In normal development vascular endothelial growth factors (VEGFs) are crucial regulators of vascular development during embryogenesis (vasculogenesis) and blood-vessel formation in the adult (angiogenesis). In tumor progression, activation of VEGF pathways promotes tumor vascularization, facilitating tumor growth and metastasis. Abnormal VEGF function is also associated with inflammatory diseases including atherosclerosis, and hyperthyroidism. The members of the VEGF and VEGF-receptor protein families have distinct but overlapping ligand-receptor specificities, cell-type expression, and function. VEGF-receptor activation in turn regulates a network of signaling processes in the body that promote endothelial cell growth, migration and survival (Hicklin and Ellis, 2005; Shibuya and Claesson-Welsh, 2006). Molecular features of the VGF signaling cascades are outlined in the figure below (from Olsson et al. 2006; Nature Publishing Group). Tyrosine residues in the intracellular domains of VEGF receptors 1, 2,and 3 are indicated by dark blue boxes; residues susceptible to phosphorylation are numbered. A circled R indicates that phosphorylation is regulated by cell state (VEGFR2), by ligand binding (VEGFR1), or by heterodimerization (VEGFR3). Specific phosphorylation sites (boxed numbers) bind signaling molecules (dark blue ovals), whose interaction with other cytosolic signaling molecules (light blue ovals) leads to specific cellular (pale blue boxes) and tissue-level (pink boxes) responses in vivo. Signaling cascades whose molecular details are unclear are indicated by dashed arrows. DAG, diacylglycerol; EC, endothelial cell; eNOS, endothelial nitric oxide synthase; FAK, focal adhesion kinase; HPC, hematopoietic progenitor cell; HSP27, heat-shock protein-27; MAPK, mitogen-activated protein kinase; MEK, MAPK and ERK kinase; PI3K, phosphatidylinositol 3' kinase; PKC, protein kinase C; PLCgamma, phospholipase C-gamma; Shb, SH2 and beta-cells; TSAd, T-cell-specific adaptor. In the current release, the first events in these cascades - the interactions between VEGF proteins and their receptors - are annotated. Details of signaling events and their biological outcome, concisely illustrated in the image below, will be available in future versions of this pathway.
View original pathway at:Reactome.
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The RAS-RAF-MEK-ERK pathway regulates processes such as proliferation, differentiation, survival, senescence and cell motility in response to growth factors, hormones and cytokines, among others. Binding of these stimuli to receptors in the plasma membrane promotes the GEF-mediated activation of RAS at the plasma membrane and initiates the three-tiered kinase cascade of the conventional MAPK cascades. GTP-bound RAS recruits RAF (the MAPK kinase kinase), and promotes its dimerization and activation (reviewed in Cseh et al, 2014; Roskoski, 2010; McKay and Morrison, 2007; Wellbrock et al, 2004). Activated RAF phosphorylates the MAPK kinase proteins MEK1 and MEK2 (also known as MAP2K1 and MAP2K2), which in turn phophorylate the proline-directed kinases ERK1 and 2 (also known as MAPK3 and MAPK1) (reviewed in Roskoski, 2012a, b; Kryiakis and Avruch, 2012). Activated ERK proteins may undergo dimerization and have identified targets in both the nucleus and the cytosol; consistent with this, a proportion of activated ERK protein relocalizes to the nucleus in response to stimuli (reviewed in Roskoski 2012b; Turjanski et al, 2007; Plotnikov et al, 2010; Cargnello et al, 2011). Although initially seen as a linear cascade originating at the plasma membrane and culminating in the nucleus, the RAS/RAF MAPK cascade is now also known to be activated from various intracellular location. Temporal and spatial specificity of the cascade is achieved in part through the interaction of pathway components with numerous scaffolding proteins (reviewed in McKay and Morrison, 2007; Brown and Sacks, 2009). The importance of the RAS/RAF MAPK cascade is highlighted by the fact that components of this pathway are mutated with high frequency in a large number of human cancers. Activating mutations in RAS are found in approximately one third of human cancers, while ~8% of tumors express an activated form of BRAF (Roberts and Der, 2007; Davies et al, 2002; Cantwell-Dorris et al, 2011).
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.
The cofactor tetrahydrobiopterin (BH4) ensures endothelial nitric oxide synthase (eNOS) couples electron transfer to L-arginine oxidation (Berka et al. 2004). During catalysis, electrons derived from NADPH transfer to the flavins FAD and FMN in the reductase domain of eNOS and then on to the ferric heme in the oxygenase domain of eNOS. BH4 can donate an electron to intermediates in this electron transfer and is oxidised in the process, forming the BH3 radical. This radical can be reduced back to BH4 by iron, completing the cycle and forming ferrous iron again. Heme reduction enables O2 binding and L-arginine oxidation to occur within the oxygenase domain (Stuehr et al. 2009).
Inositol 1,4,5-triphosphate (IP3) is a second messenger produced by phospholipase C (PLC) metabolism of phosphoinositol 4,5-bisphosphate (PIP2) (Canossa et al. 2001).
The IP3 receptor (IP3R) is an IP3-gated calcium channel. It is a large, homotetrameric protein, similar to other calcium channel proteins such as ryanodine. The four subunits form a 'four-leafed clover' structure arranged around the central calcium channel. Binding of ligands such as IP3 results in conformational changes in the receptor's structure that leads to channel opening.
Activated p38 MAPK is known to activate the Ser/Thr protein kinase MAP kinase-activated protein kinase 2 (MAPK2/MAPKAPK2) and a closely related kinase, MAPKAP kinase 3. MAPK2 is phosphorylated on T222, S272, and T334 (Ben-Levy et al. 1995). MAPK3 shows 75% sequence identity to MAPK2 and, like MAPK2, is phosphorylated by p38 but the exact phosphorylation sites are not determined. According to some authors, NGF does not induce any significant activation of MAPKAPK2 activity in PC12 cells. Potential p38 signaling effectors include transcription factors, such as cAMP-response element-binding protein and MEF2, cytoskeleton modulators, and a number of protein kinases. After activation, MAPKAP kinase 2 and 3 move to the nucleus.
