Catalytic control in the EGF Receptor and its connection togeneral kinase regulatory mechanisms

Natalia Jura1,3,9, Xuewu Zhang6, Nicholas F. Endres1,3, Markus A. Seeliger7, ThomasSchindler8, and John Kuriyan1,2,3,4,5,10

1Department of Molecular and Cell Biology University of California, Berkeley Berkeley, California94720 2Department of Chemistry University of California, Berkeley Berkeley, California 947203California Institute for Quantitative Biosciences University of California, Berkeley Berkeley,California 94720 4Howard Hughes Medical Institute University of California, Berkeley Berkeley,California 94720 5Physical Biosciences Division, Lawrence Berkeley National LaboratoryBerkeley, California 94720 6Department of Pharmacology and Department of BiochemistryUniversity of Texas Southwestern Medical Center Dallas, Texas 75390 7Department ofPharmacological Sciences State University of New York at Stony Brook Stony Brook, NY 117948Pharma Research and Early Development F. Hoffmann – La Roche, AG4070 Basel, Switzerland

SummaryIn contrast to the active conformations of protein kinases, which are essentially the same for allkinases, inactive kinase conformations are structurally diverse. Some inactive conformations are,however, observed repeatedly in different kinases, perhaps reflecting an important role incatalysis. In this review, we analyze one of these recurring conformations, first identified in CDKand Src kinases, which turned out to be central to understanding of how kinase domain of the EGFreceptor is activated. This mechanism, which involves the stabilization of the active conformationof an α helix, has features in common with mechanisms operative in several other kinases.

IntroductionProtein kinases are crucial elements of the signaling pathways that control cellular function.Kinases with specificity for either serine/threonine or tyrosine share a highly conservedcatalytic domain that adopts a conformation when active that is also highly conserved(Hanks et al., 1988; Hubbard and Till, 2000; Knighton et al., 1991; Manning et al., 2002).What differentiates one kinase from another is the diversity of input signals that impinge onthe catalytic domain, and a rich variation in the mechanisms that convert inactive forms ofthe kinase to active ones. These differences have been the key to the ability to target specifickinases by small molecules, underlying their growing importance in cancer therapy.

In an insightful review Louise Johnson and colleagues paraphrased the opening line ofTolstoy's Anna Karenina as a metaphor for understanding kinase regulation: “All activekinases are alike but an inactive kinase is inactive after its own fashion” (Noble et al., 2004).Free from the constraints of catalyzing the phosphate transfer reaction, the inactive forms ofkinases can adopt radically different conformations around the active site, each uniquelyspecialized for responding to input signals. That the inactive conformations could be

10To whom correspondence should be addressed kuriyan@berkeley.edu.9current address: Cardiovascular Research Institute Department of Molecular and Cellular Pharmacology University of California,San Francisco San Francisco, California 94158

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Published in final edited form as:Mol Cell. 2011 April 8; 42(1): 9–22. doi:10.1016/j.molcel.2011.03.004.

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targeted specifically by small molecules was first visualized for a MAP kinase (Wang et al.,1998) and was highlighted by the discovery that the cancer drug imatinib (Gleevec,Novartis) recognizes a distinctive inactive conformation of its targets Abl and c-Kit, and thatthis feature underlies its specificity (Mol et al., 2004; Schindler et al., 2000).

In the few years that have passed since the Johnson review the number of protein kinasestructures that have been determined has exploded (Eswaran and Knapp, 2010). From thisharvest of molecular detail a new realization has emerged: the inactive conformations ofkinases may fall into a relatively small number of classes, within each of which certain keyfeatures of the inactivation mechanism are conserved. This is not, in retrospect, surprising.Because protein kinases are subject to the physical constraints of the same protein fold, thereare perhaps only a limited number of ways in which the fold can be distorted away from theactive structure. It may even be that various “inactive” structures represent the stabilizationof conformations that are intermediates in as yet poorly understood aspects of catalyticmechanism, such as nucleotide release, and so are very broadly conserved because they havea fundamental role in the phosphate transfer reaction.

Despite the presence of some common features in classes of inactive structures, it is still thecase that because the structure need not be catalytically competent, each individual inactivekinase conformation is different in detail from other structures. Compounds targetinginactive conformations therefore provide increased opportunity for specificity compared tothose that target the active conformation. Most kinase-driven diseases, such as cancers,typically involve the inappropriate activation of a kinase and it might seem counterintuitiveto target inactive conformations. But kinases are highly dynamic, and are constantlyswitching between different conformations, and this process is further stimulated by theaction of phosphatases that undo the action of activating phosphorylation events. Inhibitionof the kinase can therefore be achieved by trapping it either in an active conformation (e. g.dasatinib (Tokarski et al., 2006)) or an inactive one (exemplified by imatinib).

One drug that targets the inactive conformation of a kinase is lapatinib, which inhibits theepidermal growth factor (EGF) receptor and is in current clinical use for breast cancer(Spector et al., 2005). Indeed, it was the elucidation of the structure of lapatinib bound to theEGF receptor kinase domain, by scientists at GlaxoSmithKline, that led to the realizationthat the EGF receptor could adopt this particular inactive conformation (Wood et al., 2004).This conformation was first identified in cyclin dependent kinases (CDKs) (De Bondt et al.,1993) and the Src family of kinases (Sicheri et al., 1997; Xu et al., 1997). This finding setthe stage for unraveling how the kinase domain of EGF receptor is activated, which turnedout to be quite different from the way that other receptor tyrosine kinases are controlled(Jura et al., 2009a; Red Brewer et al., 2009; Zhang et al., 2006).

