The Roundabout (ROBO) family encodes transmembrane receptors that regulate axonal guidance and cell migration. The major function of the Robo receptors is to mediate repulsion of the navigating growth cones. There are four human Robo homologues, ROBO1, ROBO2, ROBO3 and ROBO4. Most of the ROBOs have the similar ectodomain architecture as the cell adhesion molecules, with five Ig domains followed by three FN3 repeats, except for ROBO4. ROBO4 has two Ig and two FN3 repeats. The cytoplasmic domains of ROBO receptors are in general poorly conserved. However, there are four short conserved cytoplasmic sequence motifs, named CC0-3, that serve as binding sites for adaptor proteins. The ligands for the human ROBO1 and ROBO2 receptors are the three SLIT proteins SLIT1, SLIT2, and SLIT3; all of the SLIT proteins contain a tandem of four LRR (leucine rich repeat) domains at the N-terminus, termed D1-D4, followed by six EGF (epidermal growth factor)-like domains, a laminin G like domain (ALPS), three EGF-like domains, and a C-terminal cysteine knot domain. Most SLIT proteins are cleaved within the EGF-like region by unknown proteases (reviewed by Hohenster 2008, Ypsilanti and Chedotal 2014, Blockus and Chedotal 2016). NELL2 is a ligand for ROBO3 (Jaworski et al. 2015).
SLIT protein binding modulates ROBO interactions with the cytosolic adaptors. The cytoplasmic domain of ROBO1 and ROBO2 determines the repulsive responses of these receptors. Based on the studies from both invertebrate and vertebrate organisms it has been inferred that ROBO induces growth cone repulsion by controlling cytoskeletal dynamics via either Abelson kinase (ABL) and Enabled (Ena), or RAC1 activity (reviewed by Hohenster 2008, Ypsilanti and Chedotal 2014, Blockus and Chedotal 2016). While there is some redundancy in the function of ROBO receptors, ROBO1 is implicated as the predominant receptor for axon guidance in ventral tracts, and ROBO2 is the predominant receptor for axon guidance in dorsal tracts. ROBO2 also repels neuron cell bodies from the floor plate (Kim et al. 2011).
In addition to regulating axon guidance, ROBO1 and ROBO2 receptors are also implicated in regulation of proliferation and transition of primary to intermediate neuronal progenitors through a poorly characterized cross-talk with NOTCH-mediated activation of HES1 transcription (Borrell et al. 2012).<p>Thalamocortical axon extension is regulated by neuronal activity-dependent transcriptional regulation of ROBO1 transcription. Lower neuronal activity correlates with increased ROBO1 transcription, possibly mediated by the NFKB complex (Mire et al. 2012).<p>It is suggested that the homeodomain transcription factor NKX2.9 stimulates transcription of ROBO2, which is involved in regulation of motor axon exit from the vertebrate spinal code (Bravo-Ambrosio et al. 2012).<p>Of the four ROBO proteins, ROBO4 is not involved in neuronal system development but is, instead, involved in angiogenesis. The interaction of ROBO4 with SLIT3 is involved in proliferation, motility and chemotaxis of endothelial cells, and accelerates formation of blood vessels (Zhang et al. 2009).
View original pathway at Reactome.</div>
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The Nonsense-Mediated Decay (NMD) pathway activates the destruction of mRNAs containing premature termination codons (PTCs) (reviewed in Isken and Maquat 2007, Chang et al. 2007, Behm-Ansmant et al. 2007, Neu-Yilik and Kulozik 2008, Rebbapragada and Lykke-Andersen 2009, Bhuvanagiri et al. 2010, Nicholson et al. 2010, Durand and Lykke-Andersen 2011). In mammalian cells a termination codon can be recognized as premature if it precedes an exon-exon junction by at least 50-55 nucleotides or if it is followed by an abnormal 3' untranslated region (UTR). While length of the UTR may play a part, the qualifications for being "abnormal" have not been fully elucidated. Also, some termination codons preceding exon junctions are not degraded by NMD so the criteria for triggering NMD are not yet fully known (reviewed in Rebbapragada and Lykke-Andersen 2009). While about 30% of disease-associated mutations in humans activate NMD, about 10% of normal human transcripts are also degraded by NMD (reviewed in Stalder and Muhlemann 2008, Neu-Yilik and Kulozik 2008, Bhuvanagiri et al. 2010, Nicholson et al. 2010). Thus NMD is a normal physiological process controlling mRNA stability in unmutated cells. Exon junction complexes (EJCs) are deposited on an mRNA during splicing in the nucleus and are displaced by ribosomes during the first round of translation. When a ribosome terminates translation the A site encounters the termination codon and the eRF1 factor enters the empty A site and recruits eRF3. Normally, eRF1 cleaves the translated polypeptide from the tRNA in the P site and eRF3 interacts with Polyadenylate-binding protein (PABP) bound to the polyadenylated tail of the mRNA. During activation of NMD eRF3 interacts with UPF1 which is contained in a complex with SMG1, SMG8, and SMG9. NMD can arbitrarily be divided into EJC-enhanced and EJC-independent pathways. In EJC-enhanced NMD, an exon junction is located downstream of the PTC and the EJC remains on the mRNA after termination of the pioneer round of translation. The core EJC is associated with UPF2 and UPF3, which interact with UPF1 and stimulate NMD. Once bound near the PTC, UPF1 is phosphorylated by SMG1. The phosphorylation is the rate-limiting step in NMD and causes UPF1 to recruit either SMG6, which is an endoribonuclease, or SMG5 and SMG7, which recruit ribonucleases. SMG6 and SMG5:SMG7 recruit phosphatase PP2A to dephosphorylate UPF1 and allow further rounds of degradation. How EJC-independent NMD is activated remains enigmatic but may involve competition between PABP and UPF1 for eRF3.
