Special Issue: Wiring and Rewiring in Signal Transduction

Dimerization-induced allostery inprotein kinase regulationHugo Lavoie1*, John J. Li2,3*, Neroshan Thevakumaran2,4*, Marc Therrien1,5, andFrank Sicheri2,3,4

1 Institute for Research in Immunology and Cancer, Universite de Montre al, Montre al, Que bec, H3C 3J7, Canada2 The Lunenfeld–Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, M5G 1X5, Canada3 Department of Molecular Genetics, University of Toronto, Toronto, Ontario, M5S 1A8, Canada4 Department of Biochemistry, University of Toronto, Toronto, Ontario, M5S 1A8, Canada5 De partement de Pathologie et Biologie Cellulaire, Universite de Montre al, Montre al, Que bec, H3C 3J7, Canada

Review

The ability of protein kinases to switch between inactiveand active states is critical to control the outputs ofcellular signaling pathways. In several protein kinases,the conformation of helix aC is a key hub on whichregulatory inputs converge to induce catalytic switch-ing. An emerging mechanism involved in regulatinghelix aC orientation is the allosteric coupling with kinasedomain surfaces involved in homo- or heterodimeriza-tion. In this review, we discuss dimerization-mediatedregulation of the rapidly accelerated fibrosarcoma (RAF)and eIF2a kinase families and draw parallels with theanalogous behavior of the epidermal growth factor re-ceptor (EGFR) and serine/threonine-protein kinaseendoribonuclease 1 (IRE1)/ribonuclease L (RNAse L) ki-nase families. Given that resistance to RAF-targetedtherapeutics often stems from dimerization-dependentmechanisms, we suggest that a better understanding ofdimerization-induced allostery may assist in developingalternate therapeutic strategies.

Catalytic switching of eukaryotic protein kinasesAllosteric regulation of protein function mediated by self-assembly was first recognized more than 50 years ago inhemoglobin [1,2]. Since then, the notion that homotypicprotein–protein interactions can modulate protein func-tion has been extended to numerous other biological sys-tems [3]. In the past 10 years, structural and functionalstudies have revealed that select members of the eukary-otic protein kinase super-family also exploit dimerizationand/or multimerization for direct regulation of their kinasedomains. These studies revealed a great deal of diversity inthe underlying modes of dimerization that underpin allo-steric regulation of catalytic function. They also providedinsight into mechanisms of dysregulation that give rise todisease and nucleated drug design strategies that exploit

0968-0004/

� 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tibs.2014.08.004

Corresponding authors: Therrien, M. (marc.therrien@umontreal.ca);Sicheri, F. (sicheri@lunenfeld.ca).Keywords: dimerization; allosteric regulation; protein kinases; pseudokinase.*The first three authors contributed equally to this work.

or minimize the beneficial and adverse effects of theunderlying dimerization phenomenon.

Eukaryotic protein kinases catalyze the transfer of the g

phosphate of ATP onto seryl, threonyl, or tyrosyl acceptorsites on target substrates [4]. Protein phosphorylation is ahighly utilized mechanism of cellular regulation that canmodulate the localization, stability, affinity, and/or activityof up to 30% of all cellular proteins in eukaryotes [4,5]. Theprotein kinase domain possesses a bilobal architectureconsisting of a smaller N terminal (N-) lobe and a largerC terminal (C-) lobe linked through a short flexible hinge(Figure 1A). The C-lobe consists primarily of a-heliceswhile the N-lobe consists minimally of a five-strandedantiparallel b-sheet and a key regulatory helix denotedhelix aC. The active site, which houses the majorityof conserved residues dedicated to nucleotide binding(G-loop, invariant Lys–Glu ion pair), phospho-acceptor sitepositioning (activation segment), magnesium binding(DFG motif), and phospho-transfer function (HRD motif),resides within the interfacial cleft between the two lobes[6]. Inter-lobe motion imparted by the flexible hingeregion facilitates substrate binding and product release,but catalysis is restricted to a closed conformation withproductively oriented active site residues. The activationstate of the protein kinase domain can be assessed byvisualization of the alignment of two sets of spatiallyconserved hydrophobic residues, termed the catalyticand regulatory spines [7,8]. When bound to nucleotide,the spines form parallel, linear columns of interactingresidues that span the N- and C-lobes and organizethe catalytic infrastructure by restricting inter-lobemovements [7] (Figure 1B, right panel).