The structures and regulatory mechanisms of protein kinases have been reviewed in depthelsewhere (Hubbard and Till, 2000; Huse and Kuriyan, 2002; Kornev and Taylor, 2010;Lemmon and Schlessinger, 2010; Noble et al., 2004; Pearce et al., 2010). Here, we focus ontwo particular aspects of kinases, viewed through the lens of the catalytic domain of theEGF receptor. First, we highlight the connection between the inactive conformations of theEGF receptor and those of the CDKs and the Src kinases. We point out that this class ofinactive conformations is very broadly distributed, and discuss why this might be so. Wethen turn to the mechanism by which the kinase domain of the EGF receptor is activated,and make a connection to a mechanism of allosteric control that is very broadly distributedin kinases, including members of the AGC family, such as c-AMP dependent protein kinaseA (PKA) and protein kinase B (PKB/AKT).

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The Active State of Protein KinasesThere are ~500 kinases in the human genome, with specificity towards serine/threonine ortyrosine, (the histidine kinases, prevalent in bacteria, are excluded from our discussion)(Manning et al., 2002). Most kinases have multiple regulatory domains that link kinaseactivity to unique signaling inputs and outputs (Pawson and Kofler, 2009). In spite of thisbroad diversity, the phosphorylation reaction itself constitutes a highly conserved process,which is dependent on a set of structural features of the active site that are common to allkinases.

Much of what we know about the structure of the active state of protein kinases emergedfrom studies on c-AMP-dependent protein kinase (PKA), a serine/threonine kinase (Kornevand Taylor, 2010). The active site is located between two lobes of the kinase domain (the N-and C-terminal lobes; Figure 1A). In the case of PKA, a hinge motion changes the relativeorientation of these two lobes from a more open state in the inactive conformation to a moreclosed state in the active conformation. The closure of the lobes results in the properpositioning of ATP and the substrate in the active site and is dependent on phosphorylationof a threonine residue (Thr 197; the residue numbering and secondary structure notation arefrom PKA, PDB code: 1ATP; this numbering will be used in the rest of the review, unlessotherwise specified) in a centrally located activation segment or “loop” (Knighton et al.,1991; Nolen et al., 2004). The general features of the mechanism of PKA are modulated toyield the diverse set of regulatory mechanisms seen in other kinases (Huse and Kuriyan,2002).

The key structural features required for catalysis were revealed by the analysis of the crystalstructures of the active conformation of PKA and the insulin receptor tyrosine kinase boundto ATP (or ATP-analogs) and substrate peptide (or mimics) (Bossemeyer et al., 1993;Hubbard, 1997; Knighton et al., 1991; Zheng et al., 1993). These structures highlighted theimportance of the activation loop in controlling the activation state of the kinase. The N-terminal region of the activation loop contains a conserved Asp-Phe-Gly (DFG) motif. Thesidechain of the aspartate in the DFG motif points towards the phosphate groups of ATP andplays a critical role in coordinating a magnesium ion, which is required for ATP binding.The C-terminal region of the activation loop adopts an open conformation, serving as aplatform for docking the substrate peptide (Figure 1A). In some kinases, such as the C-terminal Src kinase, Csk, substrate docking involves interactions elsewhere, and the C-terminal portion of the activation loop is disordered (Levinson et al., 2008).

Another important structural element of the kinase active site is helix αC, in the N-lobe ofthe kinase, within which a conserved glutamate residue (Glu 91) is located. In the activeconformation helix αC packs closely against the rest of the N-lobe of the kinase, allowingthe glutamate residue to form a salt bridge with a conserved lysine residue (Lys 72) in strandβ3 that coordinates the α- and β-phosphate groups of the substrate ATP molecule (Figure1A). This lysine residue is commonly mutated to generate inactive forms of kinase domains(Robinson et al., 1996). Two other important interactions in the active site involve theglycine-rich P-loop and the highly conserved HRD motif (YRD in PKA) located in thecatalytic loop that directly precedes the activation loop. The glycine-rich P-loop is importantfor nucleotide binding in the active site by making interactions with the β and γ-phosphatesof ATP. The HRD aspartate (Asp 166) serves as a catalytic base to accept the proton fromthe hydroxyl group of the substrate residue during the catalysis (Figure 1A).

Two highly conserved and functionally important intramolecular networks between the N-lobe and the C-lobe are correlated with the activity of protein kinases (Kornev et al., 2006;Kornev et al., 2008). These networks, or “spines”, involve hydrophobic residues that can be

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assembled or disassembled depending on the presence of ATP or the substrate peptide(Figure 1B). One of these, the “regulatory spine”, is involved primarily in substrate bindingand its proper assembly depends on the conformation of the activation loop (Kornev et al.,2006). There are four residues in the regulatory spine, Leu 106 in strand β4, Leu 95 in thecatalytically important helix αC, Phe 185 in the conserved DFG motif and Tyr 164 in theconserved HRD motif. Upon activation, the change in activation loop conformation, whichin most kinases is responsive to phosphorylation of the activation loop, changes theorientation of helix αC and the HRD motif, and results in the assembly of the regulatoryspine. Alterations in the flexibility of the regulatory spine have been suggested to underliethe mechanism by which some of the frequently detected mutations in cancer patientsmediate resistance to treatment with kinase inhibitors (Azam et al., 2008).

The second spine, the “catalytic spine”, incorporates the adenine ring of ATP and establishesa connection between the N-lobe and the C-lobe upon nucleotide binding (Kornev et al.,2008). The residues in the catalytic spine are located in helix αF (Met 231 and Leu 227) andhelix αD (Met 128 and Leu 172) in the C-lobe, and the residues in the N-lobe: Ile 174 andLeu 173 in the β7 strand, Val 57 in β2 strand and Ala 70 in β3 strand. Helix αF also is alsoconnected to the regulatory spine by an aspartate residue (Asp 220), which is highlyconserved (Kornev et al., 2008). Helix αF, which is the most buried helix in the structure,emerges as an essential structural element in kinases that integrates assembly of the twohydrophobic spines with kinase activation (Figure 1B).