VEGFR-3 preferentially binds VEGF-C and -D. Mutations of the VEGFR-3 tyrosine kinase domain are seen in human lymphedema. VEGFR-3 expression has been correlated with transient lymphangiogenesis in wound healing and may modulate VEGFR-2 signaling in maintaining vascular integrity (Hicklin and Ellis 2005).
VEGFR-2 binds VEGF-A, -C, -D, and -E homodimers. VEGFR-2 is the primary mediator of the physiological effects of VEGF-A in angiogenesis, including microvascular permeability, endothelial cell proliferation, invasion, migration, and survival. In endothelial cells, these effects are mediated via activation of a phospholipase gamma-protein kinase C-Raf-MAPK signaling pathway for proliferation and PI3K and focal adhesion kinase for survival and migration. VEGFR-2 is the important receptor among VEGFR protiens and its activation and signaling may be positively or negatively regulated by co-expression and activation of various factors and other VEGF receptors like VEGFR-1 (Hicklin and Ellis 2005).The regulatory events of this receptor will be annotated in subsequent modules.
VEGFR-1 binds VEGF-A, VEGF-B, and PLGF homodimers. This interaction is required for normal angiogenesis and hematopoiesis, although many of the detailed molecular steps from binding to these physiological consequences remain unclear (Hickins and Ellis, 2005). VEGFR-1 is made up of 1338 aa and has three regions: an extracellular region consisting of 7 immunoglobin-like domains, a transmembrane (TM) domain and a cytosolic tyrosine kinase (TK) domain. An alternatively spliced form, soluble VEGFR-1 (sVEGFR1), also binds VEGF proteins and may serve in the body to down-regulate VEGF activation of membrane-bound receptors. Overexpression of sVEGFR1 (VEGF121) is associated with preeclampsia, a major disorder of pregnancy (Shibuya and Claesson-Welsh 2006; Levine et al. 2004).
VEGF proteins bind their receptors as homodimers. Heterodimers with PLGF and among different VEGF proteins have been observed but have no known function.
Plasma membrane-associated Neuropilin-1 (NRP1) binds vascular endothelial growth factor (VEGF) family members. NRP1 has three distinct extracellular domains, a1a2, b1b2, and c but lacks a distinct intracellular domain. VEGF165 mediates the formation of complexes containing VEGFR-2 and NRP-1, enhancing VEGF165-receptor binding on the endothelial cell membrane (Soker et al. 2002). The role of heparin, a critical component of NRP-1 interactions with VEGF proteins, will annotated in detail in future.
NRP-2 associates with VEGFR-1 on the plasma membrane. As NRP-2 lacks an intracellular domain, this association may be the means by which NRP-2 participates in VEGF-induced signaling. This interaction requires VEGF to bridge between NRP and the receptor.
Once AKT is localized at the plasma membrane, it is phosphorylated at two critical residues for its full activation. These residues are a threonine (T308 in AKT1) in the activation loop within the catalytic domain, and a serine (S473 in AKT1), in a hydrophobic motif (HM) within the carboxy terminal, non-catalytic region. PDPK1 (PDK1) is the activation loop kinase; this kinase can also directly phosphorylate p70S6K. The HM kinase, previously termed PDK2, has been identified as the mammalian TOR (Target Of Rapamycin; Sarbassov et al., 2005) but several other kinases are also able to phosphorylate AKT at S473. Phosphorylation of AKT at S473 by TORC2 complex is a prerequisite for PDPK1-mediated phosphorylation of AKT threonine T308 (Scheid et al. 2002, Sarabassov et al. 2005).
Under conditions of growth and mitogen stimulation S473 phosphorylation of AKT is carried out by mTOR (mammalian Target of Rapamycin). This kinase is found in two structurally and functionally distinct protein complexes, named TOR complex 1 (TORC1) and TOR complex 2 (TORC2). It is TORC2 complex, which is composed of mTOR, RICTOR, SIN1 (also named MAPKAP1) and LST8, that phosphorylates AKT at S473 (Sarbassov et al., 2005). This complex also regulates actin cytoskeletal reorganization (Jacinto et al., 2004; Sarbassov et al., 2004). TORC1, on the other hand, is a major regulator of ribosomal biogenesis and protein synthesis (Hay and Sonenberg, 2004). TORC1 regulates these processes largely by the phosphorylation/inactivation of the repressors of mRNA translation 4E binding proteins (4E BPs) and by the phosphorylation/activation of ribosomal S6 kinase (S6K1). TORC1 is also the principal regulator of autophagy. In other physiological conditions, other kinases may be responsible for AKT S473 phosphorylation.
Phosphorylation of AKT on S473 by TORC2 complex is a prerequisite for AKT phosphorylation on T308 by PDPK1 (Scheid et al. 2002, Sarabassov et al. 2005).
HSP90 serves as a scaffold to promote productive interaction between AKT1 and eNOS. Due to the proximity of these proteins once complexed with HSP90, AKT1 phosphorylates eNOS at Ser1177. When Ser1177 is phosphorylated, the level of NO production is elevated two- to three-fold above basal level.
Nitric oxide (NO) is produced from L-arginine by the family of nitric oxide synthases (NOS) enzymes, forming the free radical NO and citrulline as byproduct. The cofactor tetrahydrobiopterin (BH4) is an essential requirement for the delivery of an electron to the intermediate in the catalytic cycle of NOS.
