Integrating Signals from Rtks to ERK/MAPK

MM McKay and DK Morrison

Laboratory of Cell and Developmental Signaling, NCI-Frederick, Frederick, MD, USA

Signals received at the cell surface must be properly intracellular compartments to process, integrate transmitted to critical targets within the cell to achieve the and transmit information that will ultimately specify appropriate biological response. This process of signal a particular biological response. An essential effector transduction is often initiated byreceptor tyrosine kinases cascade required for most RTK function is the mitogen- (RTKs), which function as entrypoints for many activated protein kinase (MAPK) cascade comprised of extracellular cues and playa critical role in recruiting the Raf, MEK and extracellular signal-regulated the intracellular signaling cascades that orchestrate a kinase (ERK) kinases (known as the ERK cascade). particular response. Essential for most RTK-mediated Here, we will examine the signaling routes used by signaling is the engagement and activation of the mitogen- RTKs to mediate ERK activation (Figure 1), and we activated protein kinase (MAPK) cascade comprised of will highlight recent studies that further elucidate the the Raf, MEK and extracellular signal-regulated kinase mechanisms regulating the activity and subcellular (ERK) kinases. For manyyears,it was thought that localization of various RTK/ERK pathway compo- signaling from RTKs to ERK occurred onlyat the plasma nents. In addition, we will describe the ERK scaffolds membrane and was mediated bya simple, linear Ras- and signaling modulators that contribute to RTK/ERK dependent pathway. However, the limitation of this model signaling. became apparent with the discoverythat Ras and ERK can be activated at various intracellular compartments, and that RTKs can modulate Ras/ERK signaling from RTK activation and the recruitment of enzymes, signaling these sites. Moreover, ERK scaffolding proteins and adaptors and docking proteins signaling modulators have been identified that playcritical RTKs are membrane-spanning, cell surface proteins roles in determining the strength, duration and location that contain an N-terminal extracellular ligand binding of RTK-mediated ERK signaling. Together, these factors domain and a C-terminal intracellular tyrosine kinase contribute to the diversityof biological responses gener- domain (reviewed in Schlessinger, 2000; Pawson, 2002). ated byRTK signaling. Most RTKs exist as inactive monomers and form dimers Oncogene (2007) 26, 3113–3121. doi:10.1038/sj.onc.1210394 following binding to their ligands, the vast majority of which are soluble peptides. Dimerization induces Keywords: RTKs;ERK/MAPK;Ras;Raf;ERKscaffolds receptor activation and the autophosphorylation of tyrosine residues in the RTK intracellular domain that serve as specific binding sites for proteins that contain Src-homology 2 (SH2) or phosphotyrosine binding (PTB) domains. Proteins recruited to the activated RTK typically fall into two categories: enzymes whose activity is stimulated by the activated receptor (e.g., Src, Introduction PLCg, Shp-2 and PI3K) and adaptors that contain additional protein interaction motifs (e.g., Grb2 and Receptor tyrosine kinases (RTKs) transmit signals that Shc). Many activated RTKs also engage a third group regulate cell proliferation and differentiation, promote of molecules collectively known as docking proteins cell migration and survival and modulate cellular (e.g., IRS, FRS and the Gab/Dok proteins). Docking metabolism. They play a critical role in a wide range proteins are often recruited to the RTK via an intrinsic of biological processes, including embryonic develop- PTB domain or through an interaction with the Grb2 ment, growth of an organism, angiogenesis, synaptic adaptor. These proteins also contain a membrane plasticity and oncogenesis. No longer considered a targeting motif (either a pleckstrin homology (PH) linear pipeline, RTK pathways are now viewed as domain or a myristoylation site) and multiple tyrosine intricate signaling networks, containing modules of phosphorylation sites that can interact with additional multi-protein complexes that assemble at various binding partners. Thus, through the receptor itself, adaptors and docking proteins, numerous signaling Correspondence: Dr DK Morrison, NCI-Frederick, PO Box B, components are recruited to the cell surface, thereby Frederick, MD 21702, USA. activating the repertoire of effector cascades required for E-mail: [email protected] a specific response. Integrating signals from RTKs to ERK/MAPK MM McKay and DK Morrison 3114