An inactive conformation first observed in the cyclin-dependent kinasesand Src kinases

The discovery of the Src tyrosine kinase (initially characterized as its oncogenic form in theRous Sarcoma virus) demonstrated a link between aberrant kinase activation and cancer(Martin, 2004). Much attention was therefore focused on understanding why the loss of atyrosine residue in the C-terminal tail of c-Src (Tyr 527, chicken c-Src numbering) results inconstitutive activation of the kinase. The crystal structures of two Src family kinases (c-Srcand Hck) in the inactive conformation and the structure of active Lck revealed how the SH2domains of these proteins help keep the kinase domains in an autoinhibited state, by bindingto Tyr 527 (Sicheri et al., 1997; Xu et al., 1997; Yamaguchi and Hendrickson, 1996) (Figure2A).

It was quite a surprise to discover that the inactive conformation of the c-Src and Hck kinasedomains actually resembles the inactive conformation of the serine/threonine kinase, cyclin-dependent kinase 2 (CDK2), which was the first inactive conformation to be defined (DeBondt et al., 1993). This similarity is particularly striking because their regulatorymechanisms are so different (Figure 2A). Src kinases are autoinhibited by the regulatorySH2 and SH3 domains, in the absence of activating ligands or phosphorylation. In the caseof CDKs, the kinase is by default in the inactive state and activation is achieved by thebinding of cyclin proteins that are synthesized only at specific times during the cell cycle(Figure 2A). Because this inactive conformation was first discovered in CDKs and the Srckinases, we shall refer to it as the “CDK/Src-like” inactive conformation (Figure 2B). Thisinactive conformation has since been observed in many other serine/threonine and tyrosinekinases, such as Abl (Levinson et al., 2006), ZAP70 (Deindl et al., 2007), Wnk (Min et al.,2004), NEK2 (Rellos et al., 2007) and c-Met (Wang et al., 2006).

The CDK/Src-like conformation has the N- and C-lobes closed down over each otherrelative to active conformations (Figure 2B). In this closed conformation helix αC is swungoutward from the N-lobe. This orientation of helix αC pulls the conserved glutamatesidechain in this helix (Glu 91 in PKA, Glu 310 in c-Src) out of the active site and disrupts

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its interaction with the conserved lysine residue (Lys 72 in PKA, Lys 295 in c-Src) fromstrand β3 in the N-lobe, leading to inactivation of the kinase. The movement of helix αC alsodisrupts the regulatory spine by removing the conserved hydrophobic residue in helix αC(Met 314 in Src) away from the active site.

Another important feature of the CDK/Src-like inactive conformation is that the portion ofthe activation loop immediately following the DFG motif often forms a short (single ordouble turn) helix (Figure 2B). In c-Src and Hck this particular conformation of theactivation loop was observed in inactive structures that were determined subsequent to thefirst structures (Schindler et al., 1999; Xu et al., 1999). This helix stabilizes the swung-outconformation of helix αC by packing directly against it, using two or three conservedhydrophobic residues. The sidechains of these residues insert between Lys 295 (c-Src), instrand β3, and Glu 310 in helix αC, blocking the formation of the catalytically essential Lys295 - Glu 310 salt bridge, which switches instead to form a Lys 295 – Asp 404 salt bridgewith the DFG-aspartate (Figure 2B). Mutation of the hydrophobic residues in the helixactivates c-Src (Gonfloni et al., 2000).

In the first inactive crystal structures solved of c-Src and Hck, the activation loop is in aslightly different conformation than that described above (Sicheri et al., 1997; Xu et al.,1997). A crystal structure of the initiation factor 2α protein kinase GCN2 shows a similarconformation (Padyana et al., 2005). In this conformational variant the N-terminal portion ofthe activation loop does not form the single turn helix (Figure 2B). The glutamate in helixαC forms an ionic interaction with the conserved arginine residue in the His-Arg-Asp(HRD) catalytic loop, instead of the arginine in the N-terminal portion of the activation loop.This conformation may represent an intermediate in the transition pathway from the typicalCDK/Src-like to the active conformations, as discussed below.

The CDK/Src conformation may be coupled to the DFG flip, aconformational change in the activation loop

The prevalence of the CDK/Src-like inactive conformation among distantly related kinasesindicates that it might play some specific role in the general mechanism of kinases. Oneintriguing idea is that CDK/Src-like inactive conformation might be coupled to the “DFGflip”, a conformational change in the DFG motif in which the aspartate and phenylalaninesidechains exchange positions due to a crankshaft like motion of the peptide backbone(Figure 3A). The DFG flip from the DFG-in (active) to the DFG-out (inactive) conformationresults in disruption of the regulatory spine by removing the phenylalanine from the core ofthe spine. The catalytic spine is also disrupted due to the loss of ATP binding in thenucleotide binding pocket, which is now occupied by the flipped phenylalanine. Theresulting removal of the aspartate from the active site of the kinase prevents coordination ofthe magnesium ion that is required for catalysis. In the DFG-out conformation helix αCmaintains its inward orientation and the glutamatelysine salt bridge. Crystal structures of theAbl and c-Kit kinases in complex with imatinib show that imatinib binding requires thisDFG-out inactive conformation (Mol et al., 2004; Nagar et al., 2002; Schindler et al., 2000).

Src kinases can also readily adopt the DFG-out conformation, as demonstrated by a class ofcompounds, denoted the DSA compounds, that bind to c-Src and Hck with high affinity andrequire the DFG motif to be flipped. The DSA compounds are based on the chemicalscaffold of imatinib. In contrast to imatinib, which binds to c-Src with a significantly loweraffinity than to Abl, DSA compounds are equipotent inhibitors of c-Src and Abl (Seeliger etal., 2009). This means that the selective inhibition of Abl over c-Src by imatinib is not dueto an impeded DFG flip in c-Src. Instead, differences in the P-loop of Abl and c-Src appearto underlie the specificity of imatinib for Abl over c-Src.

