During the development process cell migration and adhesion are the main forces involved in morphing the cells into critical anatomical structures. The ability of a cell to migrate to its correct destination depends heavily on signaling at the cell membrane. Erythropoietin producing hepatocellular carcinoma (EPH) receptors and their ligands, the ephrins (EPH receptors interacting proteins, EFNs), orchestrates the precise control necessary to guide a cell to its destination. They are expressed in all tissues of a developing embryo and are involved in multiple developmental processes such as axon guidance, cardiovascular and skeletal development and tissue patterning. In addition, EPH receptors and EFNs are expressed in developing and mature synapses in the nervous system, where they may have a role in regulating synaptic plasticity and long-term potentiation. Activation of EPHB receptors in neurons induces the rapid formation and enlargement of dendritic spines, as well as rapid synapse maturation (Dalva et al. 2007). On the other hand, EPHA4 activation leads to dendritic spine elimination (Murai et al. 2003, Fu et al. 2007). EPH receptors are the largest known family of receptor tyrosine kinases (RTKs), with fourteen total receptors divided into either A- or B-subclasses: EPHA (1-8 and 10) and EPHB (1-4 and 6). EPH receptors can have overlapping functions, and loss of one receptor can be partially compensated for by another EPH receptor that has similar expression pattern and ligand-binding specificities. EPH receptors have an N-terminal extracellular domain through which they bind to ephrin ligands, a short transmembrane domain, and an intracellular cytoplasmic signaling structure containing a canonical tyrosine kinase catalytic domain as well as other protein interaction sites. Ephrins are also sub-divided into an A-subclass (A1-A5), which are tethered to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor, and a B-subclass (B1-B3), members of which have a transmembrane domain and a short, highly conserved cytoplasmic tail lacking endogenous catalytic activity. The interaction between EPH receptors and its ligands requires cell-cell interaction since both molecules are membrane-bound. Close contact between EPH receptors and EFNs is required for signaling to occur. EPH/EFN-initiated signaling occurs bi-directionally into either EPH- or EFN-expressing cells or axons. Signaling into the EPH receptor-expressing cell is referred as the forward signal and signaling into the EFN-expressing cell, the reverse signal. (Dalva et al. 2000, Grunwald et al. 2004, Davy & Robbins 2000, Cowan et al. 2004)
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ROCK I (alternatively called ROK ?) and ROCK II (also known as Rho kinase or ROK ?) were originally isolated as RhoA-GTP interacting proteins. The kinase domains of ROCK I and ROCK II are 92% identical, and so far there is no evidence that they phosphorylate different substrates. RhoA, RhoB, and RhoC associate with and activate ROCK but other GTP-binding proteins can be inhibitors, e.g. RhoE, Rad and Gem. PDK1 kinase promotes ROCK I activity not through phosphorylation but by blocking RhoE association. PLK1 can phosphorylate ROCK II and this enhances the effect of RhoA. Arachidonic acid can activate ROCK independently of Rho.
Class 2 myosins are a set of protein complexes that bind actin and hydrolyse ATP, acting as molecular motors. They consist of two myosin heavy chains , two essential light chains and two regulatory light chains (MRLCs). Smooth muscle and non-muscle myosin isoforms are a subset of Class 2 myosin complexes. The nomenclature for isoforms is misleading, as non-muscle isoforms can be found in smooth muscle. The 4 smooth muscle isoforms all have heavy chains encoded by MYH11. The non-muscle isoforms have heavy chains encoded by MYH9, MYH10 or MYH14 (NMHC-IIA, B and C). The essential light chain (LC17) common to smooth and non-muscle isoforms is encoded by MYL6. The regulatory light chain (LC20) is encoded by either MYL9, giving a slightly more basic protein that is referred to as the smooth muscle LC20 isoform, and MRLC2, giving a more acidic isoform referred to as the non-muscle LC20 isoform.
Class 2 myosins play a crucial role in a variety of cellular processes, including cell migration, polarity formation, and cytokinesis.
Nonmuscle myosin II (NMM2) is an actin-based motor protein that plays a crucial role in a variety of cellular processes, including smooth muscle contraction, cell migration, polarity formation, and cytokinesis. NMM2 consists of two myosin heavy chains encoded by MYH9, MYH10, MYH14 (NMHC-IIA, B and C) or MYH11, two copies of MYL6 essential light chain protein, and two regulatory light chains (MRLCs), MYL9 and MYL12B. Myosin II activity is stimulated by phosphorylation of MRLC. Diphosphorylation at Thr-19 and Ser-20 (commonly referred in the literature as Thr-18 and Ser-19) increases both actin-activated Mg2+ ATPase activity and the stability of myosin II filaments; monophosphorylation at Ser-20 is less effective (Ikebe and Hartshorne 1985, Ikebe et al. 1988). Kinases responsible for the phosphorylation include myosin light chain kinase (MLCK), ROCK kinase, citron kinase, myotonic dystrophy kinase-related CDC42-binding protein kinase, and Zipper-interacting protein (ZIP) kinase. ROCK activity has been shown to regulate MRLC phosphorylation by directly mono- or diphosphorylating MRLC (Amano et al., 1996, Ueda et al., 2002, Watanabe et al. 2007).