Figure 1 RTK to ERK signaling: Ras and ERK activation at various intracellular compartments. At the plasma membrane, activated RTKs promote Ras activation through the recruitment of Grb2/Sos complexes. KSR is an ERK scaffold that facilitates Ras- dependent ERK cascade activation at the plasma membrane, whereas Paxillin directs ERK activation at focal adhesions. Active signaling complexes containing internalized receptors, Grb2/Sos and Ras are also found on endosomes. MP1 is an MEK1/ERK1- speciﬁc scaffold that localizes to late endosomes through an interaction with the adaptor protein p14. Activated RTKs can also direct the activation of Golgi-associated Ras through a signaling route involving Src, PLCg and RasGRP1. Sef is a Golgi-localized scaffold that recruits activated MEK and promotes ERK activation. Active ERK is retained on the Sef/MEK complex and conﬁned to cytosolic substrates. The tyrosine phosphatase Shp-2 is another effector of RTKs that positively regulates Ras signaling by antagonizing the ability of negative regulators, such as CSK, RasGAP and Sprouty, to access and downregulate critical enzymes involved in Ras activation.

Ras activation at the plasma membrane only ensures that cytosolic Sos is maintained in an The Ras GTPase is the critical link that allows RTKs to inactive state, but also provides a positive feedback loop signal to ERK. Activation of all RTKs stimulates the that has been recently shown to affect the duration, exchange of GTP for GDP on Ras, and this nucleotide amplitude and output of Ras signaling in response to exchange allows Ras to interact directly with its target stimuli (Boykevisch et al., 2006). Significantly, muta- effectors, one of which is the initiating kinase of the tions of Sos that disrupt its autoinhibition have recently ERK cascade, Raf (Marshall, 1996). The best-charac- been identified in patients with Noonan’s syndrome, a terized route of Ras activation occurs at the plasma developmental disorder associated with dysregulation of membrane and is mediated by the Sos guanine nucleo- the Ras/ERK pathway (Roberts et al., 2007; Tartaglia tide exchange factor (Quilliam et al., 1995; Downward, et al., 2007). 1996). Sos is recruited from the cytosol to the plasma membrane as a result of its constitutive interaction with Grb2, whose SH2 domain can bind phosphotyrosine Ras signaling from other intracellular compartments sites located on one or more of the following molecules: For many years, Ras activation was thought to occur the activated RTK itself, Shc (another adaptor protein exclusively at the plasma membrane. However, the that binds activated RTKs via its PTB domain) or one isolation of active signaling complexes from endosomes of the membrane-localized docking proteins. that contain tyrosine-phosphorylated epidermal growth Placing Sos in the same locale as Ras was initially factor receptors, Grb2/Sos, Ras and Raf provided the thought to be the primary mechanism regulating the first evidence that Ras might signal from other Sos-mediated nucleotide exchange of Ras. However, intracellular sites (Di Guglielmo et al., 1994; Baass subsequent structure-function studies have revealed that et al., 1995). More recently, studies utilizing new Sos exists in an autoinhibited state, and that interactions imaging technologies, such as fluorescence resonance with Ras itself modulate Sos activity in a two-step energy transfer, have revealed that activated Ras manner. First, binding of RasGDP to an allosteric site proteins can be detected on various intracellular on Sos that is distal to the catalytic site induces low GEF membranes, including the Golgi and endoplasmic activity that generates RasGTP (Sondermann et al., reticulum as well as endosomes (Burke et al., 2001; 2004). RasGTP then binds with higher affinity to the Chiu et al., 2002; Jiang and Sorkin, 2002). Interestingly, same site, fully relieving steric occlusion of the active site the kinetics of Ras activation differs at various and inducing high GEF activity (Margarit et al., 2003; intracellular sites, and evidence is now accumulating Freedman et al., 2006). This regulatory mechanism not that compartmentalization may play an important role