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Just as the Src kinases can adopt the DFG-out conformation, Abl can adopt the CDK/Src-like inactive conformation (Levinson et al., 2006). This observation emphasizes the fact thatone kinase can access multiple inactive conformations. Factors that perturb the free energylandscape of a kinase, such as the binding of an allosteric regulator or a kinase inhibitor, cantip the energetic balance and shift the kinase from one conformation to another.

What might be the significance of DFG flipping for kinase catalysis? Long time scalemolecular dynamics simulations of the Abl kinase have yielded insights into the mechanismof the DFG flips (Shan et al., 2009). If one considers just the peptide segment spanning theDFG motif, it turns out that the preferred conformation corresponds to the flipped one(DFG-out), with the aspartate out of the active site. This is because the backbone ϕ and φvalues in the DFG-in conformation are in an entropically disfavored region of theRamachandran diagram, whereas the DFG-out conformation is in a more favorable region.The DFG-in conformation resembles a coiled spring that is ready to flip to a more favorableDFG-out conformation. The catalytic rate of protein kinases appears to be limited by the rateof ADP release (Grant and Adams, 1996; Lew et al., 1997). The nucleotide-free DFG-outconformation, which is more flexible than the DFG-in conformation, might facilitatenucleotide release and the rebinding of ATP (Shan et al., 2009).

The DFG flip results in the polar DFG-aspartate entering the hydrophobic environmentpreviously occupied by the DFG-phenylalanine, which carries a high energetic penalty.Protonation of the DFG-aspartate, driven by the increase of its pKa upon ATP hydrolysisand the release of ADP and a magnesium ion from the active site, might decrease theassociated cost in free energy (Shan et al., 2009). Due to the conservation of the DFG motifacross the kinase family tree, this protonation-dependent switch might represent a generalmechanism that facilitates the release of ADP from the active site.

The molecular dynamics simulations of the Abl kinase also point to a potential role of theCDK/Src-like conformation as an intermediate in DFG flipping (Levinson et al., 2006; Shanet al., 2009) (Figure 3B). The rotation of the bulky hydrophobic phenylalanine into theactive site of the kinase domain during the DFG flip requires large-scale motions in theactive site. Molecular dynamics simulations showed significant hinge-opening motionsbetween the N-lobe and the C-lobe, which were associated with the movement of helix αCtowards the CDK/Src-like inactive conformation (Figure 3C). These observations suggestthat adopting the CDK/Src-like inactive conformation would facilitate the DFG-flip byenabling the DFG-phenylalanine to move towards the position previously occupied by theDFG-aspartate. One interesting aspect of this analysis is that intermediate structures in thecomputational trajectories correlate with the crystal structures of different kinases (Figure3D). That is, intermediate steps in the DFG flip can be reconstructed by linking theexperimentally determined structures of different kinases.

Control of kinase activation in receptor tyrosine kinases - the curious caseof the EGF receptor family

The activity of most protein kinases is enhanced by phosphorylation of the activation loop.Protein kinases are so constructed that this reaction requires one kinase molecule tophosphorylate another, although there is one notable exception – the DYR kinases have beenshown to phosphorylate their activation loops in cis while partially unfolded duringbiosynthesis on the ribosome (Lochhead et al., 2005). Activation loop phosphorylationprovides a simple mechanism for how the activity of receptor tyrosine kinases is controlled.In receptor tyrosine kinases, the kinase domain is coupled through a transmembrane domainto the extracellular ligand-binding domain. In a prototypical mechanism for receptortyrosine kinase activation, ligand binding controls the ability of one kinase in a dimer to

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phosphorylate the other (Hubbard and Miller, 2007). This phosphorylation event is requiredto stabilize the active state of the kinase that enables efficient phosphorylation of othertyrosine residues in the receptor, primarily on the C-terminal tails of receptor itself. Thesephosphorylated tyrosine sites create binding sites for the SH2 and PTB domain-containingeffector molecules that couple the activated receptors to downstream signaling pathways.

The EGF receptor (also known as HER1, for human EGF receptor or ErbB1 after theerythroblastoma viral gene) and its three close relatives in humans: HER2/ErbB2, HER4/ErbB4 and the catalytic inactive HER3/ErbB3, are critical regulators of mitogenic responsesin cells and can elicit a potent oncogenic signal when deregulated in human diseases(Yarden and Sliwkowski, 2001). In fact, the discovery of the EGF receptor and its relativeHER2 coincided with the first demonstrations that receptor tyrosine kinases are causallylinked to cellular transformation. Following the identification of the receptor for EGF(Carpenter et al., 1975; Ullrich et al., 1984), Downward and colleagues discovered that thevErbB oncogene, carried by the avian erythroblastosis virus, is similar in sequence to theintracellular portion of the EGF receptor (Downward et al., 1984). Similarly, after humanHER2 was identified (Coussens et al., 1985; Yamamoto et al., 1986), the p185Neu oncogenein rat fibroblasts was shown to be the rat homolog of human HER2 (Bargmann et al., 1986a,b).

The EGF receptor family members are quite divergent in their C-terminal tails and activatedifferent sets of effectors proteins. One key feature of the activation of members of thisfamily is the formation of both homo- and heterodimers, some of which include the inactiveHER3, depending on the bound ligands. Since the catalytically inactive HER3 cannotactivate its partner by phosphorylation, the mechanism by which these receptors becomephosphorylated in heterodimers with HER3 was puzzling.

Another surprising aspect of the EGF receptor is the lack of requirement for activation loopphosphorylation. Although the EGF receptor contains a conserved phosphorylation site in itsactivation loop, Tyr 845 (human EGF receptor numbering), which undergoes rapidphosphorylation upon ligand binding (Biscardi et al., 1999), mutation of the activation looptyrosine does not interfere with receptor activation (Gotoh et al., 1992; Tice et al., 1999).These observations focused attention on the idea that a key step in the activation of thekinase domain of the EGF receptor must involve an alternative mechanism, in whichphosphorylation on Tyr 845 does not play a role. The fact that the kinase domains of theEGF receptor family are located closer on an evolutionary tree to those of non-receptortyrosine kinases, such as ACK1 and Janus kinases (JAKs) (Manning et al., 2002), alsosuggests that the mechanism of the EGF receptor activation might be distinct from thatdescribed for other receptor tyrosine kinases.

