Cancer Therapy Vol 3, 1-12, 2005

Biochemical ground-rules regulating c-MET receptor tyrosine kinase activation and signaling

Review Article

Payal R. Sheth and Stanley J. Watowich*

Department of Biochemistry and Molecular Biology and the Sealy Center for Structural Biology, University of Texas Medical Branch at Galveston, TX 77555-0645

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*Correspondence: Stanley J. Watowich; Department of Biochemistry and Molecular Biology, University of Texas Medical Branch at Galveston, TX 77555-0647, USA; Tel. 1 409 747 4748; Fax. 1-409 747 4745; E-mail. watowich@xray.utmb.edu

Keywords: c-MET, receptor tyrosine kinase, dimerization, phosphorylation, signaling, autophosphorylation, dephosphorylation

Abbreviations: activation loop, (AL); adenosine triphosphate, (ATP) epidermal growth factor receptor, (EGFR); epidermal growth factor, (EGF); fibroblast growth factor receptor, (FGFR); fibroblast growth factor, (FGF); Grb2-associated binder 1, (Gab1); hepatocyte-growth factor, (HGF); leukocyte common antigen-related, (LAR); phosphatidylinositol 3-kinase, (PI3K); phospholipase C, (PLC); PI3K protein kinase B, (PKB); platelet-derived growth factor, (PDGF); protein tyrosine binding, (PTP); protein-tyrosine phosphatase, (PTP); receptor tyrosine kinase, (RTK); regulator of kinase, (Crk); scatter factor, (SF); vascular endothelial growth factor receptor, (VEGFR)

Received: 7 November 2005; Accepted: 9 January 2006; electronically published: January 2006

Summary

c-MET receptor tyrosine kinase-mediated signaling governs numerous important cellular responses including cellular proliferation, differentiation, migration and apoptosis. Deregulation of these signals results in malignant behaviors, often leading to cancers. While the identity of many signaling molecules that are activated following hepatocyte-growth factor (HGF)-induced activation of c-MET have been established, little is known about the mechanism of activation of c-MET. From a therapeutic perspective, it is necessary to understand the detailed molecular mechanisms regulating c-MET activation to selectively target these molecules. Classically it has been believed that the sole role of ligand-induced dimerization was to autophosphorylate the receptor, thereby activating receptor’s kinase catalytic function. However, recent studies have shown that dimerization-induced changes in the kinetic, thermodynamic and dephosphorylation properties of c-MET work synergistically to selectively induce specific signaling from the dimeric and not the monomeric receptor. In this review, we highlight biochemical studies of c-MET and related RTKs that are consistent with a dynamic equilibrium mechanism of c-MET activation. Although, the proposed mechanism differs from the traditional view of the RTK activation, it successfully explains all the relevant experimental data in the literature.

I. Introduction

The strength and duration of numerous intracellular signaling responses are dependent on c-MET activation, defined as sustained c-MET phosphorylation and subsequent downstream signaling. c-MET activation is a critical and tightly regulated process in normal functioning of cells; aberrant signaling has been linked to pathological conditions including tumorigenesis and metastasis (Birchmeier et al, 2003). Thus, blocking c-MET activation may be an effective strategy to control these conditions. In this review, we highlight some of the significant advances towards understanding c-MET signaling, with particular emphasis on the structural and biochemical basis of c-MET activation.

II. c-MET receptor tyrosine kinase (RTK) and hepatocyte growth factor (HGF)/scatter factor (SF)

A. c-MET structure

c-MET, the receptor tyrosine kinase (RTK) for hepatocyte growth factor (HGF)/scatter factor (SF), was first identified as an oncogene mediating the chemically induced transformation of a human osteogenic sarcoma cell line (Cooper et al, 1984). Cellular physiological functions of c-MET include, but are not limited to, proliferation, differentiation, motility and survival. c-MET is single-pass transmembrane glycoproteins that consist of an extracellular region that possesses the specificity for the ligands, and a cytoplasmic region that harbors the tyrosine kinase catalytic activity (Ullrich et al, 1990; Pazin et al, 1992; Hubbard et al, 2000). Related members of the RTK family include receptors for epidermal growth factor (EGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF) and insulin. While the intracellular domains of different RTKs are highly conserved, the extracellular domains contain multiple structurally distinct regions that form the basis of further classification of RTKs (Hubbardet al, 2000; Blume-Jensen et al, 2001).

