Article

SH3 Domain-Based Phototrapping in Living Cells Reveals Rho Family GAP Signaling Complexes

Department of Cell Biology, Duke University Medical School, Durham, NC 27710, USA.
Science Signaling (Impact Factor: 7.65). 11/2011; 4(201):rs13. DOI: 10.1126/scisignal.2002189
Source: PubMed

ABSTRACT Rho family GAPs [guanosine triphosphatase (GTPase) activating proteins] negatively regulate Rho family GTPase activity and therefore modulate signaling events that control cytoskeletal dynamics. The spatial distribution of these GAPs and their specificity toward individual GTPases are controlled by their interactions with various proteins within signaling complexes. These interactions are likely mediated through the Src homology 3 (SH3) domain, which is abundant in the Rho family GAP proteome and exhibits a micromolar binding affinity, enabling the Rho family GAPs to participate in transient interactions with multiple binding partners. To capture these elusive GAP signaling complexes in situ, we developed a domain-based proteomics approach, starting with in vivo phototrapping of SH3 domain-binding proteins and the mass spectrometry identification of associated proteins for nine representative Rho family GAPs. After the selection of candidate binding proteins by cluster analysis, we performed peptide array-based high-throughput in vitro binding assays to confirm the direct interactions and map the SH3 domain-binding sequences. We thereby identified 54 SH3-mediated binding interactions (including 51 previously unidentified ones) for nine Rho family GAPs. We constructed Rho family GAP interactomes that provided insight into the functions of these GAPs. We further characterized one of the predicted functions for the Rac-specific GAP WRP and identified a role for WRP in mediating clustering of the postsynaptic scaffolding protein gephyrin and the GABA(A) (γ-aminobutyric acid type A) receptor at inhibitory synapses.