WASP family verprolin-homologous proteins (WAVEs) function downstream of RAC1 and are involved in activation of the ARP2/3 complex. The resulting actin polymerization mediates the projection of the plasma membrane in lamellipodia and membrane ruffles. WAVEs exist as a pentameric hetero-complex called WAVE Regulatory Complex (WRC). The WRC consists of a WAVE family protein (WASF1, WASF2 or WASF3 - commonly known as WAVE1, WAVE2 or WAVE3), ABI (Abelson-interacting protein), NCKAP1 (NAP1, p125NAP1), CYFIP1 (SRA1) or the closely related CYFIP2 (PIR121), and BRK1 (HSPC300, BRICK). Of the three structurally conserved WAVEs in mammals, the importance of WAVE2 in activation of the ARP2/3 complex and the consequent formation of branched actin filaments is best established. WAVEs in the WRC are intrinsically inactive and are stimulated by RAC1 GTPase and phosphatidylinositols (PIP3). The C-terminal VCA domain of WAVE2 (and likely WAVE1 and WAVE3) which can bind both the ARP2/3 complex and actin monomers (G-actin) is masked in the inactive state. After PIP3 binds to the polybasic region of WAVE2 (and likely WAVE1 and WAVE3) and RAC1:GTP binds to the CYFIP1 (or CYFIP2) subunit of the WRC, allosteric changes most likely occur which allow WAVEs to interact with the ARP2/3 complex. The interactions between WAVEs and RAC1 are indirect. BAIAP2/IRSp53, an insulin receptor substrate, acts as a linker, binding both activated RAC1 and the proline-rich region of WAVE2 (and likely WAVE1 and WAVE3) and forming a trimolecular complex. CYFIP1 (or CYFIP2) in the WAVE regulatory complex binds directly to RAC1:GTP and links it to WAVE2 (and likely WAVE1 and WAVE3) (Derivery et al. 2009, Yamazaki et al. 2006, Takenawa & Suetsugu 2007, Chen et al. 2010, Pollard 2007, Lebensohn & Kirschner 2009).
PIP3 generated by PI3K recruits phosphatidylinositide-dependent protein kinase 1 (PDPK1 i.e. PDK1) to the membrane, through its PH (pleckstrin-homology) domain. PDPK1 binds PIP3 with high affinity, and also shows low affinity for PIP2 (Currie et al. 1999).
Once phosphorylated on serine residue S473, AKT bound to PIP3 forms a complex with PIP3-bound PDPK1 i.e. PDK1 (Scheid et al. 2002, Sarabassov et al. 2005)
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).
Vav interacts directly with PIP2 and PIP3, with a fivefold selectivity for PIP3 over PIP2. PIP3 gives a twofold stimulation of Vav1 GEF activity while PIP2 leads to 90% inhibition. Binding probably occurs through the PH domain, known to bind phosphoinositides.
SHB binds focal adhesion kinase 1 (FAK1) via its PTB domain in a phosphotyrosine-dependent manner. This regulates FAK1 phosphorylation, leading to Src dependent enhanced cell spreading (Holmqvist et al. 2003). During vascular development, FAK1 is involved in the control of endothelial cell migration (Holmquist et al. 2004), vascular permeability (Chen et al. 2012) and tube formation (Bohnsack & Hirshi, 2003).
The adaptor protein SHB (Src homology 2 domain-containing adapter protein B) binds to phosphorylated tyrosine Y1175 in VEGFR2 and regulates the FAK activity and endothelial cell migration. The SH2 domain located in the C-terminus of SHB interacts with the phosphotyrosine residue in VEGFR2 (Holmqvist et al. 2004). SHB is not required for vascular development, but SHB-deficient mice shows diffects in vessel functionality (Christoffersson et al. 2012) and impaired tumor growth (Funa et al. 2009).
Phosphorylated tyrosine Y1175 of VEGFR2 provides the binding site for the adaptor protein SHC-transforming protein 2 (SHC2) also referred as Shc-like protein (SCK). SCK is plausibly involved in coupling VEGFR2 to ERK (Warner et al. 2000, Ratcliffe et al. 2002).
Binding of VEGFA to VEGFR2 induces receptor dimerization and autophosphorylation, leading to the recruitment of downstream signalling molecules. Once the two VEGFR2 receptors are cross-linked to each other, via simultaneous interaction with VEGFA dimer, their membrane-proximal Ig-like domain 7s are held in close proximity so that low-affinity homotypic interactions between these domains further stabilise the receptor dimers. This allows for the exact positioning of the intracellular kinase domains resulting in VEGFR2 autophosphorylation (Ruch et al. 2007, Holmes at al. 2007). The major tyrosine residues known to be autophosphorylated are Y801 and Y951 in the kinase-insert domain, Y1054 and Y1059 within the kinase domain, and Y1175 and Y1214 in the C-terminal tail of VEGFR (Dougher-Vermazen et al. 1994, Cunningham et al. 2007, Kendall et al. 1999, Matsumoto et al. 2005). The Y1175 (mice Y1173) is crucial for endothelial and haemopoietic cell development. Mice with muatation Y1173F die between E8.5 and E9.5 from lack of endothelial and haemopoietic development (Sakurai et al. 2005).
Activation of VEGFR2 has been shown to lead to direct binding and phosphorylation of PLC-gamma 1 (McLaughlin & Vries 2001). PLCG1 is tyrosine phosphorylated directly by VEGFR2 or through the Src kinases on four tyrosine residues, enhancing the activity of PLCG1.
TSAD (T cell-specific adapter protein/ SH2D2A) bound to VEGFR2 forms a complex with Src to regulate stress fiber formation and endothelial cell (EC) migration. This contributes to EC migration during pathological angiogenesis and thus the recruitment of TSAD is associated with cancer angiogenesis (Matsumoto et al. 2005).
Two-hybrid mapping showed that tyrosine 951 (Y951) serves as the binding site for T-cell specific adapter molecule (TSAD/ SH2 domain-containing protein 2A (SH2D2A)), also referred as VEGF-receptor-associated protein (VRAP) (Wu et al. 2000). TSAD mediates vasular permiability downstream of VEGFR2 by forming a complex with c-SRC (Sun et al. 2012). Site-directed mutation of Y951 to phenylalanine (Y951F) in the VEGFR2, or siRNA mediated silencing of TSAD expression, prevented VEGFA mediated cytoskeletal reorganisation and migration but not mitogenicity (Matsumoto et al. 2005).