EPHB receptor-induced phosphorylation of coffin is at least partially controlled by Rho-associated kinase (ROCK) and LIM domain kinase (LIMK) activities (Shi et al. 2009). ROCK structure comprises a kinase domain located at the amino terminus of the protein, a coiled-coil region containing the Rho-binding domain (RBD), and a pleckstrin-homology (PH) domain with a cysteine-rich domain (CRD). In resting cells ROCKs exist in an autoinhibition state where the kinase domain interacts with the C-terminal inhibitory region. Binding of active RHOA:GTP to RBD stimulates the phosphotransferase activity of ROCK by disrupting the interaction between the catalytic and the inhibitory C-terminal region of the enzyme (Khalil 2010).
Rho-associated kinases (ROCKs) contribute to the formation of actin filaments by inactivating cofilin via phosphorylation of LIM domain kinases (LIMKs). ROCKs phosphorylate LIMK1 at Thr508 and LIMK2 at Thr505, enhancing the ability of LIMKs to phosphorylate cofilin. LIMK1 has been shown to be involved in dendritic spine development, as LIMK1 KO mice fail to form morphologically mature dendritic spines (Meng et al. 2002).
Following ligand binding, EPH signaling is initiated through autophosphorylation. The cytoplasmic domain of EPH receptors can be divided into four functional units; the juxtamembrane region, a tyrosine kinase domain, a sterile alpha-motif (SAM) and a PDZ-domain binding motif. Multiple in vivo tyrosine phosphorylation sites were identified in the juxtamembrane region, kinase domain, and carboxy-terminal tail of EPH receptors. EPH receptors transduce forward signals into the cell through phosphorylation of these tyrosine (Y) residues. Two autophosphorylation sites within the juxtamembrane region (example phosphorylation sites being Y596 and 602 on EPHA4 and Y596 and 602 on EPHB2), and a Y residue within the kinase domain activation segment are identified as the key phosphorylation sites required for the catalytic activity of these EPH receptors. These Y residues are remarkably conserved between the EPHA and EPHB receptors. Substitution of these conserved Y residues in full-length EPHB2 leads to a reduction in ligand-induced kinase activity and EFN-stimulated tyrosine phosphorylation, suggesting that juxtamembrane Y residues may serve a regulatory function in addition to acting as docking sites for downstream targets. These autophosphorylated residues have been shown to interact with a number of proteins including Ras GTPase-activating protein (RasGAP), the p85 subunit of phosphatidylinositol 3-kinase, Src family kinases, the adapter protein NCK, and SHEP-1 (Binns et al. 2000).
The Src-family kinases (SFKs) SRC and FYN, colocalised with ephrinBs (EFNBs) at the membrane, are required for EFNB phosphorylation. The cytoplasmic domains of all three EFNBs consist of five conserved tyrosine residues of which three undergo phosphorylation. Electrospray tandem mass spectrometry and site-directed mutagenesis identified tyrosines 312, 317, and 331 (human 324, 329 and 343) of EFNB1 as phosphorylation sites (Kalo et al. 2001, Palmer et al. 2002).
Protein phosphorylation is an important mechanism modulating the function of NMDA receptors (NMDARs). SRC-family kinases that associate with EPHB are important for the phosphorylation of ionotropic glutamate receptor, NR2B (NMDAR2B, GRIN2B) Tyrosines 1252, 1336, and 1474 (1252, 1336, and 1472 in mouse) on NR2B are phosphorylated by FYN/SRC bound to EPHB2, thereby enhancing NMDA-dependent calcium influx upon glutamate stimulation (Takasu et al. 2002, Dalva et al. 2000). Tyrosine phosphorylation of NMDAR2B also regulates the surface expression of NMDARs. EPHB mediated phosphorylation of NMDARs can also increase the surface retention of NMDAR2B-containing NMDARs by preventing clathrin-dependent endocytosis. Phosphorylation at tyrosine 1474 (mouse Y1472) of NMDAR2B blocks binding of the mu2 subunit of clathrin adaptor protein 2 (AP2) complex thus preventing clathrin-dependent endocytosis (Chen & Roche 2007).
Src family kinases (SFKs) are one of the important components of EPH receptor-mediated axon guidance. Inhibition of SFK-mediated phosphorylation in retinal axons, abolishes EPHA-mediated repulsion in stripe and growth cone collapse assays (Knoll & Drescher 2004). Several members of the Src family, including SRC, FYN, LYN and YES, are expressed widely in the same places as EPH receptors. Autophosphorylated tyrosines in the juxtamembrane region of the EPH receptors have been shown to be critical for the association of the SRC and FYN SH2 domain (Ellis et al. 1996, Zisch et al. 1998). These SFKs are involved in the phosphorylation of RhoGEF ephexin1 and cortactin (Knoll & Drescher 2004). This event is deduced on the basis of experimental evidence from chicken assay studies (Knoll & Drescher 2004)
Focal adhesion kinase 1 (PTK2, FAK, FAK1) acts downstream of EPHB receptors in hippocampal neurons and the EPHB2-FAK signaling contributes to the dendritic spine morphogenesis and synapse maturation by suppressing the activity of actin severing cofilin through phosphorylation. Activation of EPHBs by ephrin-B (EFNB) stimulates the binding of FAK to EPHB. Knock out of FAK in mature neurons induces a shift of mushroom shaped mature dendritic spines to long filopodia like structures, suggesting that synapse formation or maturation is affected in FAK-/- neurons (Shi et al. 2009, Moeller et al. 2006).