c-MET sub-family of RTKs includes MET, RON, and SEA. These receptors have a short a-chain and a longer b-chain linked together by a disulfide bond (Huff et al, 1993; Ronsin et al, 1993). This heterodimeric structure results from furin cleavage of a single-chain precursor. The mature form of c-MET constitutes an extracellular 45kDa a-chain and a 190kDa membrane-spanning b-chain. The b-chain consists of an extracellular region, a membrane-spanning region and a cytoplasmic region containing the signaling elements - the juxtamembrane element, the catalytic domain and the carboxy-terminal tail (Figure 1). The extracellular region contains a sema domain with marked structural similarity to extracellular domains of semaphorins and plexins (Winberg et al, 1998), a small cysteine rich PSI domain similar to those found in plexins, semaphorins and integrins (Bork et al, 1999) and four IgG-like domains with similarity to the IgG domains present in plexins and transcription factors (Ohta K, et al, 1995). Structural and biochemical studies have shown that c-MET sema domain forms a high-affinity binding site for HGF (Gherardi et al, 2003; Stamos et al, 2004). The residues critical for HGF binding in c-MET have been mapped and are contained within the a-chain and 212 residues of the b-chain (Gherardi et al, 2003). Recent structural studies have revealed the domain architecture of the c-MET extracellular region, and have shed light on the mechanism of HGF-c-MET interactions (Kozlov et al, 2004; Stamos et al, 2004).

The cytoplasmic catalytic domain of c-MET is highly conserved amongst RTKs. Several structures of this domain in an unphosphorylated and phosphorylated state have been determined (Hubbard et al, 1994; Mohammadi et al, 1996, 1998; Hubbard, 1997; Schiering et al, 2003). The catalytic domain folds into distinct N-terminal and C-terminal lobes,connected via a flexible polypeptide linker. The N-terminal and C-terminal lobes are formed predominantly by b-sheets and a-helices respectively. The ATP substrate binds within a cleft formed between the two lobes, and the peptide substrate binds to the C-terminal lobe. Crystal structure of insulin receptor (IR) catalytic domain in the phosphorylated and unphosphorylated state provided a structural basis of the observed increase in the catalytic activity upon activation loop (AL) phosphorylation. The AL is a segment of amino acids within the catalytic domain that contains one or more tyrosine residues that upon autophosphorylation increase the kinase catalytic activity. Within apo IR, the AL traverses the cleft between the N-terminal and the C terminal lobes, thus obstructing the ATP binding site (Hubbard et al, 1994). The AL conformation in the unphosphorylated and phosphorylated forms in IR, showed dramatic differences, which impact the ability of ATP to access the kinase catalytic site (Hubbard, 1997).

In FGFR, autoinhibition occurs via a different mechanism: a proline residue in the AL together with flanking residues occlude the substrate binding site (Mohammadi et al, 1996). Crystal structures of apo and inhibitor-bound forms of the c-MET catalytic domain showed the characteristic kinase bilobal structure described above (Schiering et al, 2003). The AL loop residues in the apo c-MET were disordered. However, the structure of the c-MET: inhibitor binary complex showed

Figure 1. Domain map of c-MET. c-MET is composed of an a and b chain linked together via a single disulfide bond. The b chain includes the extracellular, transmembrane (TM), and the cytoplasmic regions. The cytoplasmic region contains the signaling apparatus consisting of the juxtamembrane region (JM), the kinase domain (KD) and carboxy-terminal tail (CT). HGF binds to the extracellular region of c-MET, and signal transduction is mediated by in part by phosphorylation of residues in the multifunctional docking site (Y1349, Y1356 and Y1365). c-MET catalytic activity is positively regulated by the phosphorylation of tyrosine residues 1231,1234 and 1235 in the catalytic domain. Phosphorylation of juxtamembrane residue Y1003 is important for c-MET degradation. The important effectors of c-MET are also shown.

a unique AL conformation. This conformation may represent a quasi-stable intermediate state along the transition pathway between phosphorylated and unphosphorylated AL conformations, although it is possible this conformation is an artifact arising from mutations to the conserved residues within the AL.

B. HGF/SF structure

The ligand for c-MET was independently identified by two different laboratories as a mitogen for hepatocytes, HGF and a SF in fibroblasts (Stoker et al, 1986, 1987; Nakamuraet al, 1989). Since its discovery, HGF has been shown to elicit plietropic cellular responses including mitogenesis, motility and morphogenesis. HGF/SF is synthesized as an inactive single-chain precursor that is proteolytically cleaved to form an active disulfide-linked heterodimer. Both the single-chain precursor as well as disulfide-linked heterodimer appear to bind c-MET with high affinity, however, c-MET activation occurs only by the cleaved mature form of the ligand (Lokker et al, 1992).