Activation of the EGF receptor: the CDK/Src-like switchBecause the EGF receptor does not require activation loop phosphorylation, it was thoughtthat this receptor is perhaps always in the active conformation and that dimerization of thereceptor by ligand binding serves simply to enable trans phosphorylation of the C-terminaltails of the receptor. The finding that the kinase domain of the EGF receptor adopts theCDK/Src-like inactive structure when bound to lapatinib suggested that the kinase domainmight be autoinhibited in some way prior to ligand binding (Wood et al., 2004). Alsosuggestive of the relevance of the CDK/Src-like inactive structure to the EGF receptorfunction was the discovery of mutations in the activation loop of the EGF receptor in somecancer patients (Lynch et al., 2004; Paez et al., 2004). These mutations, such as Leu 834 toArg, have been shown to activate the kinase, most likely by destabilization of the inactiveCDK/Src-like conformation (Yun et al., 2007; Zhang et al., 2006). We had noted earlier in

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this review that the corresponding mutations in c-Src result in its activation (Gonfloni et al.,2000).

The realization as to how the EGF receptor is activated came from analysis of several crystalstructures of its kinase domain in the active conformation (Zhang et al., 2006), determinedoriginally at Genentech (Stamos et al., 2002). In all of these structures the kinase domainsform an asymmetric, head to tail, dimer, in which the binding of one kinase domain (denotedas the activator kinase) stabilizes the active conformation of the second kinase domain(denoted the receiver kinase) (Figure 4A). The dimerization interface is largely hydrophobicand involves the bottom of the C-lobe of the activator kinase, which docks on the top of theN-lobe of the receiver kinase.

The principal interactions in the asymmetric dimer are between helix αH of the activatorkinase and helix αC of the receiver. This interaction stabilizes the swung-in conformation ofhelix αC in the receiver kinase and the extended conformation of the activation loop. Theformation of this asymmetric dimer is necessary for the activation of the EGF receptor andalso underlies the activation of other members of the EGF receptor family in homo- andheterodimers (Jura et al., 2009b; Monsey et al., 2010; Qiu et al., 2008). When theasymmetric dimer interface is disrupted, either by point mutations in the activator interface(such as Val 924 Arg) or by binding of the negative feedback inhibitor of the EGF receptorMig6 to the activator interface, the EGF receptor kinase domain adopts the CDK/Src-likeinactive conformation in crystal structures (Zhang et al., 2006; Zhang et al., 2007).

The activation of the EGF receptor kinase domain by formation of the asymmetric dimer isreminiscent of the way cyclin-dependent kinases CDKs become activated by their allostericregulators, the cyclins (De Bondt et al., 1993; Jeffrey et al., 1995) (Figure 4B). In the crystalstructure of the CDK2/cyclinA complex, cyclin binds to the active conformation of CDK,engaging the N-lobe, activation loop and the C-lobe of CDK (Figure 4B) (Jeffrey et al.,1995). One of the major interactions between cyclin and CDK is the packing of helix α5from cyclin against the top of the N-lobe of CDK, especially helix αC. Therefore, despitestructural differences between the asymmetric dimer of the EGF receptor kinase domainsand the CDK/cyclin complex, the nature of the interaction that results in activation isconceptually similar. In both cases, the adoption of the active conformation requiresreorganization in the N-lobe of the kinase that leads to the exposure of hydrophobic residues(the activator-binding patch in the EGF receptor and the cyclin-binding patch in CDKs).Binding of a cyclin or the activator kinase buries these hydrophobic residues in CDK and theEGF receptor, respectively, and stabilizes the active conformation (Figure 4A and B).

CDKs and the EGF receptor represent a group of kinases that are intrinsically stable in theCDK/Src-like inactive conformation and reach their active states only upon binding of theirrespective external allosteric activators (Jeffrey et al., 1995; Zhang et al., 2006). In contrast,the isolated kinase domains of c-Src and Hck tend to activate spontaneously, and theassociated SH2 and SH3 domains are required to stabilize their CDK/Src-like inactiveconformation (Figure 2A) (Sicheri et al., 1997; Xu et al., 1997). Other kinases may fall intothe spectrum of different relative stability of the CDK/Src-like conformation between thesetwo extreme groups.

Allosteric control of the active EGF receptor kinase dimerAlthough we have made an analogy between the asymmetric dimer of EGF receptor kinasedomains and the interaction between cyclins and CDKs, there is one crucial distinction. Thecyclins have high affinity for their target kinases, and in most of the cases can switch themon without additional help. In contrast, the interaction between EGF receptor kinasedomains is very weak, and the kinase domains do not interact in solution. How, then, is the

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asymmetric dimer stabilized? One might have thought that the answer would be that ligand-induced dimerization of the extracellular domains would be the key step in bringing kinasedomains together. Instead, it turns out that the segment of the receptor that connects thetransmembrane helices to the kinase domains, known as the juxtamembrane segment,suffices to bring two kinase domains together in the asymmetric arrangement.