The effective activation of the EPH kinase domain requires oligomerisation of EPH receptors and ephrins (EFNs) (Davis et al. 1994). Tetrameric EPH:EFN complexes aggregate into higher-ordered EPH:EFN clusters through several low-affinity EPH:EPH and EFN:EFN interactions, which may be responsible for EPH:EFN signaling (Stein et al. 1998, Pabbisetty et al. 2007).
Receptor stimulation induces translocation of RHOA from the cytosol to the plasma membrane and subsequent activation of RHOA. Rho guanine nucleotide exchange factor 28 (p190RhoGEF, ARHGEF28) is a specific activator of RHOA that stimulates the exchange of GDP for GTP. In neuronal cells p190RhoGEF binds to and activates RHOA with its Dbl homology/pleckstrin homology domain (van Horck et al. 2001).
G-protein-coupled receptor kinase-interacting protein 1 (GIT1) has been shown to be critical for spine morphogenesis and synapse formation through assembling and targeting multiprotein signaling complexes, which contain important actin regulators including PIX, Rac GEF, and PAK, between the sub-cellular compartments (Zhang et al. 2003). Activation of ephrinBs (EFNBs) by EPHB receptors may lead to the phosphorylation of GIT1 on Tyr392 (in humans Tyr385, based on sequence similarity). The Src-family kinases (SFKs) recruited to EFNB expression domains are involved in this phosphorylation. Tyr392 in the N-terminal of the SLD domain is required for GIT1s binding to the SH2 domain of GRB4 (Segura et al. 2007).
Once neural Wiskott-Aldrich syndrome protein (WASL, N-WASP) is activated its VCA region becomes available for binding to actin-related protein (ARP2/3) complex and actin monomer. The actin monomer binds to the V-domain and ARP2/3 complex binds to the CA-domain. The simultaneous binding of G-actin and the ARP2/3 complex to the VCA region contributes to the activation of ARP2/3-complex-mediated actin polymerization (Takenawa & Suetsugu 2007). Actin polymerisation via N-WASP and the ARP2/3 complex leads to spherical expansion of dendritic spine heads.
Two class A or B EPH:EFN hetrodimers tetramerise to form a 2:2 tetramer, forming a ring-like structure, in which each receptor interacts with two ligands and each ligand with two receptors (Himanen et al. 2001). In the tetrameric form the molecules are arranged so that the C-termini of both ligands are located on one side, and the C-termini of the receptors on the other (Himanen & Nikolov 2003).
FAK-mediated spine morphogenesis was shown to occur, in part through Rho guanine nucleotide exchange factor 28 (ARHGEF28, p190RhoGEF), suggesting that FAK-mediated spine maturation might proceed through a FAK-RhoGEF-RHOA mechanism. p190RhoGEF binds directly to phosphorylated PTK2 through a motif in the RhoGEF C-terminal domain, a feature not shared with other GEFs (Moeller et al. 2006, Rico et al. 2004, Zhai et al. 2003).
Neural Wiskott-Aldrich syndrome protein (N-WASP, WASL) with its extended proline-rich region binds simultaneously to several of the five SH3 domains of intersectin-1 (ITSN1). Double-label immunofluorescence confirmed the colocalization of ITSN1, N-WASP and EPHB2 in spines. N-WASP in cooperation with EPHB2 activates the GEF activity of intersectin-1 (Irie & Yamaguchi 2002).
Neural Wiskott-Aldrich syndrome protein (WASL, N-WASP) is a scaffold protein that transduces signals from cell surface receptors to the activation of the ARP2/3 complex and actin polymerization. N-WASP possesses a central GTPase binding domain (GBD) and an NH2-terminal WASP homology domain 1 (WH1). Adjacent to this is a basic region (B) and a C-terminal containing VCA region that contains a V domain (verprolin homology/WASP homology 2), a C domain (connecting), and an A motif (acidic). The VCA region is responsible for binding to and activating the ARP2/3 complex (Bompard & Caron 2004, Callebaut et al. 1998). Under resting conditions, N-WASP is maintained in an auto-inhibition state via interaction of the N-terminal GBD and the C-terminal VCA domains. This prevents access of the ARP2/3 complex to the VCA region. Activated CDC42 binds to the GBD region in N-WASP and this interaction releases the VCA region from auto-inhibition enabling binding of the ARP2/3 complex stimulating actin polymerization (Kim et al. 2000, Park & Cox 2009). Phosphoinositides (PtdIns(4,5)P2) interact with the basic (B) region in WASP and this interaction is important for activation of the WASP and ARP2/3 complex (Higgs & Pollard 2000).