RHO associated, coiled-coil containing protein kinases ROCK1 and ROCK2 consist of a serine/threonine kinase domain, a coiled-coil region, a RHO-binding domain and a plekstrin homology (PH) domain interspersed with a cysteine-rich region. The PH domain inhibits the kinase activity of ROCKs by an intramolecular fold. ROCKs are activated by binding of the GTP-bound RHO GTPases RHOA, RHOB and RHOC to the RHO binding domain of ROCKs (Ishizaki et al. 1996, Leung et al. 1996), which disrupts the autoinhibitory fold. Once activated, ROCK1 and ROCK2 phosphorylate target proteins, many of which are involved in the stabilization of actin filaments and generation of actin-myosin contractile force. ROCKs phosphorylate LIM kinases LIMK1 and LIMK2, enabling LIMKs to phosphorylate cofilin, an actin depolymerizing factor, and thereby regulate the reorganization of the actin cytoskeleton (Ohashi et al. 2000, Sumi et al. 2001). ROCKs phosphorylate MRLC (myosin regulatory light chain), which stimulates the activity of non-muscle myosin II (NMM2), an actin-based motor protein involved in cell migration, polarity formation and cytokinesis (Amano et al. 1996, Riento and Ridley 2003, Watanabe et al. 2007, Amano et al. 2010). ROCKs also phosphorylate the myosin phosphatase targeting subunit (MYPT1) of MLC phosphatase, inhibiting the phosphatase activity and preventing dephosphorylation of MRLC. This pathway acts synergistically with phosphorylation of MRLC by ROCKs towards stimulation of non-muscle myosin II activity (Kimura et al. 1996, Amano et al. 2010).
The PAKs (p21-activated kinases) are a family of serine/threonine kinases mainly implicated in cytoskeletal rearrangements. All PAKs share a conserved catalytic domain located at the carboxyl terminus and a highly conserved motif in the amino terminus known as p21-binding domain (PBD) or Cdc42/Rac interactive binding (CRIB) domain. There are six mammalian PAKs that can be divided into two classes: class I (or conventional) PAKs (PAK1-3) and class II PAKs (PAK4-6). Conventional PAKs are important regulators of cytoskeletal dynamics and cell motility and are additionally implicated in transcription through MAPK (mitogen-activated protein kinase) cascades, death and survival signaling and cell cycle progression (Chan and Manser 2012).
PAK1, PAK2 and PAK3 are direct effectors of RAC1 and CDC42 GTPases. RAC1 and CDC42 bind to the CRIB domain. This binding induces a conformational change that disrupts inactive PAK homodimers and relieves autoinhibition of the catalytic carboxyl terminal domain (Manser et al. 1994, Manser et al. 1995, Zhang et al. 1998, Lei et al. 2000, Parrini et al. 2002; reviewed by Daniels and Bokoch 1999, Szczepanowska 2009). Autophosphorylation of a conserved threonine residue in the catalytic domain of PAKs (T423 in PAK1, T402 in PAK2 and T436 in PAK3) is necessary for the kinase activity of PAK1, PAK2 and PAK3. Autophosphorylation of PAK1 serine residue S144, PAK2 serine residue S141, and PAK3 serine residue S154 disrupts association of PAKs with RAC1 or CDC42 and enhances kinase activity (Lei et al. 2000, Chong et al. 2001, Parrini et al. 2002, Jung and Traugh 2005, Wang et al. 2011). LIMK1 is one of the downstream targets of PAK1 and is activated through PAK1-mediated phosphorylation of the threonine residue T508 within its activation loop (Edwards et al. 1999). Further targets are the myosin regulatory light chain (MRLC), myosin light chain kinase (MLCK), filamin, cortactin, p41Arc (a subunit of the Arp2/3 complex), caldesmon, paxillin and RhoGDI, to mention a few (Szczepanowska 2009).
Class II PAKs also have a CRIB domain, but lack a defined autoinhibitory domain and proline-rich regions. They do not require GTPases for their kinase activity, but their interaction with RAC or CDC42 affects their subcellular localization. Only conventional PAKs will be annotated here.
SLIT2 ligand forms a complex with the ROBO1 receptor (Brose et al. 1999). The SLIT family consists of three members that are all expressed in the ventral midline (floor plate) of the neural tube. SLIT1 is predominantly expressed in the nervous system whereas SLIT2 and SLIT3 are also expressed outside the nervous system. SLIT proteins are the ligands for the ROBO receptors. In humans, there are four ROBO genes: ROBO1, ROBO2, ROBO3 and ROBO4. The extracellular domain of ROBO comprises five Ig domains and three FN domains except for ROBO4 (two Ig + two FN). Ig1 and Ig2 domains of ROBO1 and ROBO2 are highly conserved and are important for SLIT binding. The concave face of SLIT's second LRR domain accommodates the Ig1 and Ig2 domains of ROBO1 and ROBO2. ROBO3 does not bind SLITs (Camurri et al. 2005, Mambetisaeva et al. 2005, Zelina et al. 2014, Jaworski et al. 2015). SLIT binding with ROBO4 is controversial as the interaction is weak and it has been observed using the in-vitro methods (Wang et al. 1999, Brose et al. 1999, Piper et al. 2003, Andrews et al. 2007). Binding of secreted (cleaved) SLIT2 to ROBO1 and ROBO2 is involved in fasciculation (bundling) of motor axons, which facilitates axon pathfinding and muscle innervation (Jaworski and Tessier-Lavigne 2012).