HGF shows sequence homology to the plasminogen-related growth factor family: these proteins have a similar cleavage-mediated activation mechanism. The 69 kDa a-chain of HGF consists of an N-terminal domain (N), followed by four kringle domains (K1-K4) (Lokker et al, 1992). The 34 kDa b-chain forms a conserved protease-like domain; this domain is inactive due to substitution of required active site serine and histidine residues. The HGF residues that form the receptor binding site are unknown, although a number of studies indicate that the a- and b-chains have distinct roles in c-MET binding, and subsequent dimerization (Ultsch et al, 1998; Lietha et al, 2001; Gherardi et al, 2003; Stamos et al, 2004). Several truncated forms of the a-chain region, including NK1, bind c-MET with high affinity. HGF is also a high-affinity ligand for heparan sulfate proteoglycans. However, unlike fibroblast growth factor receptor (FGFR), this interaction does not appear to be critical for c-MET activation (Lokker et al, 1992; DiGabriele et al, 1998; Hartmannet al, 1998). The b-chain of HGF, which harbors the serine-protease-like catalytic domain, has also been shown to bind the sema domain within c-MET, albeit with relatively lower affinity (Stamos et al, 2004).

C. HGF-induced c-MET dimerization and activation

Normal signaling by RTKs requires ligand-induced receptor oligomerization and tyrosine phosphorylation of the cytoplasmic domains of the receptor. Although ligand-mediated receptor dimerization appears to be a common event preceding RTK activation, structures of several receptor ectodomains bound to their cognate ligands showed RTKs used different binding modes to accomplish dimerization. In vascular endothelial growth factor receptor (VEGFR), dimerization was induced by binding of dimeric ligand (Wiesmann et al, 1997), whereas FGFR bound monomeric FGF and the dimeric complex was stabilized by heparin cofactors (Schlessinger et al, 2000; Mohammadi et al, 2005). Recent crystallographic studies of epidermal growth factor receptor (EGFR) showed that the binding stoichiometry of EGF to receptor was 1:1, and the dimeric complex was stabilized solely through receptor-receptor interactions formed upon ligand binding (Ogiso et al, 2002). The mechanism utilized by HGF to induce c-MET dimerization remained largely elusive until recently, when the crystal structure of the HGF b-chain and c-MET sema domain highlighted a possible dimer interface between the ligand-receptor pair, and suggested a potential 2:2 HGF:c-MET complex (Stamos et al, 2004). Future structural studies on intact HGF and c-MET ectodomain would shed light on the structural-basis of recruitment of HGF by c-MET, and subsequent c-MET dimerization induced by HGF. Interestingly, cross-linking c-MET receptors by specific antibodies to the extracellular domain can trigger c-MET signaling implying that dimerization is sufficient to activate c-MET (Prat et al, 1998).

Irrespective of its mode of dimerization, autophosphorylation of c-MET tyrosines necessary for signaling occurs after dimerization and presumably, by transphosphorylation between the catalytic domains of dimeric c-MET. The biochemical events regulating c-MET signaling have been recently elucidated (as discussed below), although, the structural basis for c-MET autophosphorylation upon HGF binding remains largely unclear. Tyrosines Y1231, Y1234 and Y1235 in the AL of the c-MET catalytic domain have been shown to be phosphorylated in response to HGF-induced c-MET dimerization (Figure 1). The presence of three phosphotyrosine sites in the AL is also a characteristic of the insulin receptor, a disulfide-linked constitutive dimer.

While the phosphorylation of AL tyrosines is important for increased c-MET kinase activity (Rodrigues et al, 1994), the phosphorylation of carboxy-terminal tail tyrosines Y1349, Y1356 and Y1365 (Birchmeier et al, 2003) is required for the recruitment of cytoplasmic signaling proteins with Src homology-2 (SH2) and protein tyrosine binding (PTP) domains. Phenylalanine substitution at residues Y1349 and Y1356 render c-MET functionally impaired in its ability to induce proliferation, motility, differentiation and survival (Weidner et al, 1995). In addition, phosphorylation of Y1003 within the juxtamembrane region appears to be critical for receptor degradation (Peschard et al, 2001, 2004).

III. c-MET functions

A. c-MET signaling

Functional genetic studies of c-MET and HGF have conclusively revealed an indispensable role of these molecules in mammalian development. HGF -/- and c-MET -/- mice die in utero after incurring severe placental and live defects, along with disruption in the migration of myogenic precursors into the limb bud (Bladt et al, 1995; Schmidt et al, 1995; Uehara et al, 1995).