Ephexin1/NGEF (Neuronal guanine nucleotide exchange factor) is a member of the Dbl family of guanine nucleotide exchange factors (GEFs) for Rho GTPases, which interacts with cytoplasmic domain of EPHAs. NGEF is highly expressed in the CNS during development and is enriched in neuronal growth cones. NGEF binds to the kinase domain of EPHA through its Dbl homology (DH)-pleckstrin-homology (PH) domains and this binding does not require activation of the receptor. EPHA activation by ephrinA ligands increases the catalytic activity of ephexin1 resulting in enhanced RHOA activation in cortical neurons (Noren & Pasquale 2004, Shamah et al. 2001). Ephrin-A1 also induces the dispersal of acetylcholine receptors clusters at the neuromuscular junction through the activation of NGEF and RhoA (Shi et al., 2010). NGEF is involved in both axonal growth,growth cone collapse, dendritic spine elimination and stabilization of the neuromuscular junction. In the absence of ephrin stimulation, NGEF promotes actin polymerization and axonal growth by stimulating RHOA, RAC1 and CDC42. Whereas in the presence of ephrin stimulation, NGEF induces growth cone collapse by activating RHOA, but not RAC1 and CDC42 (Shamah et al. 2001, Sahin et al. 2005).
Upon ephrin (EFN)-mediated stimulation, proto-oncogene vav (VAV) is transiently tyrosine phosphorylated and activated in neurons, possibly by Src family kinases (SFKs). VAV2 and VAV3 are phosphorylated on Y172 and Y173 respectively in the acidic domain and this phosphorylation disrupts the inhibitory interaction of the acidic domain with the DH domain (Cowan et al. 2005, Marignani & Carpenter 2001).
The alternate method required for the initial EPH receptor:ephrin (EPH:EFN) adhesion into repulsion is by RAC-dependent endocytosis, an atypical endocytic mechanism by which the EPH:EFN complex and surrounding plasma membrane are internalized into one cell. Proto-oncogene vavs (VAVs) provide a molecular link between activated EPHs and RAC-dependent endocytosis. Upon the activation of EPH receptor, VAV proteins are recruited to the intracellular domain of EPHs and become transiently activated. Cowan et al. showed that VAV2 interacts with either EPHA4 or EPHB2, indicating that VAV can bind to either EPHA or EPHB subclass receptors. Autophosphorylated juxta-membrane tyrosines (Y596 and Y602 of human EPHA4) provide docking sites for VAV proteins (Cowan et al. 2007).
The EPHB2-FAK pathway partially promotes dendritic spine stability through LIMK-mediated cofilin (CFL1) phosphorylation (Shi et al. 2009). CFL1 is a member of the ADF (actin-depolymerizing factor) protein family that is involved in regulating actin dynamics in the growth cone. It binds to actin in a one-to-one molar ratio, and stimulates both the severing of actin filaments and depolymerization of actin subunits from the actin filament end. Activated LIMK phosphorylates CFL1 on the conserved serine 3 residue located near the actin-binding site. After phosphorylation, CFL1 is inactive, loses its affinity for actin and dissociates from G-actin monomers. Once freed, ADP-actin monomers can exchange ADP with cytoplasmic ATP, ready for reincorporation at the barbed end of a growing filament (Gungabissoon & Bamburg 2003).
Focal adhesion kinase 1 (PTK2, FAK, FAK1) activation plays a critical role in EPHB receptor signaling in dendritic spines. PTK2 has six tyrosine phosphorylation sites, with tyrosine 397 being the main auto-phosphorylation site present upstream of the kinase domain (Schaller et al. 1994). Activation of EPHB receptors induces long-lasting phosphorylation of PTK2 on tyrosine 397 (Shi et al. 2009). This phosphorylated tyrosine then creates a binding site for other signaling proteins that link PTK2 to downstream signaling pathways and actin cytoskeleton.
The Dbl homology (DH) and pleckstrin homology (PH) domains of kalirin (KALRN) provides the GEF activity and is activated upon binding to EPHB. Activated KALRN with its GEF domains activates RAC1, and controls spine remodelling by modulating actin cytoskeletal rearrangements (Penzes & Jones 2008, Penzes et al. 2003).
G-protein-coupled receptor kinase-interacting protein 1 (GIT1) is enriched in both presynaptic and postsynaptic terminals and after phosphorylation is targeted to the synapse by binding to cytoplasmic protein GRB4 (NCK2 aka GRB4). GRB4 binds by its SH2 domain to Tyr392 (Human Tyr383) in the synaptic localization domain (SLD) of GIT1. GIT1 acts as an adapter protein by providing docking site for beta-PIX (PAK interacting exchange factor) and serves to localize Rac activity (Segura et al. 2007, Zhang et al. 2003).
Src-family kinases (SFKs) are required for ephrinB (EFNB)-mediated axon guidance and angiogenic responses in endothelial cells. Following interaction with their cognate EPHB receptors and formation of circular tetramers and higher order clusters, EFNBs recruit SFKs and become tyrosine phosphorylated on the cytoplasmic tail. SFK recruitment and activation represents one of the first events in EFNB reverse signaling (Palmer et al. 2002).