DCC and ROBO1 heterodimerize via conserved sequence elements in their cytoplasmic domains, namely CC1 (conserved cytoplasmic region1) in ROBO1 and P3 in DCC. The formation of this complex is dependent on the previous interaction between ROBO and its ligand (SLIT). This physical interaction between ROBO:SLIT and DCC silences the attractive effect of Netrin:DCC and regulates the midline crossing of axons. From the analysis of multiple double mutant combinations of the ROBO:SLIT and Netrin:DCC receptor-ligand pairs, it was deduced that ROBO repulsion on its own is sufficient to prevent commissural axons from re-crossing the midline, and that Netrin:DCC is not the only source of attraction at the midline (Stein and Tessier-Lavigne 2001, Garbe and Bashaw 2007).
Ena/VASP proteins (ENAH, EVL and VASP) are required in part for ROBO's repulsive output. Ena/VASP proteins are drawn as effectors downstream of Robo signaling via a direct interaction with ROBO. ROBO's CC2 (LPPPP) motif is the consensus binding site for the EVH1 domain of Ena/VASP proteins. The Ena/VASP family of proteins has a universal role in control of cell motility and actin dynamics. These proteins consist of an N-terminal EVH1 domain, a central proline rich region, which acts as a ligand for the actin monomer binding protein Profilin (PFN), as well as several SH3 domain-containing proteins, such as ABL, and a C-terminal EVH2 domain involved in oligomerization and F-actin binding (Bashaw et al. 2000).
The ROBO1 receptor regulates Rho GTPase activity through a ligand-dependent association with members of a GAP protein family called SRGAPs (SLIT-ROBO GAPs), SRGAP1, SRGAP2 and SRGAP3 (Wong et al. 2001, Bacon et al. 2011). Extracellular interaction between SLIT and ROBO increases the intracellular interaction between the CC3 motif of ROBO1 and the SH3 motif of the SRGAPs (Wong et al. 2001).
The full length SLIT proteins are secreted and, when not bound to ROBO receptors, are indirectly associated with the plasma membrane via the extracellular matrix proteins. These full length SLITs undergo posttranslational modification and proteolytic processing to generate an N-terminal fragment (SLIT2-N) and a corresponding C-terminal fragment (SLIT2-C). SLIT2 is cleaved within the EGF repeats, between EGF5 and EGF6, by unknown proteases. Cleavage of SLIT proteins is evolutionarily conserved, although the molecular biological significance is unknown. The N-terminal fragment of SLIT2 stimulates growth and branching of dorsal root ganglia (DRG) axons, and this activity is opposed by un-cleaved SLIT. The stimulation of axon branching is mediated by ROBO receptors. Additional functional differences between the full-length and N-terminal forms have been discovered in their abilities to repel different populations of axons and dendrites. Finally, SLIT can attract migrating muscles in the fly, and also human endothelial cells, both via ROBO receptors (Brose et al. 1999, Wang et al. 1999).
SLIT C-terminal fragments may transduce signaling independently of ROBO receptors and Neuropilins (semaphorin receptors) by directly binding to Plexin A1 (Delloye-Bourgeois et al. 2015).
ROBO3 antagonizes ROBO1/ROBO2 function to prevent their response to SLIT, thus allowing cells that are expressing ROBO1/ROBO2 to progress towards and across the floor plate. Exactly how ROBO3 interferes with ROBO1/ROBO2 function is not yet clear (Chen et al. 2008). It was shown that ROBO3 isoform ROBO3.1 reduces the amount of ROBO1 and ROBO2 at the cell surface and suggested that ROBO3.1 acts by directing ROBO1 and ROBO2 to late endosome- and lysosome-dependent degradation pathway (Li et al. 2014). Direct binding of ROBO3.1 to ROBO2 was demonstrated (Li et al. 2014).
During commissural axon midline crossing in Drosophila, Robo1 signaling can also be antagonized with Robo2 expressed in trans. Extracellular domains of Robo1 and Robo2 may interact through their Ig domains, preventing Robo1 activation by Slits and interfering with axon repulsion (Evans et al. 2015).
SLIT stimulation recruits SH3-SH2 adaptor protein Dreadlocks (Dock) (NCK in vertebrates) to the ROBO1 receptor. This interaction involves the CC2 and CC3 motifs of ROBO1 (Fan et al. 2003, Ang et al. 2003).
Upon SLIT-mediated ROBO stimulation, SOS1 or SOS2 is recruited into the multiprotein complex consisting of SLIT2, ROBO1 and the SH3-SH2 protein NCK1 or NCK2 (orthologues of Drosophila Dock). NCK bridges the physical association between ROBO and SOS. This interaction was demonstrated in both Drosophila and human cells (Hu et al. 1995, Fritz et al. 2000, Yang and Bashaw 2006).
SLIT2 and both its natural cleavage products bind glypican-1 (GPC1), a glycosyl phosphatidyl inositol (GPI) anchored heparan sulfate proteoglycan (HSPG), through its C-terminus. Besides glypican-1, other HSPG may also be involved in SLIT2 binding. GPC1:HSPG is important for high affinity binding of SLIT to its receptor and for the repulsive activity of SLIT. SLIT-ROBO signaling strictly requires binding to heparan sulfate. HSPGs may also modulate the extracellular distribution or stability of SLIT proteins (Ronca et al. 2001, Zhang et al. 2004).