Phospholipase C-gamma 1 (PLCG1) plays a pivotal role in angiogenesis and VEGFR2 signal transduction. VEGFR2-mediated activation of PLCG1 in certain endothelial cellular backgrounds is suggested to stimulate cell proliferation and in other endothelial cells to stimulate differentiation and tubulogenesis (Rahimi 2006). Phosphorylated tyrosine 1175 of VEGFR2 provides the binding site for PLCG1, SHC-transforming protein 2 (SHC2/SCK) and SH2 domain-containing adapter protein B (SHB) (Takahashi et al. 2001). Binding of PLCG1 activates protein kinase C (PKC) and this in-turn stimulates mitogen-activated protein (MAP) kinase (MAPK)-dependent pathway and cell proliferation (McLaughlin & Vries 2001).
Following tyrosine phosphorylation and activation, PLCG1 dissociates from the VEGFR2 receptor and associates with its substrate phosphatidylinositol (4,5)-bisphosphate (PIP2) in the plasma membrane. PLCG1 hydrolyses PIP2 resulting in the generation of diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). DAG is an activator of PKC which leads to subsequent activation of MAP kinase, resulting in increased endothelial cell proliferation. IP3 acts upon receptors in the endoplasmic reticulum causing release of intracellular calcium. Elevation of cytosolic Ca2+ stimulates eNOS to produce nitric oxide (NO) causing vascular dilation. Entry of extracellular calcium through specific channels is important for the activation of certain proteins (Takahashi et al. 2001, Takahashi et al. 1999, Xia et al. 1996).
SRC is activated in vivo and in vitro in a VEGF/SH2D2A-dependent manner. VEGF induces phosphorylation of the activating Y418 residue, located on the c-SRC kinase activation loop, but also decreases phosphorylation of the negative regulatory Y527 (Sun et al. 2012).
Src family kinases (SFKs) induce transphosphorylation of tyrosyl residues Y576, Y577, Y861 and Y925. Phosphorylation of Y576 and Y577 within the catalytic domain confers maximal FAK1 enzymatic activity and signaling in response to adhesion (Calalb et al. 1995, Brunton et al. 2005). Y576 and Y861 are both phosphorylated in a Src-dependent manner in response to VEGF (Abu-Ghazaleh et al. 2001, Le Boeuf et al. 2006, 2004). Phosphorylation of FAK on Y861 contributes to the recruitment of vinculin to FAK1 (Le Boeuf et al. 2004).
Six tyrosine phosphorylation sites in focal adhesion kinase 1 (FAK1) serve to modulate FAK1 kinase activity or mediate FAK1 interaction with SH2-domain containing proteins. These are Y397, Y407, Y576, Y577, Y861 and Y925 (Mitra et al. 2005). They are differentially phosphorylated by diverse agonists and implicated in transmitting different signals and effects (Ciccimaro et al. 2006, Le Boeuf et al. 2004,2006). Y397 is the major autophosphorylation site present upstream of the FAK kinase domain (Schaller et al. 1994). In response to VEGF stimulation FAK1 is recruited and autophosphorylated at Y397. This phosphorylated tyrosine then creates a binding site for other signaling proteins that link FAK1 to downstream signaling pathways and the actin cytoskeleton (Toutant et al. 2002).
Heat-shock protein of 90 kDa (HSP90) a molecular chaperone, associates with VEGFR2 in response to VEGF. The last 130 amino acids of VEGFR2 C-terminal portion are involved in the association of VEGFR2 with HSP90. HSP90 associated with VEGFR2 is involved in regulating the activity of a Rho-associated protein kinase (ROCK) that is required to phosphorylate FAK on residue S732 (Le Bouef et al. 2004, 2006).
Autophosphorylation of Y397 on FAK1 provides a high affinity binding site for the Src homology 2 (SH2) domain of Src family kinases (SFKs) allowing their recruitment, activation and subsequent transphosphorylation of FAK1 at additional sites (Calalb et al. 1995).
In primary cultured human umbilical vein endothelial cells (HUVECs) VEGF-induced activation of SAPK2/p38MAPK, and pharmacological inhibition of p38MAPK attenuated VEGF-induced cell migration (Rouseseau et al. 1997, 2000). The p38MAPK pathway conveys the VEGF signal to microfilaments inducing rearrangements of the actin cytoskeleton. These actin structures are thought to generate the contractile force within cells that is required for endothelial cell migration. Activation of p38 requires the activity of FYN and PAK2 (Lamalice et al. 2004). However, little is known of the exact molecular events that follow activation of PAK2 and lead to p38 activation. Like all MAP kinases, p38 MAP kinases are activated by dual kinases termed the MAP kinase kinases (MKKs). There are two main MAPKKs that are known to activate p38, MKK3 and MKK6 (Zarubin & Han 2005). Along with FYN and PAK these MKKs might contribute to the activation of p38. Activation of p38 resulted in activation of MAP kinase activated protein kinase 2/3 (MAPK 2/3) and phosphorylation of the F-actin polymerization modulator, heat shock protein 27 (HSP27) (Rousseau et al. 1997).
After phosphorylation by PDK1, PKC undergoes autophosphorylation at two sites important for PKC activity, one in the turn motif (Thr-642 in PKCB) and the second in the hydrophobic phosphorylation motif (Ser-661 in PKCB). These phosphorylations render the enzyme catalytically competent but still inactive; diacylglycerol (DAG) and calcium are required for full activation.
FYN recruited to VEGFR2 is activated; this is required for VEGF-induced actin remodelling and endothelial migration. Once Y531 in the negative regulatory site is dephosphorylated by a phosphatase, FYN undergoes autophosphorylation on Y420 (Yeo et al. 2011).
Upon stimulation focal adhesion kinase (FAK1), in association with Src family kinases (SFKs) phosphorylates paxillin (PXN) at two main sites- tyrosine 31 and tyrosine 118. These phosphorylated sites provides the functional SH2-binding sites for members of the Crk family of SH2-SH3 adaptor proteins (Bellis et al. 1995, Shaller & Schaefer 2001).
The SH3 domain of CRK interacts constitutively with proline rich motifs present in Dedicator of cytokinesis (DOCK180), an exchange factor for RAC1. Unlike many GEFs, DOCK180 does not contain a conserved Dbl homology (DH) domain. Instead, it has a DHR2 or DOCKER domain capable of loading RAC1 with GTP (Brugnera et al 2002). Binding of DOCK180 to RAC1 alone is insufficient for GTP loading, a DOCK180-ELMO interaction is required. Engulfment and cell motility protein 1 (ELMO1) or ELMO2 form a complex with DOCK180 which functions as a bipartite GEF to optimally activate RAC1 (Gumienny et al 2001, Brugnera et al 2002, Birge et al. 2009).