The juxtamembrane segments play essential regulatory roles in restricting the basal activityof several receptor tyrosine kinases (Hubbard, 2004). In contrast, the juxtamembranesegment of the EGF receptor is known to be necessary for ligand-dependent activation anddownstream signaling (Aifa et al., 2005; Macdonald-Obermann and Pike, 2009; Thiel andCarpenter, 2007). The analysis of crystal structures of the EGF receptor and HER4 kinasedomains with their juxtamembrane segments demonstrated that the conserved C-terminalsegment of the juxtamembrane domain plays an important role in stabilizing the activekinase dimer (Jura et al., 2009a; Red Brewer et al., 2009; Wood et al., 2008). It does so byproviding an additional interaction between the receiver kinase, which extends its C-terminal juxtamembrane segment (denoted the juxtamembrane latch) to interact with the C-lobe of the activator kinase in the asymmetric dimer (Figure 5A and 5B). This interaction isessential for ligand-dependent EGF receptor activation and potentiates dimerization betweenisolated kinase domains in solution (Jura et al., 2009a; Red Brewer et al., 2009).

A segment of the EGF receptor inhibitor, Mig6, contains a sequence motif that is identical toa motif in the juxtamembrane latch in the EGF receptor. Thus Mig6 may preventjuxtamembrane latch formation in addition to blocking the asymmetric dimer (Jura et al.,2009a; Zhang et al., 2007). The juxtamembrane latch binding site on the activator kinase isalso occluded by the C-terminal tail of the kinase domain when the asymmetric dimer is notformed (Jura et al., 2009a), providing a mechanism for autoinhibition that is consistent withseveral studies (Bublil et al., 2010; Khazaie et al., 1988; Pines et al., 2010) (Figure 5C).

An intriguing observation is that the structures of the EGF receptor and HER4 kinasedomains on which the understanding of the activating juxtamembrane latch interaction arebased are actually both in the CDK/Src-like inactive conformation (Figure 5B). In the caseof the EGF receptor kinase, the inactive conformation is a result of a mutation in thecatalytic lysine residue (Lys 721 to Met) (Red Brewer et al., 2009). In the HER4 kinase, theinactive conformation is enforced by the presence of a covalently bound inhibitor (Wood etal., 2008). In spite of being in the CDK/Src-like inactive conformation, in both structures thekinases form an asymmetric dimer that largely resembles the active complex. A similarobservation was made for CDK4/cyclinD1 and CDK4/cylinD3 complexes, in which CDK4is found in the CDK/Src-like inactive conformation despite being bound to a cyclin andbeing autophosphorylated on the activation loop (Day et al., 2009; Takaki et al., 2009). Inthe case of the EGF receptor, the ability of the juxtamembrane segments to dimerize kinaseseven when they are in the inactive conformation might be essential for the first step ofreceptor activation, when two inactive receptors are brought to close proximity by a ligand.

The juxtamembrane latch by itself is not sufficient to fully activate the EGF receptor kinase,as evidenced by several studies showing that the segment N-terminal to the latch in thejuxtamembrane region is also necessary for EGF receptor activation (Aifa et al., 2005; Juraet al., 2009a; Thiel and Carpenter, 2007). This region forms an amphiphatic helix andfurther potentiates dimerization of the isolated kinase domain of the EGF receptor in vitro(Jura et al., 2009a; Red Brewer et al., 2009). This led to a model in which the N-terminaljuxtamembrane helices form a short coiled-coil dimer, which is coupled to the dimerizationof the transmembrane domains of the receptor (Jura et al., 2009a). The dimerization of thetransmembrane domains of the EGF receptor family of receptors upon ligand binding hasbeen documented (Chen et al., 2009; Duneau et al., 2007; Mendrola et al., 2002) and

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recently visualized by NMR analysis of transmembrane helices of the EGF receptor andHER2 (Bocharov et al., 2008; Mineev et al., 2010). These structures provide a clue as tohow the transmembrane domains couple dimerization of the extracellular domains toactivation of the kinase domains through the cooperation of the juxtamembrane segment(Figure 5D) (Jura et al., 2009a).

The allosteric role of the catalytically inactive HER3HER3 lacks two important catalytic residues: the aspartate that serves as a base (Asp 813 inEGF receptor) and the glutamate in helix αC (Glu 738 in EGF receptor). HER3 has beenshown to be an inactive receptor but it forms active dimers with the other members of theEGF receptor family (Guy et al., 1994; Sierke et al., 1997; Wallasch et al., 1995). TheHER3-containing receptor heterodimers are critical for the activation of thephosphoinositide 3-kinase (PI3K)/AKT pathway by the EGF receptor family due to theexclusive presence of multiple binding sites for the p85 subunit of PI3K in the C-terminaltail of HER3 (Prigent and Gullick, 1994). Inhibition of HER3 phosphorylation by targetingits active dimerization partners, EGF receptor and HER2, through the use of tyrosine kinaseinhibitors or therapeutic antibodies is buffered in cancer cells by HER3 overexpression(Sergina et al., 2007). This makes the inhibition of HER3-mediated signaling an importanttarget for drug discovery.

In the allosteric mechanism for activation of the EGF receptor family members the activatorkinase does not have to be catalytically active. This indicates how heterodimerization ofHER3 with the active members of the EGF receptor family leads to phosphorylation of bothreceptors, because HER3 can take the activator position in the asymmetric dimer (Figure6A). Sequence conservation in HER3 shows that only the activator but not the receiverinterface of HER3 remains intact (Zhang et al., 2006). Biochemical studies have shown thatHER3 does indeed function as an allosteric activator for other members of the EGF receptorfamily (Jura et al., 2009b; Monsey et al., 2010).

The crystal structures of the HER3 kinase domain show how sequence alterations preventHER3 from becoming the receiver kinase (Jura et al., 2009b; Shi et al., 2010). In thestructure, the HER3 kinase domain is in the CDK/Src-like inactive conformation, which isstabilized by a set of hydrophobic interactions that are not present in other members of theEGF receptor family (Figure 6B). In addition, there are significant conformational changeslocalized to the N-lobe of the HER3 kinase domain that distort the receiver interface (Jura etal., 2009b; Shi et al., 2010). These changes are primarily localized to helix αC, which inHER3 is conserved poorly relative to other HER receptors, and is partially unwound. Thedistinct conformation and packing of helix αC in the HER3 structure alters the receiverinterface substantially.