Vilse and its human homolog ARHGAP39 bind directly to the intracellular domains of the corresponding ROBO receptors and promote the hydrolysis of GTP bound to RAC1 (Lundstrom et al. 2004, Hu et al. 2005).
NCK1 or NCK2, orthologues of Drosophila Dock, bound to ROBO1 receptor, recruits PAK to specific sites at the growth cone membrane, where PAK, activated by RAC1, regulates the recycling and retrograde flow of actin filaments. In mammals, there are six PAK isoforms (PAK1-6) and PAK binds to the 2nd SH3 domain of NCK with its proline rich PxxP motif (Galisteo et al. 1996, Fan et al. 2003). PAK autophosphorylation triggered by RAC1/CDC42 activation disrupts PAK interaction with NCK proteins (Zhao et al. 2000).
SRGAP bound to ROBO's cytoplasmic tail increases the intrinsic GTPase activity of CDC42, converting the GTP-bound form of CDC42 into its GDP-bound form, therefore inactivating CDC42. Inactivation of CDC42 leads to a reduction in the activation of the Neuronal Wiskott-Aldrich Syndrome protein (NWASP), thus decreasing the level of the active Arp2/3 complex. Because active Arp2/3 promotes actin polymerization, the reduction of active CDC42 eventually decreases actin polymerization. SLIT regulates SRGAP interaction with ROBO1 and CDC42, increasing SRGAP binding to CDC42 (Wong et al. 2001, Li et al. 2006).
Ena/VASP proteins (ENAH, EVL1 and VASP) enhance actin filament elongation via the recruitment of profilin:actin complexes to the tips of spreading lamellipodia. Profilin (PFN1 or PFN2) binds to the central proline rich domain of an Ena/VASP protein (Bashaw et al. 2000).
SOS (SOS1 or SOS2), bound to Dock orholog NCK (NCK1 or NCK2), has a Rac GEF activity and activates RAC1. Son of sevenless (SOS) is a dual specificity guanine nucleotide exchange factor (GEF) that regulates both Ras and Rho family GTPases. The Ras and Rac-GEF activities of Sos can be uncoupled during ROBO-mediated axon repulsion; SOS axon guidance function depends on its Rac-GEF activity, but not its Ras-GEF activity (Yang and Bashaw 2006).
Vilse/CrossGAP (CrGAP), a conserved Rac-specific GAP in Drosophila, is involved in Robo mediated repulsion. CrGAP directly interacts with Robo, both biochemically and genetically. The biochemical interaction is mediated by the WW domains in CrGAP and the CC2 motif of Robo. The human homologue of Vilse/CrGAP, ARHGAP39 (also known as KIAA1688), was identified. ARHGAP39 shares 54.4% sequence identity with Drosophila CrGAP and is referred to as human Vilse/CrGAP protein (Lundstrom et al. 2004, Hu et al. 2005).
ABL (ABL1 or ABL2) associated with the complex of ROBO1, SLIT2, and glypican-1 (GPC1) at the plasma membrane binds CAP (CAP1 or CAP2) and regulates its activity to inhibit net actin assembly. Studies of CAP homologs from yeast, Dictyostelium, mouse, pig, and human suggest that the C-terminal actin binding domain acts to sequester actin monomers to prevent actin polymerization (Wills et al. 2002).
CLASP (CLASP1 or CLASP2) acts positively downstream of ABL (ABL1 or ABL2) as part of the repellent response initiated by activation of ROBO1. CLASP is spatially positioned to interact with ROBO receptors. SLIT mediated repulsion results in activation of CLASP, presumably through its phosphorylation by the ABL kinase. Activation of CLASP in turn results in inhibition of microtubule polymerization on the side of the growth cone receiving the repulsive signal and consequently the growth cone turns to the opposite side. A direct link between ABL and CLASP, notably the mechanism of CLASP activation, has not been demonstrated, however (Wills et al. 2002, Lee et al. 2004, Kalil and Dent 2004).
ROBO1, bound to SLIT2, binds to the RHO GAP protein MYO9B. The interaction involves all four cytoplasmic conserved (CC) motifs in the intracellular domain of ROBO1 and the RHO GAP domain of MYO9B. Binding to ROBO1 inhibits the RHOA GAP activity of MYO9B and increases the amount of active GTP-bound RHOA (RHOA:GTP). ROBO1-mediated inhibition of MYO9B and the resulting increase in RHOA activity is implicated as a negative regulator of invasiveness of lung cancer cells (Kong et al. 2015).
MYO9B (MYR5) is a RHO GAP protein that stimulates RHOA GTPase activity, resulting in hydrolysis of RHOA bound GTP to GDP, and conversion of the active form of RHOA, RHOA:GTP, to the inactive form, RHOA:GDP (Post et al. 1998, Graf et al. 2000, Kong et al. 2015). MYO9B does not act on CDC42 and RAC1. The GAP activity of MYO9B is inhibited by binding to ROBO1 receptor activated by SLIT2 (Kong et al. 2015).