Oncogene Integrating signals from RTKs to ERK/MAPK MM McKay and DK Morrison 3115 in determining not only the strength, duration and complexes, binding to either the receptor or an targets of Ras signaling, but also the ultimate biological associated docking protein (Neel et al., 2003). Shp-2 consequences of a signaling cue (Sorkin and Von contributes to Ras signaling from the Golgi by Zastrow, 2002; Plowman and Hancock, 2005; Mor stimulating the activation of Src family kinases, and in and Philips, 2006). turn, their substrate PLCg (Zhang et al., 2004). Shp-2 Amajor factor influencing Ras localization is the promotes Src activation by dephosphorylating binding processing and trafficking of the different Ras family sites on proteins that function to colocalize the members, H-Ras, N-Ras and K-Ras. Membrane target- inhibitory C-terminal Src kinase (CSK) with Src family ing of the various Ras proteins is determined by the members. sequence and post-translational modifications of their Shp-2 has also been reported to have positive effects C-terminal regions known as the hypervariable region on Ras signaling that occurs at the plasma membrane. (HVR). Although all Ras proteins undergo farnesyla- First, by dephosphorylating binding sites on some tion and carboxylmethylation, which localizes them to receptors and docking proteins that mediate binding of the endoplasmic reticulum (Choy et al., 1999), their the Ras GTPase activating protein (RasGAP), Shp-2 HVRs possess different secondary targeting motifs, prevents the localization of RasGAP to the plasma which are required for stable plasma membrane membrane and thereby, prolongs Ras activation (Klin- association. The HVR of K-Ras 4B (the ubiquitous ghoffer and Kazlauskas, 1995; Agazie and Hayman, and predominant splice variant of K-Ras and referred to 2003; Montagner et al., 2005). Second, by depho- hereafter as K-Ras) contains a stretch of polybasic sphorylating the negative signaling modulator Sprouty, residues, whereas the HVRs of H-Ras and N-Ras contain Shp-2 prevents Sprouty from binding and blocking the cysteine residues that are palymitoylated (Hancock membrane recruitment of Grb2/Sos (Hanafusa et al., et al., 1990). Processed K-Ras is confined primarily to 2004). Thus, the general underlying mechanism for the the plasma membrane; however, when the polybasic positive effects exerted by Shp-2 on Ras signaling is that region is phosphorylated or targeted by Ca2 þ /calmodu- Shp-2 antagonizes the ability of negative regulators to lin, K-Ras relocalizes to other endomembrane compart- access and downregulate critical enzymes involved in ments, including the Golgi, endoplasmic reticulum and Ras activation. mitochondria (Fivaz and Meyer, 2005; Bivona et al., 2006). In contrast, H-Ras and N-Ras constantly shuttle between the Golgi and plasma membrane as a result The Ras/MEK/ERK cascade of a constitutive depalmitoylation/repalmitoylation All activated Ras proteins can interact directly with the cycle (Goodwin et al., 2005; Rocks et al., 2005). Raf family kinases (comprised of A-Raf, B-Raf and the Ubiquitination has recently been found to be another most extensively studied C-Raf), and this interaction is a post-translation modification that distinguishes the Ras crucial first step required for Raf activation. Strikingly, proteins and contributes to their intracellular localiza- converting the Raf kinases from inactive to active tion (Jura et al., 2006). The selective mono- and di- enzymes is a highly complex process, involving mem- ubiquitination of H-Ras and N-Ras, but not K-Ras, is brane localization, cycles of phosphorylation/depho- determined by their HVRs and requires farnesylation sphorylation and protein interactions (Figure 2; and palmitoylation. Ubiquitination of these Ras pro- reviewed in Dhillon and Kolch, 2002; Wellbrock et al., teins stabilizes their association with endosomes and 2004). All Raf proteins are thought to exist in an modulates their ability to interact with Raf and to inactive state in the cytosol, with the N-terminal domain activate the ERK cascade (Jura et al., 2006). acting as an autoinhibitor of the C-terminal kinase In addition to regulating the classical Sos-mediated domain. 14-3-3 dimers bind to phosphorylation sites Ras exchange that occurs at the plasma membrane and present in both the N- and C-terminal regions, stabiliz- on endosomes, RTKs can also direct the activation of ing the autoinhibited state and concealing the N- Golgi-associated Ras through a signaling route that terminal Ras binding sites. RTK activation stimulates utilizes the RasGRP1 exchange factor (Bivona et al., the membrane recruitment of Raf via Ras and induces 2003). RasGRP1 is a C1-domain containing protein that the release of 14-3-3 from the N-terminal binding site, is activated by diacylglycerol (DAG) and Ca2 þ ,ina which together promote the phosphorylation of the manner analogous to members of the protein kinase C kinase domain on activating sites. For the mammalian (PKC) family (Ebinu et al., 1998; Brose and Rosen- C-Raf and A-Raf proteins, these sites are located in two mund, 2002). Many activated RTKs induce the Src- regions of the kinase domain – the negative-charge dependent activation of PLCg, resulting in the produc- regulatory region (N-region) and the activation segment. tion of DAG and an increase in intracellular Ca2 þ . In contrast, B-Raf appears to require only activation These second messengers then activate and direct segment phosphorylation, given that its N-region RasGRP1 to the Golgi where it acts on Ras (Bivona exhibits a constitutive negative charge owing to in- et al., 2003). Interestingly, studies of cell lines derived creased basal phosphorylation of an activating serine from Shp-2 knockout mice have revealed a role for the site and the presence of two aspartic acid residues. The Shp-2 tyrosine phosphatase in Ras activation occurring cumulative effect of these phosphorylation events as well at the Golgi (Zhang et al., 2004). Shp-2 has long been as Ras binding and membrane lipid interactions is to known to be a positive regulator of RTK-mediated Ras disrupt the N-terminal autoinhibition and stabilize the activation and is a component of many activated RTK active conformation of the kinase domain.

