Because dendritic spines are actin-rich protrusions that form the major postsynaptic sites of the excitatory synaptic input, dendrogenesis and spine morphogenesis are essential for synaptic plasticity and cognitive function. Moreover, spine abnormalities have been shown to be associated with many neurological disorders including most types of mental retardation (19
). Even though we previously reported that a hippocampal neuron overexpressing δ-catenin demonstrates an elaborate arborization of dendrites, swelling and enhanced dendritic spine maturation (15
), a complete picture of how δ-catenin affects dendrogenesis and spine morphogenesis would be very important for understanding its role in synaptic plasticity and cognitive function. In this study, we found that δ-catenin interacts with p190RhoGEF and significantly lowered the level of GTP-RhoA, and that Akt1 mediates the interaction between δ-catenin and p190RhoGEF through Thr-454 phosphorylation of δ-catenin. The formation of δ-catenin-induced dendrite-like process formation in NIH 3T3 fibroblasts was totally abolished by substitution of Thr-454 residue to Ala, which is a defective form in binding p190RhoGEF. Furthermore, δ-catenin T454A significantly reduced the length and number of mature mushroom shaped spines in primary hippocampal neurons. Overall, these results suggest that the interaction between δ-catenin and p190RhoGEF can act as a key modulator in regulating δ-catenin-induced dendrogenesis and spine formation.
Our protein-motif-scan analysis demonstrated 3 putative sites (Ser-282, Thr-454, Ser-1094) in mouse δ-catenin as a putative target phosphorylation sites for Akt and/or 14-3-3 binding sites. The precise mapping for the phosphorylation site by Akt, and subsequently, a binding site for p190RhoGEF and/or 14-3-3, is of great interest because the three molecules, δ-catenin, p190RhoGEF, and 14-3-3, may interact with each other independently. For example, a recent report demonstrated that mouse δ-catenin interacts with 14-3-3ζ through Ser-1094 in a phosphorylation-dependent manner (30
). 14-3-3 also binds to p190RhoGEF through a unique phosphorylation-independent binding site (I(1370)QAIQNL) in p190RhoGEF (29
). Hence, this study examined the possibility that δ-catenin may bind to p190RhoGEF via 14-3-3 through Ser-1094, forming a trimeric complex. The δ-catenin T454A mutant, unable to bind p190RhoGEF, still interacts with both 14-3-3 isoforms, ε and ζ, suggesting that p190RhoGEF binds directly to the domains containing Thr-454 in δ-catenin in a Akt-phosphorylation-dependent manner rather than by forming a trimeric complex. As shown in , the overexpressed δ-catenin noticeably decreased the interaction between p190RhoGEF and RhoA, supporting the direct interaction between δ-catenin and p190RhoGEF as proposed in our model in . Similar to the results of Mackie and Aitken (30
), δ-catenin ΔC207 (Ser-1094 deleted) showed very little 14-3-3ζ binding. However, we did observe a faint band, possibly due to formation of heterodimers with other 14-3-3 isoforms. In contrast, δ-catenin ΔC207 interacts well with 14-3-3ε, suggesting that δ-catenin can combine with the isoforms of 14-3-3 through different binding domains. Interestingly, p190RhoGEF has been shown to bind to the β, γ, ε and η, isoforms but not to ζ, or τ isoforms of 14-3-3 (29
). Therefore, even though our results suggests p190RhoGEF binds directly to δ-catenin in an Akt-dependent manner, the possibility that 14-3-3ε has favorable effects on the association between p190RhoGEF and δ-catenin cannot be totally excluded.