PAK2 activity via GTPases can be strongly potentiated by concurrent stimulation of cellular tyrosine kinase activity. FYN may be involved in this potentiation by phosphorylating Y130 in the N-terminal regulatory domain leading to a robust enhancement of the catalytic activity of PAK2 (Renkema et al. 2002).
PKC contains a N-terminal C2 like domain, a pseudosubstrate (PS), DAG binding (C1) domain and a C-terminal kinase domain. The PS sequence resembles an ideal substrate with the exception that it contains an alanine residue instead of a substrate serine residue. It is bound to the kinase domain in the resting state. As a result, PKC is maintained in a closed inactive state, inaccessible to cellular substrates. On stimulation of receptors there is an increase in intracellular calcium and diacylglycerol (DAG) levels which leads to the activation of PKC and its translocation from the cytosol to the plasma membrane. PKCs tether to the plasma membrane through DAG binding to the C1 domain. This confers a high-affinity interaction between PKC and the membrane, leading to a massive conformational change that releases the PS domain from the catalytic site, the system becomes both competent and accessible (Colon-Gonzalez & Kazanietz 2006).
PAK2 undergoes autophosphorylation on serine and threonine residues, which maintains PAK2 in a catalytically active state. PAK is autophosphorylated at several sites but S141 flanking the kinase inhibitor region and T402 within the catalytic domain are the two conserved sites that regulate the catalytic activity (Chong et al. 2001, Gatti et al. 1999).
In response to VEGF, the increased actin polymerization required to trigger actin based motility involves the recruitment of adapter protein NCK to VEGFR2 (Lamalice et al. 2007). Phosphorylated tyrosine 1214 in VEGFR2 is the binding site for NCK. NCK later recruits FYN and PAK2, which are required for the activation of SAPK2/p38 activation, formation of stress fibers, and endothelial cell migration (Lamalice et al. 2006, Stoletov et al. 2004, Lu et al. 1997). NCK also participates in a signaling pathway leading to actin nucleation and polymerization through its interactions with N WASP and WAVE1 (Stoletov et al. 2004, Rhoatgi et al. 2001).
Several receptor tyrosine kinases (RTKs) are known to associate with integrins, and it has been suggested that focal adhesion kinase (FAK) is at the crossroads of these signaling pathways. On endothelial cells integrin alphaVbeta3 acts as a regulator of VEGFR2 signaling and shown to be necessary for angiogenic response (Hood et al. 2003). In mouse endothelial cells VEGF stimulated complex formation between VEGFR2 and beta3 integrin. This association between alphaVbeta3 with VEGFR2 appears to be synergistic, because VEGFR2 activation induces beta3 integrin tyrosine phosphorylation, which, in turn, enhances the phosphorylation of VEGFR2 and mediates the activation of mitogenic pathways involving focal adhesion kinase (FAK) and stress-activated protein kinase-2/p38 (SAPK2/p38) (Masson-Gadais et al. 2003, Mahabaleshwar et al. 2006, Somanath et al. 2009). This promotes activation of alphaVbeta3 and results in the increase of ligand binding ability (integrin activation), integrin ligation, and phosphorylation of beta3 integrin by cSrc.
PI3-kinase (PI3K) catalyzes the phosphorylation of inositol phospholipids at the 3 position to generate phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate. PIP2 and PIP3 generated serve as lipid substrates where they recruit guanine nucleotide exchange factors (GEFs) like VAV (Proto-oncogene vav) that catalyze the exchange of GDP for GTP on Rac, activating it (Han et al. 1998). VAV2 acts downstream of VEGF to activate Rac1 (Garretta et al. 2007).
Following VEGF treatment, VAV2 phosphorylation on tyrosine 172 stimulates its GEF activity for RAC1 (Garrett et al. 2007) and thus plays an important role in linking VEGFR2 to endothelial migration. VAV exists in an auto-inhibitory state, folded in such a way as to inhibit the GEF activity of its DH domain. This folding is mediated through binding of tyrosines in the acidic domain to the DH domain and through binding of the calponin homology (CH) domain to the C1 region. Activation of VAV is thought to involve three events which relieve this auto-inhibition: phosphorylation of tyrosines in the acidic domain causes them to be displaced from the DH domain; binding of a ligand to the CH domain may cause it to release the C1 domain; binding of the PI3K product PIP3 to the PH domain may alter its conformation (Aghazadeh et al. 2000). VAV is phosphorylated on a tyrosine residue (Y174 in VAV1, 172 in VAV2, 173 in VAV3) in the acidic domain. This is mediated by Src and related family tyrosine kinases (Deckert et al. 1996, Schuebel et al. 1998).
Protein kinase C (PKC) activation enhances angiogenesis by participating in the intracellular signaling of vascular endothelial growth factor (VEGF) in endothelial cells. VEGF can activate several PKC isoforms including alpha, beta, delta and zeta isoforms. Their activation is preceded by the activation of PLC gamma (Suzuma et al. 2002, Xia et al. 1996, Takahashi et al. 1999, Wellner et al. 1999). Before Protein kinase C (PKC) is competent to respond to second messengers it must first be phosphorylated at three conserved positions: the activation loop and two positions at the carboxyl terminus of the protein (Dutil et al. 1998). The phosphorylation of the activation loop appears to occur first and is mediated by phosphoinositide dependent protein kinases (PDKs). PDK1 phosphorylates PKCs at a critical Thr (T) residue in the activation loop, a requirement for PKC to gain catalytic competency (Toker 2003).
CRK (CT10 Regulator of Kinase) is composed of one SH2 and one or two SH3 domains. This adaptor protein binds with phosphorylated tyrosine motifs found in proteins involved in cell spreading, actin reorganisation, and cell migration. Paxillin (PAX) and p130CAS are the two major focal adhesion components that binds with CRK to form multiprotein signaling complexes and regulate cell migration (Klemke et al. 1998, Valles et al. 2004, Lamorte et al. 2003).