The conformational plasticity of the kinase domainallows for great diversity in how phospho-transfer functionis regulated. Although the active conformations ofprotein kinases appear highly similar, inactive conforma-tions vary drastically. This reflects the tight constraints ofa shared catalytic mechanism compared to the ease anddiversity by which catalytic function can be perturbed. HelixaC plays a recurring role in protein kinase regulation as firstdelineated for the cyclin-dependent kinase (Cdk) family[9,10] (Figure 1B,C). In the absence of cyclin binding, helix

Trends in Biochemical Sciences, October 2014, Vol. 39, No. 10 475

Ac�ve PKA (PDB 1ATP)

Helix αC

Invariant Lys-Glu ion pair

ATP

Hinge region

Ac�va�on segment

C-lobe

N-lobe

Key:

Magnesium

G-loop

DFG mo�f

Substrate pep�de mimic

N-lobe

C-lobe

(A)

C-spine R-spine

Ac�ve CDK2linear spines

N-lobe

C-lobe

(B)

Helix αC

Ac�ve CDK2closed conforma�on

Invariant Lys33-Glu51

ATP

Hinge region

Ac�va�on segment

DFG loop Phe146

N-lobe

C-lobe

Substrate pep�de

C-spine R-spine

Inac�ve CDK2broken spines

Inac�ve CDK2open conforma�on

18.6A2.7A

(C)

Cyclinbinding

Key:

TiBS

Figure 1. General architecture and prototypical activation mechanism of a protein kinase domain. (A) Cartoon representation of the protein kinase A (PKA) kinase domain

bound to ATP (red) (PDB: 1ATP) with conserved functional elements highlighted. (B) Conformational changes that control catalytic switching of cyclin-dependent kinase

2 (CDK2) upon cyclin binding. Hydrophobic spine alignment of CDK2 in the inactive (left; PDB: 1HCL) and active (right; PDB: 1QMZ) conformations. The catalytic spine

(C-spine) residues are shown with red surface rendering while regulatory spine (R-spine) residues are shown in yellow. (C) Conformational motion of key structural

elements in the inactive (left) and active (right) states of the CDK2 protein kinase domain.

Review Trends in Biochemical Sciences October 2014, Vol. 39, No. 10

aC of the Cdk kinase domain resides in a laterally displacedconformation [11]. This manifests as a broken regulatoryspine (Figure 1B, left panel), prohibiting the establishmentof an essential salt-bridge interaction between subdomainresidues Glu51 (residing on helix aC) and Lys33 (residing on

476

b-strand 3) that productively coordinates the phosphatemoieties of ATP in the active site (residue numbering fromhuman CDK2) [11] (Figure 1C, right panel). In response tocyclin binding, helix aC transitions to an inward positionthat restores integrity of the regulatory spine and facilitates

Review Trends in Biochemical Sciences October 2014, Vol. 39, No. 10

formation of the Lys33–Glu51 salt interaction, therebyenabling catalytic function (Figure 1B,C, right panels).The modulation of helix aC conformation is employed byother protein kinases to regulate catalytic function includ-ing the sarcoma family kinases (Src) [12,13], feline sarcoma/Fujinami avian sarcoma kinase (Fes/Fps) [14] and TCR-zetaassociated protein kinase 70/spleen tyrosine kinase (ZAP70/Syk) [15]. However, the precise mechanisms by which helixaC modulation is achieved are highly divergent.

An emerging mechanism to modulate the position ofhelix aC and thus regulate catalytic output is the self-association of protein kinase domains in precise config-urations (reviewed previously by Pellicena and Kuriyan,2006 [16]). As first uncovered for the eukaryotic initiationfactor 2a (eIF2a) family kinase PKR (double-strandedRNA-dependent protein kinase), and further exemplifiedby distantly related protein kinases such as the EGFRand RAF/kinase suppressor of Ras (RAF/KSR) families(Figure 2), this mode of regulation involves the allosterictransition of the kinase domain from a variable non-productive state to a specific productive dimer state. Inthis review, we focus on the RAF and eIF2a proteinkinase families, for which X-ray crystallographic struc-tures have provided insight into the opposing enzymestates (Figure 2, bottom panels; and Table 1). We discusshow allostery arising by the adoption of a specific dimerconfiguration may have contributed to the evolution ofpseudo-kinases in these families, and finally, the patho-logical and therapeutic implications associated withaberrant dimerization. We note that allosteric regulationof the EGFR family kinases has been extensivelyreviewed elsewhere [17,18] and that the principlesdiscussed here are highly complementary to thosediscerned for that protein kinase family.