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    • "It is very interesting to test if srGAP3 interacts with 14-3-3 proteins to block nucleocytoplasmic shuttling, like DLC1 in a phosphorylation-dependent manner (Scholz et al., 2009). Some binding partners of srGAP3 are in the nuclei, such as ZAP3/YLPM1 (YLP motif–containing protein 1) (Okada et al., 2011), APP (Amyloid Precursor Protein) (Bai et al., 2008; Cao and Sudhof, 2001), Robo1 ICD(Seki et al., 2010) and Abi-1 (Abelson interacting protein 1) (Seki et al., 2010). Especially, cytoplasmic Abi-1 can also be translocated into the nuclei on depolarization in cultured hippocampal neurons (Proepper et al., 2007). "
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    ABSTRACT: The Slit-Robo GTPase activating protein 3 (srGAP3) is an important modulator of actin cytoskeletal dynamics and has an important influence on a variety of neurodevelopmental processes. Mutations in the SRGAP3 gene on chromosome 3p25 have been found in patients with intellectual disability. Genome-wide association studies and behavioral assays of knockout mice had also revealed SRGAP3 as a risk gene for schizophrenia. We have recently shown that srGAP3 protein undergoes regulated shuttling between the cytoplasm and the nucleus during neuronal development. It is shown here that nuclear-localized srGAP3 interacts with the SWI/SNF remodeling factor Brg1. This interaction is mediated by the C-terminal of srGAP3 and the ATPase motif of Brg1. In the primary cultured rat cortical neurons, the levels of nuclear-localized srGAP3 and its interaction with Brg1 have a significant impact on dendrite complexity. Furthermore, the interaction between srGAP3 and Brg1 was also involved in valporic acid (VPA) -induced neuronal differentiation of Neuro2a cells. We then show that GTP-bound Rac1 and GAP-43 may be potential mediators of nuclear srGAP3 and Brg1. Our results not only indicate a novel signaling pathway that contributes to neuronal differentiation and dendrite morphology, but also implicate a novel molecular mechanism underlying srGAP3 regulation of gene expression.
    Molecular and Cellular Neuroscience 05/2014; 60. DOI:10.1016/j.mcn.2014.02.005 · 3.73 Impact Factor
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    • "Acting as nuclear and cytoplasmic scaffold proteins, paralogous srGAPs proteins are likely to share other binding partners [17], [39], [40], such as YLPM1/ZAP3, Palladin, Gephyrin, DVL3, Lamellipodin, TNIK and Disc1. Among them, at least YLPM1/ZAP3, TNIK and Disc1 were reported to localize to nucleus [41]–[43]. "
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    ABSTRACT: The inverse F-BAR (IF-BAR) domain proteins srGAP1, srGAP2 and srGAP3 are implicated in neuronal development and may be linked to mental retardation, schizophrenia and seizure. A partially overlapping expression pattern and highly similar protein structures indicate a functional redundancy of srGAPs in neuronal development. Our previous study suggests that srGAP3 negatively regulates neuronal differentiation in a Rac1-dependent manner in mouse Neuro2a cells. Here we show that exogenously expressed srGAP1 and srGAP2 are sufficient to inhibit valporic acid (VPA)-induced neurite initiation and growth in the mouse Neuro2a cells. While ectopic- or over-expression of RhoGAP-defective mutants, srGAP1R542A and srGAP2R527A exert a visible inhibitory effect on neuronal differentiation. Unexpectedly, knockdown of endogenous srGAP2 fails to facilitate the neuronal differentiation induced by VPA, but promotes neurite outgrowth of differentiated cells. All three IF-BAR domains from srGAP1-3 can induce filopodia formation in Neuro2a, but the isolated IF-BAR domain from srGAP2, not from srGAP1 and srGAP3, can promote VPA-induced neurite initiation and neuronal differentiation. We identify biochemical and functional interactions of the three srGAPs family members. We propose that srGAP3-Rac1 signaling may be required for the effect of srGAP1 and srGAP2 on attenuating neuronal differentiation. Furthermore, inhibition of Slit-Robo interaction can phenocopy a loss-of-function of srGAP3, indicating that srGAP3 may be dedicated to the Slit-Robo pathway. Our results demonstrate the interplay between srGAP1, srGAP2 and srGAP3 regulates neuronal differentiation and neurite outgrowth. These findings may provide us new insights into the possible roles of srGAPs in neuronal development and a potential mechanism for neurodevelopmental diseases.
    PLoS ONE 03/2013; 8(3). DOI:10.1371/journal.pone.0057865 · 3.23 Impact Factor
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    • "There are several cytoskeletal regulatory proteins that have been found to interact with gephyrin: some of those proteins are GEFs or GAPs of the Rho family of small GTPases: the Cdc42-GEF “ARHGEF9” (collybistin) (see below), the Rho-GAP “SRGAP2” (SLIT-ROBO Rho GTPase-activating protein 2) and the Rac-GAP “WRP” (WAVE-associated Rac GAP). WRP affects gephyrin cluster size and plays a role in a human 3p-syndrome mental retardation and mutations of this protein are also linked to schizophrenia (Endris et al., 2002; Lewis et al., 2008; Addington and Rapoport, 2009; Okada et al., 2011). "
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    ABSTRACT: GABA(A) receptors are clustered at synaptic sites to achieve a high density of postsynaptic receptors opposite the input axonal terminals. This allows for an efficient propagation of GABA mediated signals, which mostly result in neuronal inhibition. A key organizer for inhibitory synaptic receptors is the 93 kDa protein gephyrin that forms oligomeric superstructures beneath the synaptic area. Gephyrin has long been known to be directly associated with glycine receptor β subunits that mediate synaptic inhibition in the spinal cord. Recently, synaptic GABA(A) receptors have also been shown to directly interact with gephyrin and interaction sites have been identified and mapped within the intracellular loops of the GABA(A) receptor α1, α2, and α3 subunits. Gephyrin-binding to GABA(A) receptors seems to be at least one order of magnitude weaker than to glycine receptors (GlyRs) and most probably is regulated by phosphorylation. Gephyrin not only has a structural function at synaptic sites, but also plays a crucial role in synaptic dynamics and is a platform for multiple protein-protein interactions, bringing receptors, cytoskeletal proteins and downstream signaling proteins into close spatial proximity.
    Frontiers in Cellular Neuroscience 05/2012; 6:23. DOI:10.3389/fncel.2012.00023 · 4.18 Impact Factor
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