The RHOA-ROCK-myosin pathway mediates neurite retraction by increasing the phosphorylation of the regulatory chain of myosin (Amano et al. 1998). Non-muscle myosin II (NMM2) is an actin-based motor protein that plays a crucial role in a variety of cellular processes, including cell migration, polarity formation, and cytokinesis. NMM2 consists of two myosin heavy chains encoded by MYH9, MYH10 or MYH14 (NMHC-IIA, B and C), two copies of MYL6 essential light chain protein, and two regulatory light chains (MRLCs), MYL9 and MYLC2B. Myosin II activity is stimulated by phosphorylation of MRLC. Diphosphorylation at Thr-19 and Ser-20 increases both actin-activated Mg2+ ATPase activity and the stability of myosin II filaments; monophosphorylation at Ser-20 is less effective. Kinases responsible for the phosphorylation include myosin light chain kinase (MLCK), ROCK kinase, citron kinase, myotonic dystrophy kinase-related CDC42-binding protein kinase, and Zipper-interacting protein (ZIP) kinase. ROCK activity has been shown to regulate MRLC phosphorylation directly by mono- (Amano et al. 1996) or di- (Ueda et al. 2002) phosphorylation of MRLC.
Penzes et al. showed that the Kalirin-Rac1-PAK pathway is another signaling cascade downstream of EPHB2 that leads to dendritic spine maturation (Penzes et al. 2003). Kalirin (KALRN) is a Rac-GEF expressed postnatally in neurons and localised to the postsynaptic densities at excitatory synapses, where it participates in the formation and maintenance of dendritic spines. In young hippocampal pyramidal neurons, KALRN links trans-synaptic signaling through ephrinB (EFNB) and EPHB receptors to spine formation and maturation. Upon EFNB activation, KALRN binds EPHB2 and translocates to the postsynaptic membrane.
Serine/threonine-protein kinase PAK1 (PAK1) exists as homodimer in a trans-inhibited conformation. PAK1 translocates from cytosol to plasma membrane and comes in close proximity to RAC1. The kinase inhibitory (KI) domain of one PAK1 molecule binds to the C-terminal catalytic domain of the other and inhibits catalytic activity. RAC1:GTP bind to the GBD domain of PAK1 thereby altering the conformation of the KI domain, relieving inhibition of its catalytic domain, and allowing PAK1 autophosphorylation that is required for full kinase activity (Parrini et al. 2002, Zhao & Manser 2005, Sells et al. 2000).
Autophosphorylation of serine/threonine-protein kinase PAK1 (PAK1) is required for complete activation. PAK1 is autophosphorylated at several sites, but serine 144 (S144) in the GTPase binding domain and threonine 423 (T423) in the activation loop are the two conserved sites that regulate catalytic activity. As a result, active PAK1 phosphorylates substrates like LIMK (LIM domain kinase) that are involved in remodelling of the actin cytoskeleton.
EPHBs are involved in spine synapse formation and also for the recruitment and clustering of glutamate receptors to synapses. At the time of synaptogenesis, EPHBs are localized to the postsynaptic region of excitatory synapses. These postsynaptic EPHBs, upon activation by presynaptic ephrinBs (EFNBs), directly interact with ionotropic glutamate receptor, NR1 and NR2B (NMDAR1and 2B aka GRIN1and 2B). The interaction between EPHB and NMDARs is mediated by the extracellular domains of these two proteins. This interaction promotes clustering of NMDARs at synaptic locations and leads to the formation of functional presynaptic release sites. Activated EPHBs function as tyrosine kinases and may also indirectly potentiate NMDAR-mediated calcium influx (Dalva et al. 2000, Takasu et al. 2002).
The first step in the initiation of EPH-mediated signaling is the high affinity interaction between EPH receptors and ephrin (EFN) ligands of the same subclass located on closely opposed cell surfaces. Membrane bound EPHs bind to EFNs with high affinity and this results in the development of close contacts between the cells. This association between EPHs and EFNs on opposing cells is called trans-interaction and this close contact is required for signaling. This high affinity interaction usually result in contact-mediated repulsion. With some exceptions, B-type EPH receptors (EPHB1-B6) interact with the B-subclass ephrins (EFNB1-B3). EPH and EFN initially form a 1:1 high affinity heterodimer, where EFN inserts its extended loop into a channel at the surface of the receptor (Himanen et al. 2007, Himanen & Nikolov 2003, Himanen et al. 2001).
PAK is also locally activated in the synapse. PAK1 needs autophosphorylation for complete activation. PAK1 is autophosphorylated at several sites, but serine 144 (S144) in the GTPase-binding domain and threonine 423 (T423) in the activation loop are the two conserved sites that regulate the catalytic activity (Bokoch 2003).
Once recruited to the EPHB:NMDAR complex, T-lymphoma invasion and metastasis-inducing protein 1 (TIAM1) is phosphorylated on tyrosine 829 by either EPHB or a kinase that associates with activated EPHB. TIAM1 may then activate Rac1, leading to actin cytoskeletal remodelling required for spine development and morphogenesis (Tolias et al. 2007).