The Rho signaling pathway has attracted considerable attention for several reasons. First, the dendrite-like process formation and cytoskeletal remodeling induced by overexpressed δ-catenin will greatly mimic those shown by the use of the C3 toxin from Clostridium botulinum
, in which ADP-ribosylates and specifically inactivates Rho (46
). We previously reported that rhodamine phalloidine staining of NIH 3T3 fibroblasts produced lower levels of stress fiber-associated actin filaments (15
), which is indicative of reduced Rho signaling in fibroblasts. Even though Martinez et al. (12
) reported that δ-catenin enhances the effects of Rho inhibition on neurite branching, there is no evidence showing that δ-catenin reduces the level of GTP-RhoA in cells. This report provides the first biochemical evidence exhibiting that overexpressed δ-catenin indeed reduces the levels of GTP-RhoA but not those of GTP-Cdc42 or -Rac1, along with the mechanism that Akt phosphorylates the Thr-454 residue in δ-catenin and enables its association of p190RhoGEF.
In contrast to the few reports on δ-catenin, studies on p120ctn
, a prototype of the p120ctn
family protein, have revealed several lines of evidence that may be unique to p120ctn
or related to δ-catenin. p120ctn
has been shown to primarily enhance the development of spine-like protrusions, which were abolished by deletions in the Arm domain (48
overexpression in CHO cells increased the activity of endogenous Cdc42 (~3.1 fold) and Rac1 (~1.9 fold) activity, but diminished the Rho activity by ~45% through the Rho family exchange factor Vav2, which has activity for RhoA, RhoG, Cdc42, and Rac1 (50
). Consistent with this data, a deletion of the p120ctn
gene in hippocampal pyramidal neurons reduced the GTP-Rac1 level (~40%) and increased the RhoA level (~160%) (51
). In contrast, another report demonstrated that p120ctn
null epidermis showed markedly higher GTP-RhoA levels but similar GTP-Rac1 and -Cdc42 levels (52
). Our results demonstrate that overexpressed δ-catenin reduces the level of GTP-RhoA but not those of GTP-Cdc42 or -Rac1. It might be possible because p190RhoGEF is a brain-enriched and RhoA specific GEF (24
). In contrast to the published result that p120ctn
directly interacts with Rho, we could not observe such interaction between δ-catenin and RhoA, indicating that the interaction between δ-catenin and p190RhoGEF might be a unique mechanism for δ-catenin to lower RhoA activity. The mechanism for δ-catenin binding of p190RhoGEF and subsequent alteration of RhoA activation could be through a sequestration model as suggested in this report. However, we can not yet rule out the possibility that δ-catenin directly alters the exchange activity of the p190RhoGEF.
Compared to the explicit mechanism for δ-catenin T454A inability to lower active RhoA and modulate dendrogenesis, δ-catenin ΔC207 interactions are more complex and needs further investigation. As shown in and , δ-catenin ΔC207 binds to p190RhoGEF better than δ-catenin FL wt but displays less capability in lowering RhoA-GTP. This suggests that the C-terminus in δ-catenin also plays a pivotal role in lowering RhoA activity. We speculate two possibilities based on published results. First, in addition to the association between δ-catenin and p190RhoGEF, another protein(s) bound to the C-terminus of δ-catenin can be important for inactivating p190RhoGEF to a full extent. δ-Catenin binds to the last PDZ domain of S-SCAM through its C-terminus, which can affect the association between δ-catenin and NMDA receptors and other glutamate receptors. There was a recent report showing that GRIP, a direct binding protein of AMPA receptor, also binds to the C-terminal region of δ-catenin, whose interaction can affect LTD (53
, Ochiishi et al., Proceedings of 36th
Annual Neuroscience meeting, 2006). The association between Erbin and the Cadherin and Catenin complex was mediated by an interaction between the C-terminal binding motifs (DSWV-COOH) of δ-catenin and ARVCF (54
). Martinez et al. (12
) showed that a deletion of 99 amino acid (aa) residues from the δ-catenin COOH terminal (ΔC99), but not a deletion of 205 aa (ΔC205), retains its ability to interact with Cortactin. As RhoA combines with the glutamate receptors at the spine plasma membrane and NMDA receptor activation decreases the RhoA activity (55
), the C-terminus of δ-catenin may affect the Rho activity by interacting with one or more of these newly identified proteins. It also would be interesting to examine whether Cortactin or 14-3-3ζ mediates δ-catenin–dependent RhoA inactivation and dendritic spine morphogenesis. Alternatively, the C-terminus in δ-catenin may be important by being specifically localized to a certain membrane junction. Our results showed that δ-catenin ΔC207, which can interact with p190RhoGEF but possess a relatively low level of GTP-RhoA, showed total abolishment of dendrite-like processes in NIH 3T3 fibroblasts and a significantly lower number of dendrites in cultured hippocampal neurons, highlighting the important role of the C-terminus of δ-catenin plus its intact Thr-454 residue in regulating dendrite morphogenesis. Compared with the significantly diminished dendrite formation, δ-catenin ΔC207 has less affect on the formation of mature mushroom shaped spine in terms of the number and length than δ-catenin T454A. This indicates that δ-catenin contributes to dendrogenesis and spine formation through some overlapping and unique signaling pathways for each process. The different contribution of the C-terminus in δ-catenin in dendrogenesis and spine formation will require future study to determine its precise mechanism.