Oncogene Integrating signals from RTKs to ERK/MAPK MM McKay and DK Morrison 3116

Figure 2 Membrane recruitment of Raf and KSR. ‘Inactive’: 14-3-3 dimers bind and retain inactive B-Raf and C-Raf proteins in the cytosol. The inactive KSR1 scaffold constitutively interacts with MEK, CK2 and the catalytic subunits of PP2A, and is sequestered from the plasma membrane by interactions with 14-3-3 and IMP. ‘Active’: C-Raf and B-Raf are recruited to the plasma membrane as a result of RasGTP binding and the dephosphorylation of N-terminal 14-3-3-binding sites, mediated by either PP1 or PP2A. Raf proteins are activated at the membrane through a complex process that involves phosphorylation events and protein/lipid interactions. Heterodimerization with B-Raf also contributes to C-Raf activation and requires the binding of 14-3-3 to the C-terminal Raf sites. Ras activation also mediates the translocation of the KSR1 scaffold to the plasma membrane. RasGTP disrupts the IMP/KSR1 interaction by recruiting IMP to the cell surface and promoting its autoubiquitination. Ras activation also induces binding of the PP2Aregulatory B subunit to the KSR1-associated PP2Acatalytic core complex. B subunit binding stimulates the dephosphorylation and release of 14- 3-3 from one of the KSR1 sites, thereby unmasking the C1 domain required for membrane targeting as well as the ERK docking site. At the plasma membrane, KSR1 interacts with Raf and in doing so colocalizes the Raf kinases with MEK and ERK. In addition, KSR1 brings the active CK2 holoenzyme, which can function as a Raf family N-region kinase.

