. Furthermore, we demonstrate that the ubiquitination of the NR2B subunit by Mib2 is also Fyn kinase-dependent. Importantly, we show that Mib2 colocalizes with the NMDAR at postsynaptic sites and interacts with the NR2B subunit within hippocampal neurons. Finally we present data to suggest that the activity of the NMDAR is negatively regulated by Mib2 in a ubiquitin-proteasome-dependent manner.
In this study, we identify Mib2 as a protein whose association with the NR2B subunit is increased by Fyn phosphorylation. Relatively few studies have studied how NMDAR-protein interactions are dynamically regulated, such as by phosphorylation. PI 3-kinase and PLC-γ
are two proteins that have been identified as interacting with NR2B subunits in a tyrosine phosphorylation-dependent manner (24
), while serine phosphorylation of NR2B has been demonstrated to regulate the interaction of PSD-95 with the NMDAR (38
). These phosphorylation-dependent interactions are likely to play important roles in altering signaling via the NMDAR during processes such as synaptic plasticity (38
) or during pathophysiological events (20
). One plausible explanation for our current findings is that tyrosine phosphorylation of the NR2B subunit by Fyn leads to increased binding of Mib2 binding. The NR2B subunit is a known substrate for Fyn, and overexpression of FynCA was observed to lead to a significant increase in the level of tyrosine phosphorylation on the NR2B subunit. Furthermore, we observed association of Mib2 with NR2B in hippocampal slices after forskolin treatment, which induced the tyrosine phosphorylation of the NR2B subunit. Thus, Mib2 may serve a novel role in down-regulating the function of phosphorylated NR2B-containing NMDARs.
Tyrosine phosphorylation has generally been thought of as positively modulating NMDAR function (7
), however the ubiquitination and down-regulation of NMDAR function that we observed by Mib2 occurred in a Fyn kinase-dependent manner. We hypothesize that this negative regulation may be one mechanism that neurons use to avert the pathophysiological consequences of overactivation of the NMDAR, under circumstances in which Fyn activation and subsequent phosphorylation and positive modulation of the NMDAR would be detrimental if allowed to continue chronically. For example, to counterbalance excessive amounts of calcium entry through the channel that occurs during ischemia, due to up-regulation of tyrosine phosphorylation of NMDARs (20
). Interestingly, recently Wu et al.
) reported that Fyn phosphorylation of the tyrosine 1336 residue on NR2B leads to calpain-mediated degradation of the subunit. Another possibility is that Mib2 may also be regulated by Fyn phosphorylation. The tyrosine phosphorylation of other E3 ligases, such as Cbl, has been shown to lead to enhanced binding and ubiquitination of the epidermal growth factor receptor (41
). Furthermore, kinases such as cAMP-dependent protein kinase (PKA) and Src family PTKs are also regulated by the UPS (42
). Hence, multiple levels of complexity in this system are possible, with our current findings not excluding the possibility that Mib2 is a target for regulation via Fyn phosphorylation, in addition to the NR2B subunit. Furthermore, Fyn may also potentially be modulated by ubiquitination. Dynamic regulation of the function of Mib2 or Fyn could affect the degree to which NR2B and Mib2 associate, and hence could be additional mechanisms that allow the level of NMDAR ubiquitination to be regulated in a temporally and spatially restricted manner.
Our finding that the NR2B subunit of the NMDAR is a substrate for Mib2 is interesting in light of other substrates that have previously been identified as associating with Mib2. The Notch ligand, Delta, is an integral membrane protein that associates with Mib2, resulting in its ubiquitination and down-regulation (37
). Mib1, an E3 ligase that shares significant similarity to Mib2, has also been shown to ubiquitinate Delta (43
). Furthermore, a recent article has reported that Mib1 is localized to the PSD in hippocampal neurons (45
). Here, we show that Mib2 is also localized to the PSD of hippocampal neurons. Thus both proteins are likely to play important roles in the ubiquitination of several neuronal proteins. Our identification of Mib2 as an E3 ligase that ubiquitinates the NR2B subunit does not rule out the possibility that other E3 ligases, in addition to Mib2, also function in this capacity, as five other E3 ligases are known to act in coordination with Mib2 to regulate the Notch-Delta signaling pathway (43
). Other E3 ligases, including Mdm2 and Parkin, have been demonstrated to reside at the PSD (30
). Furthermore, the ubiquitin-proteasome machinery is present in the dendrites of hippocampal neurons, with activity-dependent regulated movement of the proteasome from dendritic shafts to synaptic spines being observed (48
). In addition, several ligand-gated ion channels, including mammalian glycine, GABAA
, and glutamate receptors, are known to be regulated by ubiquitination or sumoylation, a ubiquitin-like modification (49
). However, phosphorylation-dependent ubiquitination of these receptors has not previously been reported and the exact nature of the ubiquitin machinery involved, especially the substrate specificity of synaptic E3 ligases, are questions that are only now starting to be addressed, and hence, will continue to be important issues for future research.
Abnormalities in the UPS pathway have been demonstrated in numerous neurological and neurodegenerative disorders (53
), and large-scale changes in the expression of multiple genes involved in ubiquitination have been found in studies of alcoholics (54
). Furthermore, mutations in the synaptically located E3 ligase Parkin have been associated with Parkinson’s disease (55
). The predominant localization of Mib2 at postsynaptic sites suggests that dysregulation in its function may also have significant consequences for neurological function. Conversely, regulated manipulation of Mib2 function may have important therapeutic implications, especially if selective modulation of NMDARs could be achieved. This could be particularly relevant for a number of neurological disorders, such as schizophrenia, alcoholism, neuropathic pain, or ischemia, in which dysregulation of NMDAR function occurs (56
In summary, the identification of Mib2 as an E3 ubiquitin ligase that binds to and ubiquitinates the NR2B subunit in a Fyn phosphorylation-dependent manner has significant implications for the negative regulation of NR2B-containing NMDARs.