Even though the potential roles of dendrogenesis by δ-catenin (12
) and the promotion of mature spines in cultured hippocampal neurons (15
) have been described, the effects of δ-catenin on the number, shape, and length of spines are not completely understood. Interestingly, Elia et al. (51
) recently reported a role for p120ctn
in regulating spine and synapse formation. Consistent with our previous description, cultured hippocampal neurons expressing full-length δ-catenin showed an increased number of mature mushroom shaped spines compared with the GFP control, whereas δ-catenin T454A showed noticeable increases in the number of long, thin filopodia shaped spines with a concomitant reduction in the number of mature mushroom shaped spines. δ-Catenin ΔC207 showed intermediate changes in the number and length of mature spines between δ-catenin full-length and T454A, which strongly suggests that the C-terminus plus the intact Thr-454 in δ-catenin are essential for its full effects on the head shape and length of the spine, as well as its maturation. Interestingly, the morphological changes in the spine induced by δ-catenin T454A were similar to those induced by the dominant negative form of N-cadherin, alpha N-catenin, and RhoGEF (24
). There is accumulated evidence suggesting the role of Cadherin complexes and Rho in spine formation (57
), whereas the progressive loss of F-actin induced by a treatment with latrunculin B results in a rough spine head with random protrusion (59
). Compared with the full-length δ-catenin, δ-catenin T454A showed differences in the recruitment of Akt and p190RhoGEF, and in the levels of GTP-RhoA, suggesting their important roles in δ-catenin-induced spine morphogenesis. The reduced activities of the Rho downstream effectors, ROCK, LIMK and its substrate ADF/cofilin, and Dia, in neurons promotes the formation of dendritic spines or branches (61
). Furthermore, the expression of RhoA V14, a constitutively active form, decreased the spine density and length dramatically (62
), and the inhibition of Rho using C3 toxin in cortical and hippocampal neurons has also been shown to increase the density and length of some mouse cortical and hippocampal pyramidal neurons in organotypic slices (63
). Consistently, we showed that δ-catenin FL wt, which binds to p190RhoGEF and reduces RhoA activation, significantly increased the number and length of spines while δ-catenin T454A, which cannot binds to p190RhoGEF, significantly decreased the number and length of mature spines. Therefore, it is possible to say that δ-catenin exerts its effects on spine formation through very limited spatial sites including junctions containing Cadherins or NMDA and/or AMPA glutamate receptor, which allows for local reduction of RhoA, restricted actin reorganization, and its effects on other potential binding proteins. As the stimuli that produce various forms of long-term potentiation (LTP) cause a rapid local increase in the extension of filopodia and the formation of new spines at the site of stimulation (19
), the strong effects of δ-catenin and its mutants on the shape, length, and number of spines suggest its assumptive important role in synaptic plasticity and cognitive function. However, future investigation will be needed to compare the functional roles of δ-catenin full-length, and T454A mutant in cognition using transgenic mice.