Activated proto-oncogene vav (VAV) act as guanine nucleotide exchange factors (GEFs) for RAC1, catalysing the exchange of bound GDP for GTP. Endocytosis of large EPH:EFN complexes is dependent on RAC1-regulated actin polymerisation. RAC1 signaling is positively linked to EPH:EFN internalization but the exact link to clathrin endocytosis machinery remains unclear. Inhibiting RAC1 signalling and Arp2/3-driven actin polymerization in EPH receptor-expressing cells completely blocks both endocytosis of EPH:EFN complexes and cell retraction (Marston et al. 2003). In response to repulsive guidance molecules, the function of RAC1 changes from promoting actin polymerization associated with axon growth to driving endocytosis of the plasma membrane, resulting in growth cone collapse (Jurney et al. 2002).
p-21-activated kinase (PAK) interacting exchange factor (beta-PIX, bPIX aka ARHGEF7) is a Rac guanine nucleotide exchange factor that acts downstream of GIT1 to affect spine morphology and synapse formation. Endogenous bPIX is present in hippocampal neurons and is targeted to the synapses by binding to GIT1. bPIX serves as an exchange factor for Rac and a binding protein for a Rac effector, PAK (Zhang et al. 2003).
p-21-activated kinase (PAK) interacting exchange factor (beta-PIX, bPIX aka ARHGEF7) a Rac GEF localised at the dendritic spines, activates Rac by exchanging GDP for GTP.
Unlike EPH receptors, ephrinBs (EFNBs) do not possess intrinsic catalytic activity and thus rely on the recruitment of signaling molecules to signal. Phosphorylated ephrinB (p-EFNB) provides docking site for the SH2/SH3 domain-containing adapter protein cytoplasmic protein NCK2 (NCK2 aka GRB4) (Cowan & Henkemeyer 2001). GRB4 is able to bind to phosphotyrosines in EFNBs through their SH2 domains and 'PxxP' motifs through their SH3 domains. It has been postulated that GRB4 acts as a bridge between EFNBs and G protein-coupled receptor kinase interacting protein (GIT) 1 and Rac at synapses (Segura et al. 2007).
Myosin II regulatory light chain (MLC) acts downstream of serine/threonine-protein kinase PAK (PAK) to mediate its effect on spine morphogenesis and excitatory synapse formation. PAK directly phosphorylates MLC on serine 19. MLC phosphorylation could promote dendritic spine morphogenesis by stabilizing the actin network at this site (Zhang et al. 2005).
Serine/threonine-protein kinases PAK1, 2 and 3 (PAK1,2,3) serve as downstream effectors of RACs in regulating spine and synapse formation. Once activated, Ras-related C3 botulinum toxin substrate 1 (RAC1) binds to a variety of downstream effector proteins. Among the most well characterized effectors are PAKs. In addition to binding active RAC, PAKs also directly bind p-21-activated kinase (PAK) interacting exchange factor (beta-PIX, bPIX aka ARHGEF7) (Zhang et al. 2005).
Activation of EPH receptors triggers the local activation of T-lymphoma invasion and metastasis-inducing protein 1 (TIAM1) and this in turn activates RAC1 (Yoo et al. 2010). RAC1 signaling is positively linked to EPH:ephrin (EFN) internalization but the exact link to clathrin endocytosis machinery remains unclear. TIAM1 co-localizes with the ARP2/3 complex at sites of actin polymerization. RAC1 activated by TIAM1 then subsequently activates the ARP2/3 complex proteins leading to actin polymerization (Ten Klooster et al. 2006).
Intersectin-1 (ITSN1), a guanine nucleotide exchange factor (GEF) for CDC42 is one of the binding partners of EPH receptors class B (EPHBs) in synapses. ITSN1 is expressed specifically in the brain and acts as the functional link between CDC42 and EPHB. EPHB2 receptor FORMS a complex with ITSN1 and N-WASP, triggers the activation of CDC42 to promote actin polymerisation via N-WASP and the ARP2/3 complex, leading to spherical expansion of dendritic spine heads. EPHB co-immunoprecipitates with ITSN1, mediated by the kinase domain-containing fragment of EPHB and the amino (N)-terminal region of ITSN1 (Irie & Yamaguchi 2002).
T-lymphoma invasion and metastasis-inducing protein 1 (TIAM1) is a Rac1-specific GEF, highly expressed in the nervous system, which is necessary for proper spinal and synapse development. It is one of the critical mediators of NMDA receptor (NMDAR)-dependent spine development (Tolias et al. 2005). TIAM1 might also play a role in regulating EPHB-dependent spine morphogenesis. TIAM1 appears to be required for ephrinB (EFNB)-induced increase in spine density. TIAM1 is recruited to EPHB complexes containing NMDAR after EPHB receptor activation in neurons. The PH-CC-Ex (consisting of a pleckstrin homology (PH) domain followed by a coiled-coiled (CC) domain and an adjacent region (Ex) domain) of TIAM1 is required for binding to EPHB2 (Tolias et al. 2007).