Furthermore, in adults, c-MET and HGF are widely expressed, and c-MET signaling has been shown to be important for tissue repair and organ regeneration (Michalopoulos et al, 1997; Matsumoto et al, 2001). In the recent years, extensive studies have been conducted to elucidate the mechanism by which HGF/c-MET regulate such diverse physiological responses.

HGF-activated c-MET recruits cytoplasmic signaling molecules such as Grb2-associated binder 1 (Gab1), growth factor receptor-bound protein (Grb2), phosphatidylinositol 3-kinase (PI3K), SH2-containing protein (Shc) via a unique multisubstrate docking site that is conserved in the c-MET family of RTKs (Ponzetto et al, 1994). This docking site encompasses phosphorylated Y1349, Y1356 and adjacent residues. Y1365 has also been implicated in the c-MET-initiated morphogenesis, although the signaling molecules that interact with this c-MET site are largely unknown (Weidner et al, 1995). Recruitment of the signaling molecules results in the activation of specific signaling pathways that regulate multiple cellular processes including proliferation, disruption of intracellular junctions, migration and survival. Furthermore, c-MET signaling is also involved in complex processes such as cellular differentiation and formation of branching tubules. Some of the well-characterized signaling pathways activated by c-MET are Ras-MAPK, PI3K, Src and Stat3 (Bertotti et al, 2003; Birchmeier et al, 2003). Although several researchers have tried to link individual effector molecules and/or specific signaling pathways to a particular cellular response, it is becoming increasingly apparent that HGF-induced c-MET signaling is complex and branches into distinct but interacting cascades.

The multiadapter Gab1 plays a critical role in mediating c-MET signaling by providing a scaffold for simultaneous binding several signaling molecules. The central role of Gab1 in c-MET signaling is evident from the phenotype of Gab1 -/- mice, which show the characteristic placental, liver and muscle defects seen in c-MET null mice (Sachs et al, 2000). Upon HGF stimulation, Gab1 directly interacts with phosphorylated c-MET, via a unique Met-binding domain, which is not present in other members of the Gab family, and indirectly interacts with phosphorylated c-MET via Grb2 (Lock et al, 2000). The c-MET-Gab1 interaction appears to be critical for stimulating branching morphogenesis (Maroun et al, 1999). Phosphorylation of specific Gab1 tyrosines creates sites for binding the SH2 domain of Shp2, a protein-tyrosine phosphatase (PTP) (Gu et al, 2003). The Shp2-Gab1 interaction plays an important role inactivating the Erk/MAPK pathway (Gu et al, 2003; Schaeper et al, 2000). Mutations that disrupt Gab1-Shp2 binding result in a phenotype incapable of activating the Erk/MAPK pathway.

Although Shp2 is believed to act upstream of Ras and Raf, the direct effectors of Shp2 are currently unknown. Interestingly, recent studies show that Gab1 can directly interact with Erk1/2 via its Met-binding domain and this interaction is critical for transporting Erk1/2 to the nucleus (Osawa et al, 2004). However, the significance of this interaction for c-MET signaling is unclear.

Upon HGF stimulation, Gab1 also interacts with CT10 regulator of kinase (Crk), phospholipase C (PLC), PI3K and Shc. Signaling from Gab1 and Crk appears to be important for motility (Schaeper et al, 2000), whereas the Gab1-PLC and Gab1-Shp2 interactions have been shown to be important for branching morphogenesis (Gual et al, 2000; Maroun et al, 2000).

Another important adapter molecule for c-MET is Grb2, which possesses a SH2 domain and multiple SH3 domains. Grb2 constitutively associates with Sos, a Ras-specific guanine nucleotide exchange factor. Grb2 binds phosphorylated RTKs via its SH2 domain, thereby shuttling the Sos to the plasma membrane, where Ras is localized (Ponzetto et al, 1994). This sequence of events activates Ras, which then activates the Raf1 serine threonine kinase. Raf1 activates the MAPK signaling pathway by phosphorylating MEK, which in turn phosphorylatesthe MAP kinase (Campbell et al, 1998). As mentioned earlier, Grb2 also provides a high-affinity binding site for Gab1. Grb2 has been implicated in c-MET-mediated proliferation, transformation and motility. Upon its phosphorylation Grb2 is also able to interact with Shc, which can also directly bind c-MET (Pelicci et al, 1995).