VEGF mediated activation of ERK1/2 depends on the activity of PKC. Sphingosine kinase 1 (SPHK1) has been identified as the connecting link between PKC and Ras activation. Activated SPHK1 does not activate Ras-GEF directly but rather modulates Ras-GAP activity to favour Ras activation. VEGF-mediated stimulation of SPHK1 results from the direct phosphorylation of SPHK1 by PKC (Shu et al. 2002). S225 in SPHK1 may be the target site of phosphorylation (Piston et al. 2003).
Tyrosine-1214 phosphorylation allows recruitment of cytoplasmic tyrosine kinase FYN along with NCK to VEGFR2. FYN and NCK associate with each other. This complex mediates phosphorylation of p21-activated protein kinase 2 (PAK2) and subsequent activation of p38MAPK mediating actin reorganization and cell migration (Lamalice et al. 2006).
Activated ROCK directly phosphorylates FAK1 on S732. This phosphorylation induces a conformational change that is necessary to trigger the phosphorylation of FAK on Y407 (Le Bouef et al. 2006).
NADPH oxidase (NOX) proteins are membrane-associated, multiunit enzymes that catalyze the reduction of oxygen using NADPH as an electron donor. NOX proteins produce superoxide (O2.-) via a single electron reduction (Brown & Griendling 2009). Superoxide molecules function as second messengers to stimulate diverse redox signaling pathways linked to various functions including angiogenesis. VEGF specifically stimulates superoxide production via RAC1 dependent activation of NOX2 complex. VEGF rapidly activates RAC1 and promotes translocation of RAC1 from cytosol to the membrane. At the membrane RAC1 interacts with the NOX enzyme complex via a direct interaction with NOX2 (gp91phox or CYBB) followed by subsequent interaction with the NCF2 (Neutrophil cytosol factor 2) or p67phox subunit and this makes the complex active (Bedard & Krause 2007). O2.- derived from Rac1-dependent NOX2 are involved in oxidation and inactivation of protein tyrosine phosphatases (PTPs) which negatively regulate VEGFR2, thereby enhancing VEGFR2 autophosphorylation, and subsequent redox signaling linked to angiogenic responses such as endothelial cell proliferation and migration (Ushio-Fukai 2006, 2007).
P130CAS (CRK-associated substrate/BCAR1) contains multiple protein-protein interaction domains including an N-terminal SH3 domain, an interior substrate domain (SD), a Src-binding domain (SBD) near the C-terminus and a conserved C-terminal Cas-family homology (CCH) domain. The SH3 and CCH domains mediate localization to focal adhesions (FAs) while SD and SBD are involved in initiating signaling events (Meenderink et al. 2010, Shin et al. 2004). The p130CAS SD undergoes tyrosine phosphorylation and mediates signals by recruiting downstream effectors. The SD is characterised by fifteen YxxP motifs, of which ten can be efficiently phosphorylated by Src family kinases (SFKs) (Shin et al. 2004). FAK kinase phosphorylates the nearby SBD tyrosines 664 and 666 (mouse 668/670). These SBD tyrosines provide the additional binding sites for Src-SH2 domains, stabilizing the SRC-p130CAS association (Ruest et al. 2001). Note: Phosphorylated tyrosine numbering in human p130CAS is based on similarity with the mouse p130Cas.
CDC42 is involved in the formation of filopodia with potential functions in guidance and migration in response to a VEGF gradient. CDC42 is activated downstream of VEGFR2 and involved in the formation of stress fibres by contributing to the activation of the p38 pathway. The activation of CDC42 may rely on FYN activity but the precise mechanism that leads to activation is not known (Lamalice et al. 2004, 2006).
The PAK family of serine/threonine kinases are known to be activated by binding to the GTP-bound form of CDC42 or RAC1, small GTPases of the Rho family that are involved in regulating the organization of the actin cytoskeleton. PAK exists as homodimer in a trans-inhibited conformation. The kinase inhibitory (KI) domain of one PAK molecule binds to the C-terminal catalytic domain of the other and inhibits catalytic activity. Association of GTP-bound forms of CDC42 or RAC1 with the PAK PBD/CRIB domain induces conformational changes in the N-terminal domain that no longer support its autoinhibitory function. CDC42-mediated activation primes PAK2 for superactivation by tyrosine phosphorylation (Renkema et al. 2002).
Proline tyrosine kinase 2-beta (PTK2B), also known as cell adhesion kinase-beta or related adhesion focal tyrosine kinase, is a nonreceptor protein-tyrosine kinase closely related to focal adhesion kinase (FAK1) that couples receptors, including integrins, with a variety of downstream effectors such as small G proteins belonging to the Ras and Rho families, mitogen-activated protein kinases, protein kinase C, and inositol phosphate metabolism (Avraham et al. 2000). PYK2B has been shown to play a critical role in the adhesion and migration of many cell types. PYK2B has been shown to localise to integrin and has been demonstrated to associate directly with integrin beta3 cytoplasmic tail (Butler & Blystone 2005, Duong & Rodan 2000, Le Boeuf et al. 2006).
Paxillin (PXN) is a multidomain scaffolding protein localized primarily in focal adhesions. It binds with focal adhesion kinase (FAK1) and is recruited to the focal adhesions. VEGF induced a quick and marked increase in the recruitment of both paxillin and vinculin to FAK (Abedi & Zachary 1997).
RAC1 is activated from inactive GDP-bound state to active GTP-bound form by the GEF activity of DOCK180:ELMO complex. RAC1 signaling facilitates VEGF-stimulated angiogenesis by regulating endothelial cell migration and vascular permeability. RAC1 promotes migration by stimulating actin reorganisation to form membrane ruffles and lamellipodia. RAC1 is also a critical component of endothelial NADPH oxidase promoting reactive oxygen species (ROS) prodcution. Specifically, VEGF acts through RAC1 to stimulate lamellipodia formation at the leading edge of polarized cells for directional migration, or chemotaxis. RAC1 induces vascular permeability in part by disrupting endothelial cell-cell junctions (Soga et al. 2001a, Soga et al. 2001b, Claesson-Welsh & Welsh, 2013).