The first step in the initiation of EPH-mediated signaling is the high affinity interaction between EPH receptors and ephrin (EFN) ligands of the same subclass located on closely opposed cell surfaces. Membrane bound EPHs bind to EFNs with high affinity and this results in the development of close contacts between the cells. This association between EPHs and EFNs on opposing cells is called trans-interaction and this close contact is required for signaling. This high affinity interaction usually result in contact-mediated repulsion. With some exceptions, the EPH receptor A-subclass (EPHA1-A8, A10) bind to the A-class ephrins (EFNA1-A4, A6. EPH and EFN initially form a 1:1 high affinity heterodimer, where ephrin inserts its extended loop into a channel at the surface of the receptor (Himanen et al. 2007, Himanen & Nikolov 2003, Himanen et al. 2001).
RHOA propagates downstream signals by binding to effector proteins such as Rho-associated, coiled-coil containing protein kinases (ROCKs). ROCKs consist of an amino-terminal kinase domain, followed by a Rho-binding domain (RBD) and a carboxy terminal cysteine-rich domain (CRD) located within the pleckstrin homology (PH) motif. RHOA:GTP interacts with the RBD domain and activates the phosphotransferase activity (Ishizaki et al. 1996, Amano et al. 2000).
Activation of EPHAs leads to phosphorylation of ephexin1 (NGEF) on conserved tyrosine (Y) 179 (Y87 in isoform3) by Src family kinases (SFKs). This phosphorylation preferentially activates NGEF’s GDP/GTP exchange activity specifically towards RHOA but not RAC1 and CDC42, thus switching the substrate preference of NGEF and leading to actin cytoskeletal changes that result in growth cone collapse (Sahin et al. 2005, Knoll and Drescher 2004).
The tyrosine phosphorylation of ephexin1 (NGEF)/Ephexin-1 enhances its exchange activity and specifically activates inactive RHOA:GDP to active RHOA:GTP. Activated RHOA regulates myosin II activity in neurons through Rho-associated kinase (ROCK), and leads to rapid growth cone collapse, neurite retraction, or neurite growth inhibition in axons. Through this repulsive mechanism EPH receptors and ephrins (EFNs) guide retinal axons to their targets in the visual centres in the brain (Sahin et al. 2005).
During endocytosis the EPH-ephrin (EFN) intact complex and, possibly, associated cytoplasmic proteins, together with the surrounding plasma membrane, can be internalized into the EPH- or EFN-expressing cell. The clathrin pathway has been linked to EFNB endocytosis. C-terminal portion of EFNB1 encodes a putative endocytosis signal, a sequence that is recognized by the clathrin-associated adapter proteins required for loading molecules into clathrin-coated pits and vesicles. Treatment of cells expressing GFP-tagged EFNB1 with soluble, recombinant EPHB1/Fc fusion protein were internalized and observed in clathrin-coated vesicles. Internalized EFNB1 co-localizes with EEA1, an endosomal marker. EFNB1 internalization is inhibited by a dominant-negative dynamin mutant and potassium depletion. These results suggest that classical, clathrin-dependent endocytosis is responsible for EFNB internalization. (Parker at al. 2004).
EPHB2-C-terminal fragment1 (CTF1) is subsequently cleaved within the transmembrane domain between residues 569 and 570 (mouse EPHB2) by gamma-secretase to release a shorter cytosolic fragment containing 425 C-terminal amino acids. The EPHB2 cleavage products EPHB2-CTF1 and EPHB2-CTF2 may enhance the duration of full length EPHB2 phosphorylation. These fragments lack the ephrin (EFN)-binding domain, but retain the entire cytoplasmic portion of the receptor, and may sequester phosphatases from full-length EPHB2 receptor, thus preventing its dephosphorylation (Litterst et al. 2007).
Alternatively, the EPH:ephrin (EPH:EFN) interaction can be broken by proteolytic cleavage of the EPH receptors by matrix metalloproteinases (MMPs) and gamma-secretase. Ethell and his group showed that the EPHB2 receptor is cleaved by the secreted MMPs MMP-2 and MMP-9, and this cleavage is induced by EFNB:EPHB receptor interaction. EPHB2 receptor is initially cleaved within the fibronectin (FN) type III domain or between residues 543 and 544 (mouse EPHB2) at the cell surface by MMPs to produce a long fragment that is released to the extracellular space and a membrane bound C-terminal fragment (Ethell et al. 2007, Litterst et al. 2007).
After the initial adhesion, the EPH- and ephrin (EFN)-bearing cells break away due to a repulsive response. Repulsive signaling outcomes that promote cell-cell disengagement involve removal of the cell surface EPH:EFN complexes rather than dissociation of the bound proteins (Litterst et al. 2007). Removal of EPH:EFN complexes is mediated by a mechanism involving a metalloprotease enzyme bound to EFN or EPH receptors, which is activated after a time delay to cleave EFN or EPH. Through this mechanism, the mechanical adhesion between EFN and EPH on different cell surfaces is broken, resulting in the two cells moving apart. ADAMs (a disintegrin and a metalloprotease) are proteases responsible for the regulated ectodomain shedding of protein receptors like TGF-alpha, TNF-alpha, and the notch ligand Delta, a process that is critical to their signaling function. Similarly, EFNAs have been identified as substrates for membrane-bound ADAM10/Kuzbanian/KUZ. Flanagan and colleagues showed that ADAM10 is constitutively associated with EFNA2, and mediates the cleavage of EFNA2 ectodomain upon EPHA3:EFNA2 complex formation . This EFN cleavage mechanism was proposed to occur in cis (where both the proteinase and its substrate are within the same cell membrane) (Hattori et al. 2000). Janes et al. show the alternate model to that of the Flangan group. They showed that ADAM10 constitutively associates with the EPHA3 receptor, rather than EFNA and cleaves EFNA5 from its membrane tether only in trans (where the proteinase and its substrate are on opposing cell membrane) (Janes et al. 2005). In both models, EFN is cleaved from the membrane by ADAM10 only after the interaction of EFN with EPH receptors.