Despite the wealth of information that has been is induced by Ras activation (Garnett et al., 2005). obtained regarding Raf activation, recent research In either case, however, 14-3-3 appears to mediate the reveals that there are still new details to be learned. In interaction, as mutation of the C-terminal 14-3-3 particular, the involvement of protein phosphatases binding site on C-Raf disrupts the B-Raf/C-Raf com- (PPs) in the Raf activation process has been established. plex (Weber et al., 2001; Garnett et al., 2005; Rushworth Both PP1 and PP2Ahave been shown to positively et al., 2006). It is yet to be determined whether C-Raf regulate Raf activity by dephosphorylating the inhibi- can modulate B-Raf activity and whether heterodimer- tory Raf N-terminal 14-3-3 binding site, thereby ization extends to the A-Raf kinase. In addition, promoting the interaction with Ras and membrane although active Ras proteins are localized to various recruitment (Abraham et al., 2000; Jaumot and Han- intracellular compartments, it is not clear whether the cock, 2001; Ory et al., 2003; Rodriguez-Viciana et al., Raf activation process is the same at each site or 2006). Moreover, of particular interest are studies whether some Raf family members play a more demonstrating that heterodimerization with B-Raf also specialized role in Ras signaling from certain intracel- contributes to C-Raf activation. Previously, it had been lular compartments. shown that Ras activation could induce the hetero- Once activated, all Raf family members are capable dimerization of B-Raf and C-Raf (Weber et al., 2001). of initiating the phosphorylation cascade, whereby However, it was not until kinase-impaired, oncogenic Raf activates MEK, and MEK in turn activates ERK. B-Raf proteins were found to activate ERK through However, in contrast to the complexity observed in Raf the binding and transactivation of C-Raf that the activation, MEK and ERK become fully activated signiﬁcance of Raf heterodimerization was fully appre- simply through the phosphorylation of the activation ciated (Wan et al., 2004). More recently, biochemical segments in their respective kinase domains (Payne analysis of Raf heterodimers together with RNA et al., 1991; Alessi et al., 1994; Mansour et al., 1994; interference studies has shown that heterodimerization Zheng and Guan, 1994). The ultimate goal of RTK to with B-Raf also contributes to C-Raf activation in ERK signaling is then achieved when active ERK response to normal signaling events (Garnett et al., phosphorylates critical targets in the cytoplasm and 2005; Rushworth et al., 2006). Heterodimerization with nucleus required to carry out the cellular response B-Raf is thought to induce the ‘active’ kinase conforma- speciﬁed by the initiating signal. tion of C-Raf, in a manner that requires phosphorylation of the C-Raf activation segment (Garnett et al., 2005). Whether B-Raf directly phosphorylates C-Raf, ERK scaffolds involved in RTK signaling recruits another kinase or induces C-Raf to autopho- MAPK scaffolds are now recognized to play an sphorylate is unknown. Oncogenic B-Raf proteins important role in mammalian signal transduction interact constitutively with C-Raf in a Ras-independent (Morrison and Davis, 2003; Kolch, 2005). These manner, whereas binding between wild-type B-Raf and proteins act as docking platforms that colocalize the C-Raf kinase components of a particular MAPK cascade and

Oncogene Integrating signals from RTKs to ERK/MAPK MM McKay and DK Morrison 3117

Figure 3 ERK scaffolds and signaling modulators involved in RTK to ERK signaling. facilitate MAPK activation. Moreover, they provide PP2Acatalytic core complex (Ory et al., 2003), forming specificity to MAPK signaling by insulating the module a complete holoenzyme that catalyses the dephosphor- from irrelevant stimuli and by regulating the module’s ylation of one of the KSR1 14-3-3 binding sites. The subcellular localization. Proteins that contribute to the release of 14-3-3 from this site exposes the membrane- spatiotemporal activation of ERK in RTK pathways are targeting C1 domain (Zhou et al., 2002), as well as the kinase suppressor of Ras 1 (KSR1), MEK-partner 1 docking site for ERK1/2 (Jacobs et al., 1999). At the (MP1), Sef and Paxillin (Figure 3). An emerging feature plasma membrane, KSR1 interacts with Raf and in of these ERK scaffolds is that through their distinct doing so colocalizes MEK and ERK with their upstream subcellular localizations, they contribute to the com- activator. KSR1 also transports the active CK2 partmentalization of Ras/ERK signaling (Figure 1). holoenzyme, which can contribute to Raf activation by functioning as a Raf N-region kinase (Ritt et al., 2007). Interestingly, of the ERK scaffolds utilized by RTKs, KSR. KSR is a multi-domain scaffold that interacts KSR1 is the only one whose localization changes in with Raf, MEK and ERK and facilitates ERK activa- response to signal activation. tion at the plasma membrane. KSR was identified genetically in flies and worms as a positive regulator of Ras/ERK signaling, and two family members (KSR1 MP1. MP1 is a small ERK scaffold that localizes to and KSR2) are found in mammals. Like the Raf late endosomes and selectively promotes the activation kinases, the localization of mammalian KSR1 is of ERK1. MP1 was identified in a two-hybrid screen for dynamic and controlled by a variety of protein interac- MEK1-binding partners and interacts with MEK1 and tions and phosphorylation/dephosphorylation events ERK1, but not MEK2 or ERK2. MP1 localizes to late (Figure 2). In quiescent cells, KSR1 is sequestered from endosomes through a constitutive interaction with p14, the plasma membrane by interactions with a 14-3-3 a highly conserved adaptor protein that resides on the dimer and impedes mitogenic signal propagation (IMP), cytoplasmic face of endosomes (Wunderlich et al., an E3 ubiquitin ligase. The phosphorylation-dependent 2001). Protein depletion studies using RNAi have shown binding of 14-3-3 retains KSR1 in the cytosol by that the endosomal localization of the p14/MP1 com- masking the cysteine-rich C1 domain required for plex is required for full ERK activation in response to membrane targeting (Muller et al., 2001), and when EGF stimulation (Teis et al., 2002). Interestingly, bound to IMP, KSR1 mislocalizes to a detergent- however, loss or mislocalization of the p14/MP1 insoluble cellular compartment (Matheny et al., 2004). complex was found to have no significant effect on the KSR1 also constitutively interacts with MEK1/2, the early activation of ERK at the plasma membrane, but it core catalytic subunits of PP2A, and the serine/ did alter the sustained ERK activation seen on endo- threonine kinases C-TAK1 and CK2 (Denouel-Galy somes. More recently, conditional disruption of p14 in et al., 1998; Yu et al., 1998; Muller et al., 2001; Ory mice has been found to impair late endosomal traffick- et al., 2003; Ritt et al., 2007). RTK signaling induces the ing as well as endosomal ERK activation, resulting in translocation of KSR1 to the plasma membrane by cell proliferation defects that disrupt early embryogen- promoting the exposure of the KSR1 C1 domain and by esis and tissue homeostasis (Teis et al., 2006). In freeing KSR1 from IMP. Specifically, the IMP/KSR1 addition, a causal link has recently been revealed interaction is disrupted by activated Ras, which binds between p14 deficiency and a novel human immunode- directly to IMP and promotes its autoubiquitination ficiency syndrome associated with aberrant endosomal– (Matheny et al., 2004). Ras activation also causes the lysosomal function (Bohn et al., 2007). Together, these PP2Aregulatory B subunit to bind the KSR1-associated findings demonstrate the importance of endosomal