In the crystal structures of the HER3 kinase domain nucleotide and metal ion are bound inthe active site. The possibility that HER3 might actually support catalysis was examinedrecently (Shi et al., 2010). An autophosphorylation rate estimated to be ~1000 fold lowerthan for the EGF receptor kinase domain was measured for the HER3 kinase domain when itwas brought to a sub-millimolar concentrations on lipid vesicles in vitro (Shi et al., 2010). Itis unclear at present whether this residual activity in HER3 plays a significant role insignaling by EGF receptor family members.

Regulation of other kinases by the helix αC patchActivation by binding to a hydrophobic patch in the N-lobe (the cyclin-binding patch inCDKs and the receiver interface in the EGF receptor) is a theme that is common to severalkinases, including PKA (Knighton et al., 1991), extracellular signal-regulated kinase (Erk) 2

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(Zhang et al., 1994), the p21-activated kinases (PAKs) (Lei et al., 2005) and the Ret receptortyrosine kinase (Knowles et al., 2006). These kinases have a hydrophobic patchcorresponding to the cyclin-binding patch, which we will refer to “helix αC patch” fromnow on, but rely on an intramolecular interaction for activation (Figure 7).

In these kinases, an N- (in PAKs and Ret) or C-terminal extension (in PKA and Erk2) of thekinase domain forms an extra helix, which buries and stabilizes the helix αC patch throughan intramolecular interaction. A different mechanism is operative in Aurora-A kinase, inwhich activation requires the binding of the microtubule-associated protein TPX2 to thehelix αC patch (Bayliss et al., 2003). This interaction induces a conformational change thatstabilizes the active conformation of Aurora-A kinase (Figure 7). The tyrosine kinase Fesutilizes its own SH2 domain to stabilize the “swung-in” position of helix αC in the activeconformation, through interactions at an analogous hydrophobic patch (Filippakopoulos etal., 2008). A similar interaction might also play a role in the activation of the Abl tyrosinekinase, as suggested by small angle X-ray scattering analysis and mutagenesis(Filippakopoulos et al., 2008; Nagar et al., 2006).

Activation through binding of the helix αC patch is also a shared mechanism of the subset ofthe AGC superfamily of kinases (Pearce et al., 2010), including the protein kinase B (PKB/Akt) (Yang et al., 2002), Rho-kinase (ROCK) (Yamaguchi et al., 2006), thephosphoinositide-dependent protein kinase 1 (PDK1) (Biondi et al., 2002) and proteinkinase C (PKC) (Grodsky et al., 2006). In these kinases the helix αC patch is denoted thehydrophobic motif-binding pocket (HM/PIF binding pocket). In the crystal structures ofPKB/Akt, PKC and ROCK, the HM-binding pocket is bound to a phosphorylatedhydrophobic motif (HM) in the C-terminal extensions of these kinases, and this stabilizesthe active conformation (Figure 7). In the Rho-kinase the HM motif itself is stabilized byhydrophobic interactions with the N-terminal extension that forms a helix bundle to mediatedimerization of the kinase (Figure 7). In the absence of phosphorylation, the HM motif doesnot bind to the helix αC patch, and these AGC kinases are inactive (Pearce et al., 2010).

The HM/PIF binding pocket plays a dual role in regulation of the PDK1 kinase, whichactivates many AGC kinases through phosphorylation of their activation loops. PDK1 usesits HM/PIF binding pocket to dock the substrate AGC kinases by using their HM motifs(Biondi et al., 2000). The docking interaction also results in the stabilization of the HM/PIFbinding pocket of PDK1 and allosteric activation of PDK1 activity. Small moleculecompounds have been developed that bind to the HM/PIF binding pocket in PDK1 andinduce PDK1 activation (Engel et al., 2006; Hindie et al., 2009). The discovery of thesecompounds opens a new chapter in the development of drugs targeting the AGC kinases, byuncoupling PDK1 activity from its binding to the substrate AGC kinases. Such compoundsmay also represent a general class of inhibitors that target kinases, including the EGFreceptor family, whose activation is dependent on the helix αC patch. These inhibitors arelikely to be more specific than the ATP analogs since they target a much more diverseinterface than the highly conserved nucleotide binding site. In case of the EGF receptorfamily, the idea of targeting the helix αC patch is particularly exciting as a strategy touncouple the catalytically inactive HER3 from its active dimerization partners.

ConclusionsThe CDK/Src-like inactive conformation, which is observed in structures of severalunrelated kinases, emerges as an essential and possibly conserved step in kinase catalysis. Itis possible that other inactive conformations that have been observed in crystal structuresalso represent important intermediates in the kinase catalytic cycle and they should remainan important subject of structural investigation.

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One important aspect of the analysis of kinase inactive states is that it can also shed light onthe mechanisms of kinase activation. This was important for the elucidation of the activationmechanism of the EGF receptor family of tyrosine kinases, which turns out to be verydifferent from the way other receptor tyrosine kinases are activated and more similar to howCDKs are activated by cyclins. The analogy between the activation of CDKs and the EGFreceptor family also underscores how distantly related kinases, with diverse activationmechanisms, can share common structural mechanisms. In fact, these commonalities mightbe more general and our analysis of different kinase structures demonstrates that ahydrophobic patch in the N-lobe of the kinase (the helix αC patch) serves as a binding sitefor activators in kinases representing different branches on the kinome tree. The detailedanalysis of these common themes and intrinsic differences is crucial for the advancement ofour understanding of how different kinases activate and how we can explore these specificdifferences to design selective strategies for inhibiting aberrant activation of these kinases inhuman disease.