The activated NOX2 complex generates superoxide (O2.-) by transferring an electron from NADPH in the cytosol to oxygen on the luminal or extracellular space (Bedard & Krause 2007).
Membrane-bound sphingosine (SPG) in cells attenuates basal Ras activity by stimulating the activity of Ras GTPase-activating proteins (RasGAPs). Upon its phosphorylation by SPHK1, SPG is converted to sphingosine 1-phosphate (S1P) which then displaces from GAP downregulating RASA1 (p120GAP) activity and thereby induces Ras-GTP accumulation. This overall increases the level of activated Ras-GTP leading to activation of the ERK/mitogen-activated protein kinase (MAPK) pathway and cell division (Shu et al. 2002, Wu et al. 2003, Spiegel & Milstien 2006).
P21-activated kinase 2 (PAK2) is an effector of GTP-bound CDC42. It associates with NCK and possibly links activated CDC42 to the SAPK2/p38 module (Lamalice et al. 2006, Zhao et al. 2000).
Tyrosine-phosphorylated VAVs act as guanine nucleotide exchange factors (GEFs) for RAC1, catalysing the exchange of bound GDP for GTP. RAC1 is a key regulator for actin cytoskeleton and cell migration and is also a critical component of endothelial NADPH oxidase (Wittmann et al. 2003, Tan et al. 2008, Ushio–Fukai 2007, Ushio–Fukai et al. 2002). Activated RAC1 then stimulates actin polymerisation to form lamellipodia through a number of proteins such as WASP-family veroprilin homologous protein (WAV). WAVE proteins stimulate the formation of a branched actin network by binding to the p21 subunit of the ARP2/3 nucleating complex, which is located on the sides of the pre-existing filaments.
Phosphorylation of S732 in FAK1 changes its conformation making Y407 accessible to Proline tyrosine kinase 2-beta (PTK2B). pY402-PTK2B then triggers the phosphorylation of FAK1 on Y407 (Le Boeuf et al. 2006). Phosphorylation of Y407 is required to recruit paxillin and vinculin to FAK1 and to ensure formation of focal adhesions and cell migration (Le Boeuf et al. 2004).
Activation of VEGFR2 results in the activation of phosphatidylinositol 3-kinase (PI3K) which plays an important role in regulating endothelial proliferation, migration and survival (Jiang et al. 2000). Activation of PI3K is also essential for VEGF-stimulated nitric oxide (NO) production from endothelial cells via protein kinase B (PKB/AKT) signaling to eNOS (Nitric oxide synthase, endothelial) (Blanes et al. 2007). Upon stimulation by VEGF the p85 regulatory subunit of PI3K is recruited to phosphorylated tyrosine-801 of VEGFR2 (Dayanir et al. 2001).
FYN has multiple phosphorylation sites which can affect its kinase activity. Among these phosphorylation sites, serine 21 (S21) has been identified as a target site for protein kinase A (PKA). The phosphorylation of FYN S21 is critical for both FYN's tyrosine kinase activity and its focal adhesion targeting (Yeo et al. 2007).
P130CAS (Crk-associated substrate/BCAR1) is an adaptor protein which upon phosphorylation recruits additional signaling proteins that link the scaffold to the actin cytoskeleton of the cell (Klemke at al. 1998). The C-terminal proline-rich region of Focal adhesion kinase (FAK1) spanning amino acids 712-718 binds the SH3 domain-containing region of p130CAS (Polte & Hanks 1995). P130CAS also interacts with Src-family kinases (SFKs) via its C-terminal Src-binding domain (SBD). Though FAK1 has no tyrosine kinase activity towards p130CAS, it contributes to p130CAS phosphorylation by interacting with SFKs (Ruest et al. 2001).
Activated MAP kinase-activated protein kinase (MAPK/MAPKAPK) 2 and 3 in turn phosphorylate heat shock protein beta 1 (HSPB1, HSP27). HSP27 is an actin-capping protein. Its phosphorylation has been proposed to release it from actin filaments, thus allowing addition of actin monomers and elongation of filaments. Phosphorylation-induced conformational changes causes disaggregation of oligomeric complexes of HSP27 and subsequent disassociation from actin filaments, which may result in a higher rate of actin polymerization (Lamalice et al. 2007, Rousseau et al. 2000, Lavoie et al. 1995).
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).
VEGFA-dependent activation of VEGFR2 causes autophosphorylation and activation of the Axl receptor tyrosine kinase via Src-1:SH2D2A-dependent reaction. Phosphorylation of Axl tyrosine residues 772 and 814 (773 and 815 in mouse) is required for VEGFA-dependent binding of the p85-subunit of PI3K and activation of PI3K (Ruan & Kazlauskas, 2012).
AXL/UFO (Tyrosine-protein kinase receptor UFO) is a member of the TAM (Tyro3/Axl/Mer) family of receptor tyrosine kinases (RTKs). AXL has been implicated in angiogenesis because of its ability to promote angiogenically related cellular responses in endothelial cells (Holland et al, 2005). AXL is required for VEGFA-dependent activation of PI3K. Activated Src family kinases recruit AXL via its juxtamembrane domain and thereby trigger ligand-independent autophosphorylation of AXL that promotes association with PI3K and activation (Ruan & Kazlauskas 2012).
Increased PAK1-3 catalytic activity is associated with autophosphorylation of key residues, including one site in the regulatory portion (S144 in PAK1) and another in the so-called activation loop (T423 in PAK1).
Activated PAK then phosphorylates a serene residue (S665) within a conserved motif in the cytoplasmic tail of VE-cadherin. VE-cadherin is also phosphorylated by c-Src in a manner dependent on TSAD (Sun et al. 2012, Lambeng et al. 2005). Serine-phosphorylated VE-cadherin recruits beta-arrestin 2 which promotes the internalization of VE-cadherin into clathrin-coated pits. This process leads to the disassembly of endothelial-cell junctions, resulting in the enhanced permeability of the blood-vessel wall (Gavard & Gutkind 2006).