Oncogene Integrating signals from RTKs to ERK/MAPK MM McKay and DK Morrison 3118 ERK signaling and provide further evidence that spatial site on Raf (Rodriguez-Viciana et al., 2006). These and temporal regulation of ERK signaling has impor- findings have led to the model that Sur-8 acts to enhance tant biological consequences. the strength of ERK signaling by facilitating events that promote Raf activation. Sef. Another ERK scaffold with unique subcellular Multiple CNK proteins (CNK1, CNK2A, CNK2B, targeting properties is Sef, a transmembrane protein that CNK2C and CNK3) are found in mammals and they resides on the Golgi apparatus (Torii et al., 2004). Sef possess known protein interaction domains including a selectively binds activated MEK proteins and allows sterile a motif, a conserved region in CNK domain, a MEK to phosphorylate and activate ERK. Once PDZ domain and a PH domain. The most ubiquitously activated, ERK remains associated with the MEK/Sef expressed mammalian CNK protein, CNK1, has been complex and is prevented from entering the nucleus. As reported to augment C-Raf activation by colocalizing a result, active ERK bound to Sef can only phosphor- the Src tyrosine kinase with membrane-localized C-Raf, ylate cytosolic substrates. Amajor question yet to be thereby promoting the phosphorylation of the C-Raf N- addressed is how Ras activation at the Golgi impacts region by Src (Ziogas et al., 2005). CNK1 has also been ERK signaling from Sef. found to interact with various Ral and Rho pathway components and to influence Rho-mediated gene Paxillin. Paxillin is an integral component of focal transcription and activation of the JNK/MAPK path- et al adhesion complexes and is a scaffold that directs ERK way (Jaffe ., 2004, 2005). To date, studies examining activation at sites of focal adhesions (Ishibe et al., 2003). the mammalian CNKs suggest that these proteins have Paxillin interacts constitutively with MEK and, in multiple functions, facilitating ERK cascade activation response to activation of the hepatocyte growth factor as well as mediating crosstalk between various effector receptor (HGFR), also binds activated Raf and inactive cascades. ERK. Interestingly, the binding of inactive ERK is dependent on the Src-dependent phosphorylation of Negative modulators: Erbin, Sprouty/SPRED and paxillin at Y118. Once bound and activated, ERK then RKIP. Negative modulators that specifically antago- phosphorylates paxillin on S83, which promotes binding nize RTK- and Ras-mediated signaling have also been of focal adhesion kinase (FAK) and, in turn, activation identified and include Erbin, Sprouty, Sprouty-related of PI3K and Rac. Analysis of paxillin mutants defective proteins with EVH1 domains (SPRED) and Raf kinase either in ERK binding or ERK-mediated phosphoryla- inhibitor protein (RKIP). Erbin is a member of the LAP tion indicates that the localization of active ERK to (leucine rich-repeat and PDZ domain) protein family focal adhesions is critical for the turnover of focal that interacts with Ras and precludes the binding of Raf adhesion complexes and the initiation of epithelial to activated Ras (Huang et al., 2003; Dai et al., 2006). morphogenesis (Ishibe et al., 2004). The Sprouty proteins can inhibit, in a context-dependent manner, ERK signaling by binding and sequestering the Modulators of RTK to ERK signaling Grb/Sos complex, thus preventing Ras activation Because diverse extracellular cues are transduced (Hanafusa et al., 2002). In addition, both the Sprouty through the RTK/Ras/ERK cascade, qualitative differ- and SPRED proteins can interact with the Raf catalytic ences in properties such as duration and amplitude of domain and, depending on the biological context, the ERK signal are important for achieving the interfere with the phosphorylation of Raf on activating appropriate biological response. These aspects of ERK sites (Wakioka et al., 2001; Sasaki et al., 2003). RKIP signaling are influenced by signaling modulators that interacts with the kinase domain of both MEK and Raf have been identified and found to augment the efficiency and prevents the Raf/MEK interaction (Yeung et al., and level of ERK signaling, provide regulated inhibition 2000). Acommon feature for these proteins is that they and/or mediate crosstalk with other signaling cascades all interfere with critical protein interactions involved in (Figure 3). RTK to ERK signaling.