T-lymphoma invasion and metastasis-inducing protein 1 (TIAM1) is a GEF exhibiting highest specificity for RAC1 and is critically involved in EPH/ephrin (EFN)-mediated neurite outgrowth and dendritic spine development (Tanaka et al. 2004, Tolias et al. 2007). TIAM1 has a role in regulating the endocytosis of EPHA receptors, by regulating the RAC1 signaling downstream of the EPHA8 receptor. Yoo and his colleagues found that TIAM1, a RAC-specific guanine nucleotide exchange factor, co-immunoprecipitates with EPHA8 in response to EFNA5 stimulation. TIAM1 associates with EPHA8 in the juxtamembrane (JM) region. Deletion of the JM region or down-regulation of TIAM1 expression compromises EPHA8:EFNA5 endocytosis (Yoo et al. 2010).
Activation of ephrin-B1 (EFNB1) and ephrin-B2 (EFNB2) by postsynaptic EPHB receptors initiates presynaptic differentiation and causes increases in the density of presynaptic release sites. The EPHB-EFNB interaction recruits the adaptor protein syntenin-1 (SDCBP) through the PDZ-binding domain of EFNBs. SDCBP colocalizes with EFNB1 and EFNB2 at synaptic contacts and knockdown of EFNBs leads to a reduction in the number of synaptic contacts. SDCBP provides a direct link by which EFNB can associate with a protein complex involved in the recruitment and regulation of presynaptic vesicles. McClelland et al. suggest a model whereby SDCBP recruits multidomain scaffolding molecules that enables the clustering of synaptic vesicles including ERC2/CAST1, RIM1 and Rab synaptic vesicle proteins (McClelland et al. 2009).
In addition to regulating Rho family proteins, the EPH receptors and ephrins (EFNs) also regulate the activity of Ras family proteins. Ras-MAPK pathway is a key regulator of cell proliferation, adhesion and transformation, but can also influence axon guidance (Forcet et al. 2002). EPHB receptors downregulate H-Ras and consequently its downstream effector extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway in neuronal cells (Elowe et al. 2001, Miao et al. 2000). EPHB2 signals through the SH2 domain protein p120-RasGAP (RASA1) to inhibit the Ras-MAPK pathway. p120-RasGAP binds directly through its SH2 domains to the autophosphorylated EPHB2 juxtamembrane region (Holland et al. 1997, Elowe et al. 2001).
p120-RasGAP (RASA1) binds to activated Ras (Ras:GTP) through its GAP-related catalytic domain, which is sufficient for stimulating the Ras GTPase activity. p120-RasGAP accelerates the intrinsic GTPase activity of Ras to promote Ras inactivation (Yao et al. 1995).
Syndecans (SDCs) are a major class of cell surface heparan sulphate proteoglycans (HSPGs) and the member syndecan-2 (SDC2) plays a critical role in EPHB-mediated spine formation in hippocampal neurons. Ethell et al. suggest that one of the mechanisms by which EPHB receptors regulate spine morphology is through phosphorylation of SDC2 (Ethell et al. 2001, Ethell & Yamaguchi 1999). Activated EPHB2 phosphorylates SDC2 at tyrosine residues Y189 and Y201 (Y179 and Y191 according to uniprot reference sequence) in the C1 and V regions respectively (Ethell et al. 2001).
Phosphorylated T-lymphoma invasion and metastasis-inducing protein 1 (p-TIAM1) bound to EPHB complexes containing NMDARs, promotes spine morphogenesis by activating RAC1, which triggers actin cytoskeletal remodelling that is essential for spine development (Tolias et al. 2007, Tolias et al. 2005).
The p120-RasGAP C-terminus contains a GAP domain that catalyses the activation of H-Ras by hydrolyzing GTP-bound active Ras into an inactive GDP-bound form of Ras. Inactivation of H-Ras by EPHB2 down regulates MAP kinase phosphorylation and induces neurite retraction in neuronal cells (Elowe et al. 2001, Tong et al. 2003).
Syndecan-2 (SDC2), upon phosphorylation by EPHB2, undergoes multimerization and clustering on dendrites leading to spinogenesis. Pathways downstream of SDC2 that ultimately leads to cytoskeletal rearrangement of the spine have yet to be elucidated. Ethell et al. hypothesised that EPHB2 may associate with SDC2 after clustering and localise SDC2 to sites of nascent spines. Subsequent recruitment of syntenin and CASK by SDC2 via PDZ interactions may promote spinogenesis (Ethell et al. 2001, Lin et al. 2007).
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muscle/non-muscle
myosin IIAnnotated Interactions
muscle/non-muscle
myosin II