The RAF family kinasesThe RAF family of protein kinases is one of the mostheavily studied eukaryotic enzyme families owing to itscentral role in growth factor signaling and human pathol-ogies. In vertebrates, the RAF family is composed of threeRAF paralogs (ARAF, BRAF, and CRAF) and two moredistantly related pseudo-kinase paralogs (KSR1 andKSR2), whereas in invertebrates, the family is typicallycomposed of a single ortholog of RAF and KSR [19]. At thecellular level, RAF activation results in diverse cellularoutputs including proliferation, differentiation, and sur-vival [20]. RAF family members serve as signaling relayswithin the RAS–RAF–MEK–ERK (RAS/ERK) cascade,and dysregulation of this pathway is conducive to tumorformation or developmental anomalies. Tumor analysisand high-throughput sequencing showed that, within thispathway, activating mutations in RAS genes (HRAS,KRAS, NRAS) are the most recurrent genetic lesions,while gain-of-function mutations in BRAF are arguablythe second leading defect (COSMIC database [21]). Underphysiological conditions, RAF activation is initiated at theplasma membrane through direct binding to growth factor-stimulated RAS GTPases (Figure 3A). RAF activation, inturn, triggers the sequential phosphorylation and activa-tion of the downstream kinases MEK and ERK. ActivatedERK then phosphorylates specific cellular substrates that

elicit diverse cellular responses (reviewed in Roskoski Jr.,2012 [22]).

RAF kinase activation by dimerization

An essential component of the RAF kinase activationmechanism involves the regulated transition of the kinasedomain from a non-productive monomer configuration to aspecific productive side-to-side dimer configuration,centered on the critical residue Arg509 (residue numberingfrom human BRAF) (Figure 3A,B,C). Both loss-of-functionand gain-of-function mutations have validated the biologi-cal relevance of the side-to-side dimer configuration visu-alized in crystal structures [23–25], but the absence ofstructures for ARAF, BRAF, and CRAF in their inactive,monomeric state leaves our understanding of the allostericswitching mechanism incomplete. Notwithstanding, a rolefor helix aC in the switching mechanism was hinted at byits conspicuous positioning within the side-to-side dimerinterface. It is conceivable that in the absence of dimeriza-tion, helix aC transitions to a non-productive static confor-mation, as observed for the inactive state of CDKs(Figure 1B), or to a non-productive dynamic conformation,as observed for the inactive state of AKT [26]. The struc-ture of the RAF family member KSR2 [27] provided a firstglimpse of what a non-productive conformation of a RAFkinase domain might look like. Throughout evolution, KSRhas accumulated mutations that render it severely com-promised for phospho-transfer function, relegating it to alarger group of catalytically impaired pseudo-kinases thatencompass 10% of all kinases encoded in the human ge-nome [28]. The kinase domain of KSR2, solved in complexwith MEK1 (PDB: 2Y4I), revealed a homotypic side-to-sidedimer configuration between KSR protomers (Figure 3A)using largely the same surface employed by BRAF andCRAF dimers in their active state (Figure 3B), but with a1808 difference in rotation on the interaction surface plane(Figure 3A). As in BRAF structures, helix aC of KSRcontributed to the dimer contact surface, but in contrastadopted a laterally displaced conformation that prohibitedthe essential salt interaction between subdomain IIArg692 and subdomain III Glu710 (residue numberingfrom human KSR2). If KSR was not already compromisedfor catalytic function by virtue of being a pseudo-kinase,this structural feature would be sufficient to render theKSR kinase inactive.

The available structures of BRAF, CRAF, and KSR2kinase domain dimers provide support for the followingallosteric model for catalytic regulation (Figure 3A). Acatalytically productive state is achieved by BRAF andCRAF by adoption of a common side-to-side dimer config-uration in either a homotypic or heterotypic manner withany other family members including the pseudo-kinasesKSR1 and KSR2 [19,29]. BRAF and CRAF also likely adoptan inactive monomer conformation (as yet to be deter-mined crystallographically), possibly involving a non-pro-ductive position of helix aC. KSR, for its part, can adoptan inactive-like dimer configuration also involving a non-productive orientation of helix aC. We speculate that, inaddition to its elusive monomeric configuration, BRAFand CRAF kinase domains could demonstrate a KSR-likedimeric conformation in their inhibited state.