Based on mouse experiments, SLIT1 binds to ROBO1 and/or ROBO2 to stimulate cortical dendrite branching (Round and Sun 2011). SLIT1-mediated activation of ROBO1 and/or ROBO2 may also be involved in regulation of midline crossing in the spinal cord (Mambetisaeva et al. 2015). SLIT1 is expressed by new neurons in the adult brain which migrate from the subventricular zone to the olfactory bulb through the rostral migratory stream. Astrocytes in the rostral migratory stream express ROBO receptors, mostly ROBO2, but also ROBO1 and ROBO3. These ROBO-expressing astrocytes are repelled by SLIT1-expressing young migrating neurons, which results in the formation and maintenance of the astrocytic tunnels. Astrocytic tunnels allow rapid directional migration of new neurons in the adult brain (Kaneko et al. 2010). Signaling through SLIT1-ROBO2 is implicated in regulation of peripheral nerve regeneration (Zhang et al. 2010).
Based on mouse experiments, SLIT1-activated ROBO receptors ROBO1 and/or ROBO2 bind to NCK2. The interaction involves three SH3 domains of NCK2 and the PxxP-rich region between CC2 and CC3 motifs in the cytoplasmic domain of ROBO. While NCK1 can also associate with ROBO receptors, only NCK2 is implicated in SLIT1-induced cortical dendrite branching (Round and Sun 2011).
The ubiquitin protease USP33 deubiquitinates ROBO1, thus stabilizing it and increasing the concentration of ROBO1 at the plasma membrane (Yuasa-Kawada et al. 2009, Huang et al. 2015). USP33 is frequently downregulated in colorectal cancer, which is associated with lymph node metastasis and poor survival (Huang et al. 2015). USP33 is required for SLIT-ROBO1-mediated inhibition of breast cancer cell migration (Yuasa-Kawada et al. 2009). Ubiquitin ligases that ubiquitinate ROBO1 are not known.
SLIT2-activated ROBO1 receptor can form a complex with a G-protein coupled receptor (GPCR) CXCR4 (Prasad et al. 2007), resulting in downregulation of CXCR4 signaling (Prasad et al. 2004, Prasad et al. 2007). Formation of the complex between ROBO1 and CXCR4, which involves the CC3 motif of ROBO1, does not interfere with CXCR4 binding to its ligand, CXCL12 (Prasad et al. 2007). SLIT-ROBO signaling may also downregulate CXCR4 expression (Marlow et al. 2008). Downregulation of CXCL12-CXCR4 signaling is thought to contribute to SLIT-ROBO-mediated inhibition of cell migration (Prasad et al. 2004, Prasad et al. 2007, Marlow et al. 2008).
Based on studies in mice, ROBO3 isoforms ROBO3.1 and ROBO3.2 bind to a secreted ligand NELL2 (neural epidermal growth factor-like-like 2). This interaction involves the EGF-like domains of NELL2 and the FN3 (FNIII) domain of ROBO3. Pre-crossing commissural axons which express ROBO3.1 are repelled by NELL2. Post-crossing axons, which express ROBO3 isoform ROBO3.2 are not repelled by NELL2, despite interaction between ROBO3.2 and NELL2. NELL1 can also bind to both ROBO3.1 and ROBO3.2, but since NELL1 is not expressed in mouse spinal cord during commissural axon guidance, these interactions are not considered to be physiologically relevant. ROBO1 and ROBO2 do not interact with NELL proteins (Jaworski et al. 2015).
Based on studies in mice, ROBO1, activated by SLIT1 binding, forms a complex with FLRT3. This interaction involves the intracellular domains of FLRT3 and ROBO1. FLRT3 is a member of the fibronectin leucine-rich repeat transmembrane protein family. The interaction of FLRT3 and ROBO1 in the presence of SLIT1 increases Netrin-1 attraction of thalamocortical axons by increasing the amount of DCC receptors at the plasma membrane via an unknown mechanism that may involve PKA activation (Leyva-Diaz et al. 2014).
Based on studies in zebrafish, SLIT1 binds to a type IV collagen COL4A5, which forms the basement membrane on the surface of the optical tectum. COL4A5 and HSPGs may act synergistically to anchor SLIT1 in the basement membrane. ROBO2 receptor is required for lamina-specific axon pathfinding of retinal ganglion cells in the optical tectum (Xiao et al. 2011).
Based on studies in mice, the homeobox transcription factor HOXA2 binds to an evolutionarily conserved (also present in the human gene) HOX-PBX binding site in the second intron of the ROBO2 gene (Geisen et al. 2008). The heterodimerization partner of HOXA2 at the ROBO2 gene binding site is not known.
Based on studies in mice, the homeobox transcription factor HOXA2, which directly binds to an evolutionarily conserved site in the second intron of the ROBO2 gene, is needed for the maintenance of ROBO2 expression during pontine neuron migration (Geisen et al. 2008).
Also based on mouse studies, LHX2, a LIM-homeodomain transcription factor, directly represses transcription of the ROBO2 gene by binding to evolutionarily conserved LHX2 binding sites about 50 kb downstream from the ROBO2 gene transcription start site. LHX2 is involved in thalamocortical axon guidance (Marcos-Mondejar et al. 2012). In commissural relay neurons of the dorsal spinal cord, however, ROBO2 expression is not affected by LHX2 (Wilson et al. 2008).
In zebrafish, transcription of Robo2 is directly stimulated by Mecp2 (Leong et al. 2015).
Based on studies in mice, the transcription factor ISL1, in complex with either LHX3 or LHX4, directly stimulates transcription of the SLIT2 gene. ISL1-mediated regulation of SLIT2 gene transcription in branchiomotor (BM) neurons and somatic motor (SM) neurons involves LHX4 and LHX3, respectively (Kim et al. 2016). Slit2 expression is diminished in Isl1 mutant mice (Lee et al. 2015).