Axl with its two phopshorylated YxxM motifs associates with the p85 subunit of PI3K and mediates VEGFA mediated activation of PI3K/AKT pathway (Ruan & Kazlauskas, 2012).
Activated Rac1 binds to and stimulates the kinase activity of PAK1-3 (p21 activated kinases 1-3). PAK dimers are arranged in head-to-tail fashion, in which the kinase domain of one molecule is inhibited by the regulatory domain of the other molecule and vice versa. Binding of activated Rac1 breaks the PAK dimer and removes the trans-inhibition (Knaus et al. 1998, Parrini et al. 2002). PAK activated by Rac1 in turn phosphorylates VE-cadherin thereby promoting the beta-arestin-dependent endocytosis of VE-cadherin. This consequently disassemblies intracellular junctions leading to vascular permeability (Gavard & Gutkind 2006).
Tyrosine-phosphorylated VAVs bind RAC1:GDP as RAC1 guanine nucleotide exchange factors (GEFs), catalysing the exchange of bound GDP for GTP. RAC1 is a key regulator for actin cytoskeleton and cell migration and is also a critical component of endothelial NADPH oxidase (Wittmann et al. 2003, Tan et al. 2008, Ushio–Fukai 2007, Ushio–Fukai et al. 2002).
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x p-VAV
family:PIP3:RAC1:GTP:PAK 1-3The importance of the RAS/RAF MAPK cascade is highlighted by the fact that components of this pathway are mutated with high frequency in a large number of human cancers. Activating mutations in RAS are found in approximately one third of human cancers, while ~8% of tumors express an activated form of BRAF (Roberts and Der, 2007; Davies et al, 2002; Cantwell-Dorris et al, 2011).
dimer:p-6Y-VEGFR2
dimer:NCK1,NCK2:FYNdimer:p-6Y-VEGFR2
dimer:NCK1,NCK2:p-S21,Y420-FYN:PAK2dimer:p-6Y-VEGFR2
dimer:NCK1,NCK2:p-S21,Y420-FYNdimer:p-6Y-VEGFR2
dimer:NCK1,NCK2:p-Y420-FYNdimer:p-6Y-VEGFR2
dimer:NCK1,NCK2dimer:p-6Y-VEGFR2
dimer:PI3K/VEGFA:p-6Y-VEGFR2:TSAd:p-Y418-SRC-1:p-Y772,Y814-AXL:PI3Kdimer:p-6Y-VEGFR2
dimer:PI3Kdimer:p-6Y-VEGFR2
dimer:PLCG1dimer:p-6Y-VEGFR2
dimer:SH2D2A:SRC-1dimer:p-6Y-VEGFR2
dimer:SH2D2Adimer:p-6Y-VEGFR2
dimer:SHBdimer:p-6Y-VEGFR2
dimer:p-4Y-PLCG1dimer:p-6Y-VEGFR2
dimer:p-S-SHBdimer:p-6Y-VEGFR2
dimerAnnotated Interactions
x p-VAV
family:PIP3:RAC1:GTP:PAK 1-3x p-VAV
family:PIP3:RAC1:GTP:PAK 1-3Phosphorylation of AKT on S473 by TORC2 complex is a prerequisite for AKT phosphorylation on T308 by PDPK1 (Scheid et al. 2002, Sarabassov et al. 2005).
dimer:p-6Y-VEGFR2
dimer:NCK1,NCK2:FYNdimer:p-6Y-VEGFR2
dimer:NCK1,NCK2:FYNdimer:p-6Y-VEGFR2
dimer:NCK1,NCK2:FYNdimer:p-6Y-VEGFR2
dimer:NCK1,NCK2:p-S21,Y420-FYN:PAK2dimer:p-6Y-VEGFR2
dimer:NCK1,NCK2:p-S21,Y420-FYN:PAK2dimer:p-6Y-VEGFR2
dimer:NCK1,NCK2:p-S21,Y420-FYNdimer:p-6Y-VEGFR2
dimer:NCK1,NCK2:p-S21,Y420-FYNdimer:p-6Y-VEGFR2
dimer:NCK1,NCK2:p-Y420-FYNdimer:p-6Y-VEGFR2
dimer:NCK1,NCK2:p-Y420-FYNdimer:p-6Y-VEGFR2
dimer:NCK1,NCK2dimer:p-6Y-VEGFR2
dimer:NCK1,NCK2dimer:p-6Y-VEGFR2
dimer:PI3K/VEGFA:p-6Y-VEGFR2:TSAd:p-Y418-SRC-1:p-Y772,Y814-AXL:PI3Kdimer:p-6Y-VEGFR2
dimer:PI3Kdimer:p-6Y-VEGFR2
dimer:PLCG1dimer:p-6Y-VEGFR2
dimer:PLCG1dimer:p-6Y-VEGFR2
dimer:SH2D2A:SRC-1dimer:p-6Y-VEGFR2
dimer:SH2D2A:SRC-1dimer:p-6Y-VEGFR2
dimer:SH2D2A:SRC-1dimer:p-6Y-VEGFR2
dimer:SH2D2Adimer:p-6Y-VEGFR2
dimer:SH2D2Adimer:p-6Y-VEGFR2
dimer:SHBdimer:p-6Y-VEGFR2
dimer:SHBdimer:p-6Y-VEGFR2
dimer:p-4Y-PLCG1dimer:p-6Y-VEGFR2
dimer:p-4Y-PLCG1dimer:p-6Y-VEGFR2
dimer:p-S-SHBdimer:p-6Y-VEGFR2
dimer:p-S-SHBdimer:p-6Y-VEGFR2
dimerdimer:p-6Y-VEGFR2
dimerdimer:p-6Y-VEGFR2
dimerdimer:p-6Y-VEGFR2
dimerdimer:p-6Y-VEGFR2
dimerdimer:p-6Y-VEGFR2
dimerdimer:p-6Y-VEGFR2
dimerdimer:p-6Y-VEGFR2
dimerdimer:p-6Y-VEGFR2
dimer