477

TiBS

BRAF ERBBPKR

C-lobe

C-lobe

N-lobe

N-lobeC-lobe

N-lobe

C-lobe

N-lobe

C-lobe

N-lobe

C-lobe

N-lobe

EGFR kinases

RAF kinases

EGFR kinasesRAF kinases

eIF2α kinases

eIF2α kinases

IRE1 /RNAse L

TK

CMGC

(A)

(B)

Side-to-side dimer Back-to-back dimer Head-to-tail dimer

TKL

STE

CK1

AGC

CAMK

Figure 2. Kinase domain regulation by dimerization is observed in diverse branches of the kinome tree. (A) Dendrogram depicting the conservation and main families found in

the human kinome. The rapidly accelerated fibrosarcoma (RAF), eukaryotic initiation factor 2a (eIF2a), serine/threonine-protein kinase endoribonuclease 1 (IRE1)/ribonuclease L

(RNase L), and epidermal growth factor receptor (EGFR) families of protein kinases are highlighted. Illustration reproduced courtesy of Cell Signaling Technology, Inc. (B) The

RAF, eIF2a, and EGFR kinase domains engage along different dimerization interfaces and form geometrically distinct assemblies in their active states: RAF kinases dimerize in a

side-to-side fashion; eIF2a kinases form back-to-back dimers; active EGFR kinase domains for their part adopt an asymmetrical head-to-tail dimerization mode.

Review Trends in Biochemical Sciences October 2014, Vol. 39, No. 10

The ability of the pseudo-kinase KSR to participate inthe allosteric regulation of active RAF kinase family mem-bers provides an explanation of how some pseudo-kinasesmay have evolved. The primordial ancestor of the RAF

478

family possessed both catalytic and allosteric regulatoryfunctions. Following gene duplication, one duplicatedmember dispensed with catalytic function, but still partic-ipated in the allosteric regulation of other members by

Table 1. Helix aC conformations of selected dimer regulated kinases

Protein kinase PDB ID aC conformation

eIF2a Family PERK 3QD2 In

4G31, 4G34 Out (inhibitor induced)

PKR 2A1A, 2A19, 3UIU In

GCN2 1ZYC, 1ZYD, 1ZY4,

1ZY5, 1ZXE

Out

HRI – –

RAF Family BRAF 4JVG, 1UWH, 1UWJ,

2FB8, 3D4Q, 3IDP, 3II5,

3PPJ, 3PPK, 3PRF, 3PRI,

3Q4C, 3Q96, 3PSD,

4DBN, 4E26, 4G9R,

4H58, 3PSB, 4SKQ, 4FC0

In

CRAF 3OMV In

KSR 2Y4I Out

ARAF – –

Kinase-Ribonuclease Family IRE1 2RIO, 3LJ0, 3LJ2, 3FBV,

3SDM, 3LJ1, 3SDJ

In

3P23 Out

RNase L 4OAU, 4OAV, 4O1O,

4O1P

In

Bacterial Ser/Thr protein kinase PKnB 2FUM, 1MRU In

3F61, 4EQM, 3ORM,

3ORI, 3ORK, 3ORL,

3ORO, 3ORP, 3ORT

Out

Review Trends in Biochemical Sciences October 2014, Vol. 39, No. 10

maintenance of the ability to dimerize. This model mayreadily explain the evolution and function of pseudo-kinases that reside in families with bona fide activemembers regulated allosterically through dimerization.ERBB3/ERBB2 dimers in the EGFR kinase family(reviewed in Baselga, 2009 [30]), ribonuclease L-dependent(RNase L) kinase dimers [31,32] and possibly the intramo-lecular assembly of the two eIF2a kinase domains ofgeneral control non-derepressible 2 kinase (GCN2) repre-sent examples of pseudokinase/active kinase domain pairslikely involved in a trans-activation process similar to theKSR/RAF system.

Physiological and pharmacological regulation of RAF

dimerization

The cellular mechanisms that regulate RAF kinase domaindimerization remain the subject of much speculation andresearch. Three interconnected mechanisms uncovered forRAF thus far include relocalization to membrane surfaces,relief of intra-molecular auto-inhibitory interactions thatmay restrain dimerization, and binding to proteins withintrinsic dimerization potential.