The SH2 domain of the effector protein PI3K has been shown to bind phosphorylated c-MET (Ponzetto et al, 1994). In addition, PI3K indirectly interacts with c-MET via Gab1 (Holgado-Madruga et al, 1996). Several studies have concluded that PI3K mediates most of the MET-induced signaling responses namely- mitogenesis, motility, and morphogenesis (Royal et al, 1995, 1997; Khwaja et al, 1998; Potempa et al, 1998). Furthermore the PI3K protein kinase B (PKB)/Akt pathway, which mediates MET-induced scattering and branching morphogenesis (Royal et al, 1997), is also the main mediator for cell survival (Xiao et al, 2001).

Other proteins reportedly recruited to c-MET phosphotyrosine docking sites include Shc, Src and Stat3. Shc and Src are involved in cellular proliferation and motility, Stat3 is involved in branching morphogenesis, and Stat3 and Src are also involved in cellular transformation (Ponzetto et al, 1993; Pelicci et al, 1995; Boccaccio et al, 1998; Rahimi et al, 1998; Zhang et al, 2002). Phosphorylation of c-MET Y1003 is important for recruitment of c-Cbl, a member of the E3 ubiquitin ligase family (Preschard et al, 2001). c-Cbl has also shown to be recruited indirectly to the MET-signaling complex via interactions with Grb2. The c-Cbl-c-MET interaction appears to be critical for MET ubiquitination and degradation. Finally, several transmembrane proteins namely a6b4 integrin (Trusolino et al, 2001), Plexin B1 (Giordano et al, 2002; Basile et al, 2005), and CD44 (Orian-Rousseau et al, 2002) have also been shown to associate with c-MET, although the significance of these interactions for c-MET signaling in vivo is unclear. Thus HGF-activated c-MET triggers complex cellular responses by activating interacting signaling pathways.

B. Aberrant c-MET regulation and human malignancies

Aberrant regulation of c-MET signaling has emerged as a likely causative element for a number of human malignancies. Abnormal activation of c-MET can occur via different mechanisms, some of the reported mechanisms include c-MET or HGF overexpression, and c-MET mutations (Figure 2). c-MET activation and signaling is clearly deregulated in several osteosarcomas, glioblastomas and melanoma, where c-MET and HGF have been observed to be constitutively overexpressed (Koochekpouret al, 1997; Fukuda et al, 1998; Hendrix et al, 1998; Birchmeier et al, 2003). These observations are further strengthened by the evidence of c-MET and/or HGF expression in carcinomas, and other types of human solid tumors and their metastasis (Birchmeieret al, 2003). Furthermore, mouse and human cells that ectopically overexpress HGF or c-MET become tumorigenic and metastatic in athymic nude mice (Rong et al, 1994). A large number of sporadic and germline mutations of c-MET have been identified in human renal papillary carcinomas (Danilkovitch-Miagkova et al, 2002) and homologous c-MET mutations produce distinct tumor profiles in mice (Graveel et al, 2004). These mutations occur within the c-MET kinase domain, often making it capable of constitutive signaling. Mutations in the c-MET juxtamembrane domain, a region important for c-Cbl binding have been observed in gastric and lung cancers (Lee et al, 2000; Ma et al, 2003). Recently, mutations in extracellular semaphorin domain that is important for HGF binding, were identified in lung cancers (Ma et al, 2003; Ma et al, 2005). The role of c-MET in physiological processes such as proliferation, survival, invasion and angiogenesis could point to its involvement in corresponding stages during tumor progression.

c-MET signaling has also repeatedly emerged as a pathway that is exploited by several pathogens including Listeria monocytogenes, Plasmodium spp. and Helicobacter pylori (Figure 2) (Shen et al, 2000; Carrolo et al, 2003; Churin et al, 2003). InlB, a listerial protein was identified as a bacterial agonist for c-MET and shown to mimic HGF-induced c-MET activation, endocytosis (Ireton et al, 1999; Li et al, 2005) and signaling (Shen et al, 2000). H. pylori CagA protein also activated c-MET, although by a distinct mechanism. The CagA- induced c-MET signaling could be important for H. pylori-induced cancer onset and tumor progression (Churinet al, 2003). Contrary to Listeria and H pylori, Plasmodium, the causative agent for malaria, did not directly interact with c-MET, but exploited HGF-c-MET signaling to make the host cell more susceptible to infection (Carrollo et al, 2003).