Positive modulators: Sur-8 and CNK. Two proteins Concluding comments identiﬁed by genetic studies as positive regulators of The past two decades have witnessed tremendous RTK/Ras-mediated ERK activation are suppressor of advances in our understanding of how RTKs mediate ras-8 (Sur-8) and connector enhancer of KSR (CNK). ERK activation. Following the initial discoveries of the Sur-8 is a leucine-rich repeat protein, and initial studies individual core components of the pathway, such as characterizing mammalian Sur-8 found that when over- RTKs, Ras, Raf, MEK and ERK, many years of study expressed, Sur-8 could enhance Raf activation by were devoted to the elucidation of their biochemical promoting the Ras/Raf interaction (Li et al., 2000). activities and their relationships within the cascade. The More recently, Sur-8 has been reported to function as a accelerating pace of research in recent years has revealed regulatory protein for the catalytic subunit of PP1, and that RTK to ERK signaling can occur at other through an interaction with M-Ras (a relative of the intracellular compartments in addition to the plasma prototypical H-, N- and K-Ras proteins), contributes to membrane, and that numerous auxiliary factors, such as the Raf activation process by promoting the depho- ERK scaffolding proteins and signaling modulators, sphorylation of the inhibitory N-terminal 14-3-3 binding play key roles in determining the strength, duration and

Oncogene Integrating signals from RTKs to ERK/MAPK MM McKay and DK Morrison 3119 location of ERK signaling. Although much remains to diseases, including cancer, further identiﬁcation of be clariﬁed, it has already become apparent that RTK to proteins and mechanisms regulating this signaling process ERK signaling is controlled by highly dynamic and may reveal novel targets for therapeutic intervention. complex regulatory mechanisms. Elucidating the details of these events and how ERK signaling from various Acknowledgements compartments integrates with other RTK effector cascades will be crucial in understanding how this We thank M Fortini, D Ritt and I Daar for comments and important pathway mediates such diverse cellular critical reading of the manuscript. This project has been responses. Moreover, because aberrant RTK to ERK funded in whole or in part by Federal funds from the National signaling has been implicated in a variety of human Cancer Institute, National Institutes of Health.

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