SLIT2 is one of gene suggested to be repressed by the transcription factor ARX, involved in neuronal proliferation, migration, maturation and differentiation, as well as axon guidance (reviewed by Friocourt and Parnavelas, 2011).
Based on studies in mice, the transcription factor ISL1, in complex with either LHX3 or LHX4, binds to an evolutionarily conserved LIM-HD binding site in the enhancer of the SLIT2 gene, located in the sixth intron of the SLIT2 gene. The complex of ISL1 and LHX4 regulates SLIT2 expression in branchiomotor neurons, while the complex of ISL1 and LHX3 regulates SLIT2 expression in somatic motor neurons (Kim et al. 2016). From the previous structural studies of the ISL1 complex with LHX3, conducted using mouse and rat proteins, it is known that LDB1 is also part of this complex (Thaler et al. 2002).
Based on studies in mice, AKAP5 (also known as AKAP79) recruits protein kinase A (PKA) to the ROBO2 receptor, by interacting with the PKA regulatory subunit RIIalpha (Samelson et al. 2015).
Based on studies in mice, AKAP5 (also known as AKAP79) recruits activated protein kinase C - PRKCA and possibly other isoforms - to the ROBO2 receptor. This interaction was also confirmed using recombinant human AKAP5 and mouse Robo2 (Samelson et al. 2015).
Based on studies in mice, AKAP5 (also known as AKAP79) recruits protein phosphatase PP2B subunit B (PPP3CB) to ROBO2 receptor. It is not known whether prior binding of PPP3CB to calcium-activated calmodulin is needed for this interaction to occur (Samelson et al. 2015).
AKAP5 (also known as AKAP79) recruits activated protein kinase C - PRKCA and possibly other isoforms - to the ROBO3 receptor isoform ROBO3.1 (Samelson et al. 2015).
Based on studies in mice, AKAP5 (also known as AKAP79), an A-kinase anchoring protein, forms a complex with ROBO2. The interaction involves the intracellular domain of ROBO2. It has not been investigated whether SLIT-mediated activation of ROBO2 is needed for this interaction (Samelson et al. 2015).
PKC (PRKCA, and possibly other isoforms), recruited to ROBO3 receptor isoform ROBO3.1 by AKAP5 (AKAP79) phosphorylates ROBO3.1 on serine residue S1330 (Samelson et al. 2015).
SLIT2 is expressed by corneal epithelial cells and able to bind to keratan sulfate, which is part of the corneal stroma extracellular matrix. This interaction may influence corneal nerve growth cone penetration. SLIT3 may also interact with keratan sulfate, as well as ROBO receptors ROBO1 and ROBO2 (Conrad et al. 2010).
SLIT2 binds to dystroglycan (DAG1). The interaction involves the C-terminal region of human SLIT2. The species origin of the DAG1 construct was not specified and is assumed to be human. Dystroglycan is required for proper SLIT2 localization within the basement membrane and the floor plate. Dystroglycan glycosylation, mediated at least in part by B4GAT1 (B3GNT1) and ISPD, is likely required for its interaction with SLIT2, but it has not been annotated. Mice mutant for B4gat1, Ispd or Dag1 have axon guidance defects similar to those observed in Slit or Robo mutant mice (Wright et al. 2012).
Based on studies in mice, ROBO1 binds semaphorin receptor NRP1. Other NRP1-binding proteins, such as NRP2, Plexin A1 and Plexin A2, also co-immunoprecipitate with ROBO1, but it is thought that the direct interaction involves ROBO1 and NRP1 only. ROBO1 binds to NRP1 via Ig1and Ig2 domains in the extracellular region of ROBO1. Interaction with ROBO1 may increase the stability of NRP1 and NRP1-associated proteins, or increase their abundance at the plasma membrane. Semaphorins direct the migration of cortical interneurons, and mice deficient in Robo1 function show reduced responsiveness of cortical interneurons to semaphorins (Hernandez-Miranda et al. 2011).
Based on studies in mice, a LIM-homeodomain transcription factor LHX2 binds to evolutionarily conserved LHX2 binding elements about 30 kb downstream from the ROBO1 gene transcription start site (Marcos-Mondejar et al. 2012).
Based on studies in mice, a LIM-homeodomain transcription factor LHX2 binds to evolutionarily conserved LHX2 binding elements about 50 kb downstream from the ROBO2 gene transcription start site (Marcos-Mondejar et al. 2012).
Based on studies in mice, LHX2, a LIM-homeodomain transcription factor, directly represses transcription of the ROBO1 gene by binding to evolutionarily conserved LHX2 binding sites upstream of the ROBO1 gene promoter region. LHX2 is involved in thalamocortical axon guidance (Marcos-Mondejar et al. 2012). In commissural relay neurons of the dorsal spinal cord, however, ROBO1 expression is not affected by LHX2 (Wilson et al. 2008).
Transcription factors GBX2 and LMO3 may be indirectly involved in ROBO1 gene expression regulation by LHX2 (Chatterjee et al. 2012).
Based on studies in mice, expression of the ROBO3.1 isoform from the ROBO3 gene is directly stimulated by LHX2 and possibly LHX9. LHX2/9-mediated regulation of ROBO3.1 levels is involved in midline crossing by commissural relay neurons of the dorsal spinal cord (Wilson et al. 2008). ROBO3.1 levels, however, seem to be unaffected by LHX2 in thalamocortical neurons (Marcos-Mondejar et al. 2012).