IV. Regulating c-MET enzymatic activity

HGF-mediated dimerization facilitates c-MET autophosphorylation. The kinetics of c-MET autophosphorylation has not been extensively studied, although the phosphotyrosine sites and their role in c-MET signaling is well-characterized (as reviewed above). Phosphorylation of Y1231, Y1234 and Y1235 in the kinase domain AL has been reported to modulate c-MET catalytic activity (Rodrigues et al, 1994). The correlation between AL phosphorylation and increased kinase catalytic activity has been extensively documented in a number of RTKs (Cobbet et al, 1989; Parast et al, 1998; Murray et al, 2001). In IR, where insulin binding induces receptor activation of a constitutive dimeric receptor, autophosphorylation kinetics were observed to follow a two phase model where the ligand activated receptor had a prolonged fast phase compared to the non-ligand stimulated receptor (Kohanski, 1993). Murray et al, determined the kinetic parameters for phosphorylated and unphosphorylated Tie2 cytoplasmic kinase domain and showed that phosphorylation resulted in a 2-5-fold decrease in substrate KM relative to the unphosphorylated kinase (Murray et al, 2001). Parast et al also showed an order of magnitude increase in the catalytic activity of the phosphorylated VEGFR2 tyrosine kinase domain versus unphosphorylated receptor (Parast et al, 1998).

The crystal structure of IR in its phosphorylated and unphosphorylated forms provided a structural interpretation for how AL phosphorylation might modulate kinase activity (Hubbard et al, 1994; Hubbard, 1997). In the phosphorylated state, the AL adopted a

Figure 2. c-MET signaling and function. HGF-mediated c-MET signaling is important for several physiological processes including cell proliferation, differentiation and survival. Aberrant regulation of c-MET signaling by HGF/c-MET overexpression or c-MET mutations is associated with tumorigenesis and metastasis. Furthermore, c-MET signaling has been shown to be exploited by several pathogens, including Listeria monocytogenes, Helicobacter pylori and malarianparasite Plasmodium spp for tissue invasion and pathogen dissemination.

conformation that was more amenable to binding ATP and tyrosine-containing peptide substrate (Hubbard, 1997). These studies demonstrated the importance of the phosphorylation state of RTKs in modulating their kinase activity. However, these studies but did not address whether oligomerization could impact these parameters, although Hubbard et al, hypothesized that dimer formation could stabilize the “flipped out” activation loop conformation in a catalysis favorable position (Hubbard, 1997).

The mechanism of autophosphorylation within the oligomeric RTK is still elusive, although evidence for intramolecular (i.e. cis) (Weber et al, 1984; Bertics et al, 1985; Biswas et al, 1985; Villalba et al, 1989), intermolecular (i.e. trans) mechanism (Yarden et al, 1987; Cobb et al, 1989; Treadway et al, 1991; Sherrill, 1997) as well as sequential cis/trans mechanisms (Iwasaki et al, 1997) exist. Structural studies support a trans mechanism of AL autophosphorylation within IR. In this system the AL tyrosine is believed to bind to the kinase catalytic site in a cis fashion, but cannot be phosphorylated due to steric constraints that prevent simultaneous binding of MgATP when the tyrosine is bound to the IR active site (Hubbard et al, 1994). The kinetic properties of several RTKs have been characterized and the reaction model is dependent on the purification process and the constructs used. Both the EGF receptor (Posner et al, 1992; Ward et al, 1994) and the IR (Walker et al, 1987; Yuan et al, 1990) were consistent with a rapid equilibrium random order mechanism, while the TrkA receptor showed an ordered sequential scheme (Angeles et al, 1998). In contrast, kinetic studies on the Rous sarcoma virus pp60src supported a steady state ordered bi-bi mechanism with ATP binding occurring first (Wong et al, 1984). The VEGFR was characterized as a hybrid of the rapid equilibrium random order and sequential mechanisms (Parast et al, 1998). Unfortunately, in some studies, an isolated kinase domain was used and in other studies, an immunoprecipitated whole receptor or kinase domain was used, making the direct comparison between these studies difficult. This difficulty was highlighted by Cheng and Koland, who showed that the binding properties of the EGF receptor were dependent on the form of the receptor studied, as the whole cytoplasmic domain had 10-fold greater affinity for ATP relative to the isolated kinase domain (Cheng et al, 1996).