RAS, the best-characterized upstream regulator of RAF,may induce RAF activation in part by all three mecha-nisms. First, RAS is localized to membrane surfaces andupon binding to RAF, causes RAF relocalization from thecytoplasm to the inner leaflet of the plasma membrane.This increases the effective concentration of RAF and couldpromote dimerization by mass action, but could also bringit into contact with lipids that modulate its activity and/ordimerization potential. Consistent with this model, fusinga CAAX box to the isolated kinase domain of RAF issufficient to strongly promote its activity and dimerization[29,33] (Figure 3A). Second, RAF is thought to adopt ahigher-order auto-inhibited state arising from intra-molec-ular association between its N terminal regulatory region,

including the RAS binding domain (RBD), with its kinasedomain [34,35]. Binding of RAS to the RBD displaces thepredicted auto-inhibitory interactions, thereby contribut-ing to activation. This model could explain why targetingthe full-length protein to membranes using a CAAX boxdoes not induce maximal RAF catalytic activation and stillrequires RAS stimulation [33]. Third, RAS itself possessesintrinsic oligomerization potential. as evidenced by itsability to form nanoclusters on cellular membranes [36]and to form dimers in vitro and in cells [37–39]. If this modeof oligomerization is compatible with binding RAF in itsproductive dimer configuration, then it too would contrib-ute to RAF dimerization and activation.

The 14-3-3 protein family are bivalent phospho-recogni-tion modules with a dimeric architecture that regulate RAFfunction on two levels. In the quiescent state, 14-3-3proteins promote the auto-inhibited state of RAF that isincompatible with kinase domain dimerization by binding totwo phospho-epitopes, p-Ser365 and p-Ser729 (residue num-bering from human BRAF) on a single RAF monomer[19]. Upon EGF signaling, Ser365 is rapidly dephosphory-lated resulting in destabilization of the auto-inhibited state.In addition, 14-3-3 is then thought to stabilize the dimericassembly of RAF by simultaneously binding the p-Ser729epitopes of two RAF protomers [19] (Figure 3A).

Given the prominent role of BRAF in oncogenic signal-ing, numerous ATP-competitive kinase inhibitors havebeen developed to attenuate BRAF catalytic output(reviewed in Baska, 2014 [40]). Surprisingly, and greatlylimiting their utility in the clinic, virtually all RAF kinaseinhibitors display the opposite effect in vivo, counter-intui-tively activating total RAF enzymatic activity, increasingERK signaling and as a result increasing cell proliferation[40–42]. The basis for this paradoxical effect is traceable tothe ability of BRAF inhibitors to promote RAF familykinase domain dimerization in cells and tissues where

479

N-lobe

C-lobe

Extracellular matrix

Cytosol

Inac�ve monomer Inac�ve dimerAc�ve dimer

N-lobe

C-lobe

RAS-bindingdomain

Cysteine-richdomain

RAS-GDP RAS-GTP

N C

NC

14-3-3 proteins

RTK and RASac�va�on

RAF N

C

N

CNC

KSR protomer 2KSR protomer 1KSR protomer 1

BRAF protomer 1

BRAF protomer 2

BRAF DWWHGRKTRHVLFMGQDYAKELTE CRAF YWWHGRKTRHVLFMGQDYAKELTE ARAF YWWHGRKTRHVLFMGQDYAKELTE KSR1 QWWHGRQTRHVLFMGSGYAKDGKD KSR2 QWWHGRQTRHVLFMGSGYAKDGKD

448

450

476

477

478

506

507

508

509

510

511

515

516

517

518

530

565

566

569

570

586

588

589

715

helix αC

C-spine R-spineDimer interface

helix αC

C-spine R-spineDimer Interface

R509

BRAF KSR

Ser/Thr-richdomain

(A)

(C)

(B)

KSR

Residue numbering from human BRAF

TiBS

Figure 3. Dimerization and activation of the rapidly accelerated fibrosarcoma (RAF) family kinases. (A) In the resting state, RAF is maintained in a yet unresolved inactive

conformation through intramolecular interactions between N terminal regulatory elements and the protein kinase domain. Additionally, the pseudokinase kinase

suppressor of Ras (KSR) may exist as an inactive dimer (red inlet) in resting cells. Upon pathway activation by receptor tyrosine kinase (RTK) ligands, post-translational

modifications and direct membrane recruitment by the activated, GTP-bound rat sarcoma (RAS) small GTPase lead to RAF side-to-side dimerization (blue inlet) and catalytic

switching. (B) Sequence alignment of the residues participating in side-to-side dimer interface shows strong conservation between human RAF family members (identical

residues are shaded in dark grey while similar residues are in pale grey). (C) The dimerization surface and the hydrophobic spines of RAF in active (BRAF; PDB 3Q4C; left;

blue box) and inactive (KSR2; PDB 2Y4I; right; red box) state dimers. Side-to-side dimer interface residue, R509 (Residue number from human BRAF), is shown (yellow). The

dimer surface buried underneath the stripped out protomer is colored blue (left) or yellow (right).