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Figure 1.Activation of protein kinases. (A) Crystal structure (PDB ID: 1ATP) and cartoonrepresentation of the active state of the protein kinase A (PKA). The inset selectivelydisplays the catalytically critical components of the kinase active site: the activation loop,helix αC, P-loop, ATP, magnesium ion and the catalytic residues: DFG-aspartate (Asp 184),HRD-aspartate (Asp 166), catalytic lysine (Lys 72) catalytic glutamate (Glu 91), and theautophosphorylation site in the activation loop (Thr 197). (B) The cartoon representation ofthe assembly of the hydrophobic spines (the regulatory and the catalytic spine) in the activestate of a kinase and of their disassembly in the inactive state. The insets to the right of thecartoons represent surface representation of the residues corresponding to the regulatory andcatalytic spines in the inactive Abl kinase (PDB ID: 1OPJ) and in the active Abl kinase(PDB ID: 2G2I). The DFG-phenylanine is depicted, as well as the catalytic lysine (K) andthe catalytic glutamate in helix αC (E).

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Figure 2.CDK/Src-like inactive conformation. (A) The activation mechanisms of the Src family ofkinases and cyclin-dependent kinases (CDKs). When not bound to SH2 and SH3 domains,Src kinases adopt an active conformation. In contrast, CDKs are inactive when not bound totheir regulators cyclins. Activation of CDKs requires cyclin binding. (B) Crystal structuresof the Hck kinase (PDB ID: 1QCF) in the CDK/Src-like inactive conformation, Lck kinase(PDB ID: 3LCK) in the active conformation and the Hck kinase (PDB ID: 2HCK) in thealternate CDK/Src-like inactive conformation. For clarity the SH2 and SH3 domains are notshown in the inactive structures. The insets underneath the structures selectively display theactivation loop, helix αC, P-loop and the selected residues to highlight major conformationalchanges between different states.

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Figure 3.Coupling of the DFG flip to the CDK/Src-like inactive conformation. (A) Two distinctconformations of the DFG motif found in the crystal structures of the Abl kinase domain. Inthe active structure (PDB ID: 2F4J), DFG adopts the DFG-in conformation, in which theDFG-aspartate is positioned towards the active site. In the inactive, DFG-out conformation(PDB ID: 1OPK), DFG-phenylalanine flips towards the active site. The DFG residues aredepicted as single letter amino acid codes (D, F, G), as is the catalytic lysine (K), thecatalytic glutamate in helix αC (E) and the activation loop tyrosine (Y). (B) CDK/Src-likeinactive conformation is proposed as an intermediate conformation adopted by kinasesduring DFG flipping. (C) A DFG-in to DFG-out flip observed in long time scale moleculardynamics simulations. Starting from conformation 1 (active Abl kinase crystal structurePDB: 2F4J), the DFG-aspartate migrates away from the active site, whereas DFG-phenylalanine enters from above. The accompanying displacement of helix αC is illustratedwith conformations from different simulation times (figure adapted with permission from(Shan et al., 2009). (D) The series of kinase structures captures stepwise progression of theDFG flipping from the DFG-in to the DFG-out conformation, adapted with permission from(Shan et al., 2009). These steps are associated with the significant movement of helix αCand a transient adoption of the CDK/Src-like inactive conformation (PDB ID: IHCK (CDK2kinase) and PDB ID: 1R1W (c-Met kinase)). The insets selectively highlight helix αC, thecatalytic lysine (K), the catalytic glutamate (E) and the DFG-aspartate (D) and DFG-phenylalanine (F) residues.

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Figure 4.The CDK/Src-like switch underlies the activation of the EGF receptor kinase domain. (Aand B) Both CDKs and the EGF receptor (EGFR) kinase are stable in the inactive CDK/Src-like conformation due to a network of hydrophobic residues in the N-lobe (shown as dotrepresentation in magenta color). During activation, the hydrophobic residues becomeexposed creating an interface (an activator-binding patch or a cyclin-binding patch) thatbinds allosteric activators of these kinases. In case of the EGF receptor (A), one kinasedomain (the activator kinase) becomes an activator of the other (the receiver kinase) byforming a head to tail asymmetric dimer. CDKs are subject to the allosteric activation bycyclins (B).

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Figure 5.Allosteric control of the EGF receptor dimerization. (A) Schematic representation of theEGF receptor domain organization. (B) The crystal structure and cartoon representation ofthe EGF receptor kinase (EGFR) domain in the presence of its full juxtamembrane segment(PDB ID: 3GOP) depicts binding of the juxtamembrane latch of the receiver kinase to theactivator kinase. (C) The cartoon representation of the overlapping interactions involvingthe juxtamembrane latch-binding site, based on the structures of the active EGF receptorkinase domain dimer in the presence of its juxtamembrane segment (PDB ID: 3GOP), theinactive EGF receptor kinase dimer (PDB ID: 3GT8) and the inactive EGF receptor kinasebound to an inhibitor Mig6 (PDB ID: 2RFE). (D) The model for ligand-dependent activationof EGF receptor at the plasma membrane, which depicts contribution of the extracellular,transmembrane and the juxtamembrane domains to receptor dimerization.

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Figure 6.HER3 receptor kinase domain is a specialized allosteric activator of other EGF receptorfamily kinases. (A) Cartoon representation of possible dimerization scenarios in the EGFreceptor family of kinases. (B) The crystal structures of the inactive EGF receptor kinasedomain (PDB ID: 3GT8) and the HER3 kinase domain (PDB ID: 3KEX). The HER3 kinasedomain is in the CDK/Src-like inactive conformation and has altered conformation of helixαC.

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Figure 7.A recurring mode for activation of kinases by engaging the helix αC patch. Crystalstructures and cartoon representations of PKA (PDB ID: 1ATP), the Ret receptor kinasedomain (PDB ID: 2IVT), the Aurora kinase (PDB ID: 1Ol5), the AKT/PKB kinase (PDBID: 1O6K), the Rho kinase (PDB ID: 2V55) and the Fes kinase (PDB ID: 3CD3) depictdifferent modes by which these kinases engage the helix αC patch (HM-binding pocket inAKT/PKB) during activation.

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