PTPs catalyze dephosphorylation of ligand-stimulated and unstimulated c-MET (Villa-Moruzzi et al, 1993; Sheth et al, 2005). Recent studies have shown that RTK phosphorylation was dynamically regulated by competing autophosphorylation and dephosphorylation rates (Posner et al, 1994; Bohmer et al, 1995; Baxter et al, 1998; Shethet al, 2005). Regulation of c-MET by PTPs is poorly understood, although studies using substrate trapping mutants, antisense RNA, and phosphotyrosine peptides have proposed DEP-1 (CD148/PTP-h), PTP-S and leukocyte common antigen-related (LAR) to be potentially involved in c-MET dephosphorylation (Kulas et al, 1996; Villa-Moruzzi et al, 1998; Palka et al, 2003).

Moreover, DEP-1 was observed to preferentially dephosphorylate the carboxyl-terminal Y1349 and Y1365 in c-MET, suggesting that phosphatase site-specific preferences might be an additional mechanism for regulating receptor signaling (Palka et al, 2003). Detailed animal model or cell culture studies have yet to substantiate a role of these putative PTPs in c-MET signaling.

The classical RTK activation model consists of dimerization-mediated RTK autophosphorylation, which in turn activates kinase activity of the receptor. Thus, activation of the kinase activity of RTKs has been synonymously used for RTK activation. However, in vitro studies using isolated kinase domains and ex vivo studies using phosphatase inhibitors have shown conclusively that monomeric receptors can be rapidly phosphorylated on tyrosine residues involved in intracellular signal propagation (Posner et al, 1994; Baxter et al, 1998; Sheth et al, 2005). Thus, it is clear that ligand-induced receptor oligomerization is not necessary for kinase activity. Furthermore our previous studies have shown that oligomerization modifies the thermodynamic and kinetic properties of the MET receptor independent of the autophosphorylation reaction, such that dimeric phosphorylated MET more efficiently phosphorylates substrate molecules than the similarly phosphorylated monomeric MET (Figure 3) (Hays et al, 2003, 2004). Given the conformational flexibility observed in kinase structures, it is reasonable to postulate that observed biochemical differences between monomeric and dimeric MET result from dimerization-induced conformational changes, although structural data to unequivocally support this hypothesis has not been obtained. Kinase activity is additionally regulated by receptor phosphorylation levels, in particular the phosphorylation state of tyrosines within the receptor activation loop (Parast et al, 1998; Murray et al, 2001). Moreover, the extent of receptor phosphorylation is regulated by competing autophosphorylation and dephosphorylation reactions which in turn are modulated by the receptor’s oligomeric state (Kohanski, 1993; Baer et al, 2001; Shimizu et al, 2001; Sheth et al, 2005). Thus, receptor oligomerization can directly modulate kinase activity and can indirectly modulate kinase activity by modulating autophosphorylation and dephosphorylation rates which impact receptor phosphorylation levels and in turn affect kinase activity (Figure 4). There exists a complex feed-forward loop between phosphorylation state, oligomerization state, and kinase activity which can effectively work to amplify and sharpen the separation between inactive and active c-MET states. A model that includes these oligomerization-dependent changes is necessary to provide a complete understanding of the molecular events that control c-MET (or RTK) activation, where activation is defined as receptor phosphorylation and subsequent downstream signaling.

We have recently built a mathematical model that incorporates the above mentioned synergistic feed-forward reactions for RTK activation. Although, the details of the model will be described elsewhere (Sheth et al, 2005), the model was described based on several intermediates occurring in the c-MET activation process including the monomeric and dimeric c-MET in their phosphorylated

Figure 3. Biochemical parameters for monomeric and dimeric c-MET. The kinetic and thermodynamic parameters that regulate c-MET phosphorylation (MET) and subsequent phosphorylation of substrate molecules (S) by c-MET are tabulated. The rate constants (k), catalytic efficiency (kcat) and substrate affinity (K_D) for c-MET autophosphorylation and substrate phosphorylation reactions are shown. Dephosphorylation negatively regulates c-MET phosphorylation. The kinetics of PTP b-catalyzed dephosphorylation of c-MET monomer and dimer substrates has been studied, and the corresponding K_M and V_MAX for this reaction are shown.