Based on studies in mice, a LIM-homeodomain transcription factor LHX2, and possibly LHX9, binds to conserved LHX2 binding elements in the promoter region of the ROBO3 gene (Wilson et al. 2008).
Based on studies in mice and biochemical studies with human DCC and mouse Robo3.1, ROBO3.1 (also known as ROBO3A.1) can bind to DCC both in the presence and absence of netrin-1 (NTN1). The two proteins interact via their intracellular domains through the P3 domain of DCC and the CC2 and CC3 motifs of ROBO3. Binding of ROBO3.1 to DCC apparently contributes to NTN1-mediated attraction of commissural axons to the midline (Zelina et al. 2014).
In response to NTN1 (netrin-1)-mediated activation of DCC, based on mouse studies, SRC phosphorylates ROBO3.1 on a conserved tyrosine Y1019. The mechanism of SRC recruitment to ROBO3.1 in response to NTN1 is not known (Zelina et al. 2014). The biological significance of SRC-mediated phosphorylation of ROBO3.1 is not known.
Based on studies in C. elegans and with recombinant human and mouse proteins, ZSWIM8 and its C. elegans orthologue EBAX-1 are predicted to be an E3 ubiquitin ligase component of the CUL2 ubiquitin ligase complex. ZSWIM8 co-immunoprecipitates with the CUL2 ubiquitin ligase complex components Elongin-B (ELOB) and Elongin-C (ELOC). The BC-box and Cul2-box of ZSWIM8 are needed for interaction with ELOB and ELOC, implying the presence of other CUL2 complex components in the complex of ZSWIM8, ELOB and ELOC. ZSWIM8 binds to wild-type ROBO3.1, but preferentially associates with misfolded or mutant ROBO3.1 proteins, suggesting that it is involved in the quality control of ROBO3.1 (Wang et al. 2013).
Based on studies with C. elegans proteins, ZSWIM8 (orthologue of C. elegans EBAX-1) promotes ubiquitination of ROBO3.1 (orthologue of C. elegans SAX-3) (Wang et al. 2013).
Based on studies using recombinant C. elegans proteins expressed in human 293T cells, ZSWIM8-mediated ubiquitination of ROBO3.1 targets ROBO3.1 for proteasome-mediated degradation (Wang et al. 2013).
Based on studies in mice, binding of MSI1 to ROBO3.1 mRNA positively regulates ROBO3.1 mRNA translation, resulting in increased levels of ROBO3.1 protein. Similar to Robo3 knockout mice, Msi1 knockout mice also show sever abnormalities in axonal midline crossing and migration of precerebellar neurons (Kuwako et al. 2010).
Based on studies in mice, a transcript variant ROBO3.2 is produced from nascent ROBO3 mRNA by alternative splicing. Existence of this splicing isoform is predicted to be conserved in humans and rats. The alternative splicing results in retention of the intronic sequence between exons 26 and 27, which creates a premature stop codon. While Robo3.1 mouse mRNA is expressed in the pre-crossing and crossing commissural axons, Robo3.2 mRNA is expressed after midline crossing and thought to block midline re-crossing (Chen et al. 2008).
Based on studies in mice, ROBO3.2 mRNA, which contains a premature stop codon, is recognized by components of the nonsense mediated decay (NMD) machinery during translation. Association of ROBO3.2 mRNA with UPF2 and UPF1 was directly demonstrated in mouse cells, and presence of other translation and NMD components is assumed (Colak et al. 2013). In this step, we only show association of ROBO3.2 mRNA with the UPF2-containing exon junction complex. For detailed representation of UPF2 and UPF1 in NMD, please refer to the Reactome pathway 'Nonsense Mediated Decay (NMD)'.
Based on studies in mice, translation of ROBO3.2 mRNA in commissural axons at the floor plate is negatively regulated by nonsense mediated decay (NMD). Deficiency of NMD components results in aberrant axonal trajectories after crossing the midline (Colak et al. 2013).
Based on studies in mice, SLIT3 can bind to both ROBO1 and ROBO2 receptors (Mommersteeg et al. 2013). The interaction between SLIT3 and ROBO2 contributes to targeting of axons of olfactory sensory neurons (Cho et al. 2012).
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Junction:UPF2:UPF3
ComplexExon junction complexes (EJCs) are deposited on an mRNA during splicing in the nucleus and are displaced by ribosomes during the first round of translation. When a ribosome terminates translation the A site encounters the termination codon and the eRF1 factor enters the empty A site and recruits eRF3. Normally, eRF1 cleaves the translated polypeptide from the tRNA in the P site and eRF3 interacts with Polyadenylate-binding protein (PABP) bound to the polyadenylated tail of the mRNA.
During activation of NMD eRF3 interacts with UPF1 which is contained in a complex with SMG1, SMG8, and SMG9. NMD can arbitrarily be divided into EJC-enhanced and EJC-independent pathways. In EJC-enhanced NMD, an exon junction is located downstream of the PTC and the EJC remains on the mRNA after termination of the pioneer round of translation. The core EJC is associated with UPF2 and UPF3, which interact with UPF1 and stimulate NMD. Once bound near the PTC, UPF1 is phosphorylated by SMG1. The phosphorylation is the rate-limiting step in NMD and causes UPF1 to recruit either SMG6, which is an endoribonuclease, or SMG5 and SMG7, which recruit ribonucleases. SMG6 and SMG5:SMG7 recruit phosphatase PP2A to dephosphorylate UPF1 and allow further rounds of degradation. How EJC-independent NMD is activated remains enigmatic but may involve competition between PABP and UPF1 for eRF3.