Figure 4. Feed-forward mechanism of oligomerization-mediated c-MET activation. Ligand induced c-MET oligomerization increases kinase activity of the receptor, which results in build up of phosphorylated RTK by autophosphorylation. The levels of phosphorylated receptor generated are negatively regulated by the action of cellular phosphatases. Ligand-induced oligomerization reduces the c-MET’s susceptibility to dephosphorylation and thus affects the buildup of phosphorylated receptor. Thus, oligomerization amplifies the buildup of phosphorylated RTK via two independent mechanisms - by increasing the kinase catalytic activity and reducing c-MET’s susceptibility to dephosphorylation. The increased kinase catalytic efficiency also impacts substrate phosphorylation rates, which regulates the buildup of phosphorylated substrate. The phosphorylated receptor and substrate build up are critical determinants of RTK signaling potential.

and dephosphorylated states. This kinetic model was built on a differential equation framework with key biochemical parameters derived from our in vitro studies on MET (Figure 3). Our model provides a robust quantitative description of c-MET activation, and provides a tool for analyzing the biochemical parameters critical for c-MET activation. It is clear from the biochemical studies that the ligand-stimulated c-MET is a highly competent signaling unit, which is sensitive to changes in phosphorylation and dephosphorylation rates. Unstimulated monomeric c-MET, on the other hand, is an active kinase, but is repressed in its ability to sustain autophosphorylation and signal due to its signal its higher susceptibility to dephosphorylation, lower catalytic efficiency, and reduced substrate binding properties relative to dimeric c-MET (Figure 3). If these kinetic and thermodynamic properties are altered, for example by increasing the monomer kinase catalytic k_cat by 10-fold, and decreasing the monomer’s susceptibility to dephosphorylation by 10-fold, then the buildup of phosphorylated monomer and phosphorylated exogenous substrate could occur without extracellular ligand stimulation. Thus, the term “inactive” for monomeric unstimulated c-MET is inaccurate and can often be misleading. The “activation” that occurs in response to extracellular ligand results from the accumulation of dimeric receptors that can sustain a phosphorylated state and signal due to synergistic effects of their increased k_cat for autophosphorylation and substrate phosphorylation, higher affinities for substrate, and reduced susceptibility to dephosphorylation relative to the monomeric receptor.

A unified activation mechanism based on existing experimental data and modeling predictions is depicted in Figure 5. In this model, in absence of extracellular ligand, the monomeric c-MET exists predominantly in an inactive unphosphorylated state under steady-state conditions. The signaling from monomeric c-MET is repressed due to the inefficient catalytic properties and high dephosphorylation susceptibility associated with this state. In presence of extracellular ligand stimulation, the equilibrium of the system is shifted, favoring the buildup of dimeric c-MET. Signaling from dimeric c-MET occurs due to the increased kinase catalytic efficiency and substrate binding, and

Figure 5. Model for c-MET activation. This figure of c-MET activation integrates current experimental and modeling data. In absence of ligand stimulus, signaling from monomeric c-MET is repressed due to synergistic combination of decreased catalytic efficiency, reduced substrate binding and increased susceptibility to dephosphorylation, relative to oligomeric c-MET. Thus, monomeric c-MET exists predominantly as a dephosphorylated inactive receptor under steady-state conditions. Following extracellular ligand stimulus, c-MET dimerizes, which increases its kinase catalytic efficiency and substrate binding, and decreases its susceptibility to phosphatases, relative to monomeric receptor. Thus, dimeric c-MET exists predominantly as a phosphorylated active receptor under steady-state conditions.

decreased susceptibility to dephosphorylation relative to the monomeric receptor. Thus, dimeric c-MET exists predominantly in a phosphorylated active state under steady-state conditions. Our model clearly shows that modulating catalytic activity of c-MET is not the only parameter dictating c-MET activation.

V. Conclusions

Given the importance of c-MET in physiological processes such as cell proliferation, motility, and survival, it is not surprising that dysregulated c-MET signaling can result in the onset, progression and/or spread of tumors. c-MET signaling is also involved in other pathologies such as malaria and listeriosis. Thus, targeting c-MET signaling has significant clinical implications for treating multiple pathological conditions. A prerequisite for the development of new diagnostic and therapeutic strategies is a detailed knowledge of the activation process of c-MET. Structural, biochemical and genetic studies continue to reveal the precise molecular mechanisms underlying c-MET activation, although developing effective methods to interfere with altered c-MET remains a significant challenge. Several approaches for targeting c-MET signaling have been described (Christensen et al, 2005; Corso et al, 2005) including targeting the catalytic domain with small molecule inhibitors. Acquiring high-resolution structures of c-MET in its activated (phosphorylated dimeric) state would aid the rationale design of inhibitors capableof interfering with the catalytic activity of the activated receptor and modulating dysregulated c-MET signaling.

Acknowledgements

This work was supported by grant 4952-052 (S.W) from the Texas Higher Education Coordinating Board, Sealy Center for Structural Biology (University of Texas Medical Branch) and by a McLaughlin Predoctoral Fellowship (P.S).

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