PAK1, PAK2 and PAK3 are direct effectors of RAC1 and CDC42 GTPases. RAC1 and CDC42 bind to the CRIB domain. This binding induces a conformational change that disrupts inactive PAK homodimers and relieves autoinhibition of the catalytic carboxyl terminal domain (Manser et al. 1994, Manser et al. 1995, Zhang et al. 1998, Lei et al. 2000, Parrini et al. 2002; reviewed by Daniels and Bokoch 1999, Szczepanowska 2009). Autophosphorylation of a conserved threonine residue in the catalytic domain of PAKs (T423 in PAK1, T402 in PAK2 and T436 in PAK3) is necessary for the kinase activity of PAK1, PAK2 and PAK3. Autophosphorylation of PAK1 serine residue S144, PAK2 serine residue S141, and PAK3 serine residue S154 disrupts association of PAKs with RAC1 or CDC42 and enhances kinase activity (Lei et al. 2000, Chong et al. 2001, Parrini et al. 2002, Jung and Traugh 2005, Wang et al. 2011). LIMK1 is one of the downstream targets of PAK1 and is activated through PAK1-mediated phosphorylation of the threonine residue T508 within its activation loop (Edwards et al. 1999). Further targets are the myosin regulatory light chain (MRLC), myosin light chain kinase (MLCK), filamin, cortactin, p41Arc (a subunit of the Arp2/3 complex), caldesmon, paxillin and RhoGDI, to mention a few (Szczepanowska 2009).
Class II PAKs also have a CRIB domain, but lack a defined autoinhibitory domain and proline-rich regions. They do not require GTPases for their kinase activity, but their interaction with RAC or CDC42 affects their subcellular localization. Only conventional PAKs will be annotated here.
mRNA in complex with NMD-initiating
UPF2Annotated Interactions
Junction:UPF2:UPF3
ComplexSLIT proteins are the ligands for the ROBO receptors. In humans, there are four ROBO genes: ROBO1, ROBO2, ROBO3 and ROBO4. The extracellular domain of ROBO comprises five Ig domains and three FN domains except for ROBO4 (two Ig + two FN). Ig1 and Ig2 domains of ROBO1 and ROBO2 are highly conserved and are important for SLIT binding. The concave face of SLIT's second LRR domain accommodates the Ig1 and Ig2 domains of ROBO1 and ROBO2. ROBO3 does not bind SLITs (Camurri et al. 2005, Mambetisaeva et al. 2005, Zelina et al. 2014, Jaworski et al. 2015). SLIT binding with ROBO4 is controversial as the interaction is weak and it has been observed using the in-vitro methods (Wang et al. 1999, Brose et al. 1999, Piper et al. 2003, Andrews et al. 2007).
Binding of secreted (cleaved) SLIT2 to ROBO1 and ROBO2 is involved in fasciculation (bundling) of motor axons, which facilitates axon pathfinding and muscle innervation (Jaworski and Tessier-Lavigne 2012).
From the analysis of multiple double mutant combinations of the ROBO:SLIT and Netrin:DCC receptor-ligand pairs, it was deduced that ROBO repulsion on its own is sufficient to prevent commissural axons from re-crossing the midline, and that Netrin:DCC is not the only source of attraction at the midline (Stein and Tessier-Lavigne 2001, Garbe and Bashaw 2007).
The Ena/VASP family of proteins has a universal role in control of cell motility and actin dynamics. These proteins consist of an N-terminal EVH1 domain, a central proline rich region, which acts as a ligand for the actin monomer binding protein Profilin (PFN), as well as several SH3 domain-containing proteins, such as ABL, and a C-terminal EVH2 domain involved in oligomerization and F-actin binding (Bashaw et al. 2000).
SLIT C-terminal fragments may transduce signaling independently of ROBO receptors and Neuropilins (semaphorin receptors) by directly binding to Plexin A1 (Delloye-Bourgeois et al. 2015).
During commissural axon midline crossing in Drosophila, Robo1 signaling can also be antagonized with Robo2 expressed in trans. Extracellular domains of Robo1 and Robo2 may interact through their Ig domains, preventing Robo1 activation by Slits and interfering with axon repulsion (Evans et al. 2015).
The human homologue of Vilse/CrGAP, ARHGAP39 (also known as KIAA1688), was identified. ARHGAP39 shares 54.4% sequence identity with Drosophila CrGAP and is referred to as human Vilse/CrGAP protein (Lundstrom et al. 2004, Hu et al. 2005).
Also based on mouse studies, LHX2, a LIM-homeodomain transcription factor, directly represses transcription of the ROBO2 gene by binding to evolutionarily conserved LHX2 binding sites about 50 kb downstream from the ROBO2 gene transcription start site. LHX2 is involved in thalamocortical axon guidance (Marcos-Mondejar et al. 2012). In commissural relay neurons of the dorsal spinal cord, however, ROBO2 expression is not affected by LHX2 (Wilson et al. 2008).
In zebrafish, transcription of Robo2 is directly stimulated by Mecp2 (Leong et al. 2015).
SLIT2 is one of gene suggested to be repressed by the transcription factor ARX, involved in neuronal proliferation, migration, maturation and differentiation, as well as axon guidance (reviewed by Friocourt and Parnavelas, 2011).
Transcription factors GBX2 and LMO3 may be indirectly involved in ROBO1 gene expression regulation by LHX2 (Chatterjee et al. 2012).
mRNA in complex with NMD-initiating
UPF2mRNA in complex with NMD-initiating
UPF2