Review Trends in Biochemical Sciences October 2014, Vol. 39, No. 10

480

Review Trends in Biochemical Sciences October 2014, Vol. 39, No. 10

RAS activity is elevated due to mutations or high receptortyrosine kinase (RTK) signaling (reviewed in Gibney et al.,2013 [43]). Current models envision that drug-bound BRAFprotomers, which are incapacitated for catalytic output,heterodimerize and trans-activate drug-free paralogousRAF family protomers, leading to enhanced downstreamsignaling. In this context, the BRAF–CRAF heterodimer isthe likely culprit in subverting the desired clinical outcome[41,42]. Interestingly, the primary and acquired resistanceof human tumors to RAF-targeted therapies is influenced bydimerization both through alterations of the RAF locus or bycompensatory increases in RAS and/or RTK signaling(reviewed in Holderfield et al., 2014 [44]).

The unanticipated interplay between kinase domaindimerization, ATP-competitive inhibitor binding, andtrans-activation highlights the allosteric connection be-tween the kinase active site and the side-to-side dimeriza-tion infrastructure. In principle, inhibitor-induceddimerization could manifest for all protein kinases alloste-rically regulated by dimerization and indeed this hypothe-sis appears to hold true in some instances for the eIF2a

kinase PKR-like endoplasmic reticulum kinase (PERK),as well as EGFR family members [29,45]. Whether thisphenomenon also causes unintended activation of eIF2a

kinases or EGFR signaling remains to be determined.Mechanistic insight has recently been gleaned on the

connection between the drug-binding site within the cata-lytic cleft and the dimerization interface. One consequenceof drug binding to RAF kinases is the imposition of amore stable alignment of hydrophobic spines that leadsto kinase domain inter-lobe closure. The current view isthat this closed conformation offers a more stable interfacepropitious to dimerization [29]. The same mechanism in-volving spine alignment appears to apply to RNAse Lbound to nucleotide [31] and might very well translateto PERK bound to GSK2656157 [29]. Therapeutic strate-gies are in development to avoid the unwanted side-effectsof current ATP-competitive inhibitors. Notable proofs ofprinciple include a peptide inhibitor that directly targetsthe side-to-side dimerization surface of BRAF [46] and aVemurafenib series analog [PLX7904 or Paradox breaker04 (PB04)] that was shown to limit RAF dimerization andparadoxical activation of downstream signaling [47].

The eIF2a family kinasesIn eukaryotes, the eIF2a family of protein kinases(EIF2AKs) inhibits protein synthesis in response to specificstress stimuli to conserve resources while reconfiguring geneexpression for stress adaptation, or alternatively, to induceapoptosis [48]. The four family members, PKR, PERK, GCN2and HRI (heme regulated inhibitor kinase) harbor a diverserepertoire of regulatory domains that enable detection ofdifferent stimulatory cues [49] and highly similar proteinkinase catalytic domains that enable transmission of apotent signal that inhibits translation initiation. This isachieved by specific phosphorylation of a common cellularsubstrate, eIF2a, on Ser51 (reviewed in Dever, 2002 [50]).

Dimerization surface of the eIF2a kinase family

A shared feature of eIF2a kinase function is the prominentrole of dimerization in the regulation of phospho-transfer

function: fusion of either constitutive (glutathione S-trans-ferase) or inducible (GyrB/Fv2E) dimerization domains tothe kinase domains of PKR or PERK is sufficient for kinaseactivation and downstream signaling, even in the absence ofexogenous stress cues [49,51]. The nature of the activateddimer state was revealed first by the crystal structure of thekinase domain of PKR in complex with eIF2a [52,53], andsubsequently, for the kinase domain of PKR (PDB 3UIU)and PERK [54] in isolation. In these structures, the kinasedomains adopted a common back-to-back orientation withthe active sites facing outwards [49,52,53], completely dis-tinct from the side-to-side dimer configurations displayed byactive state RAF family kinases. The back-to-back dimerinterfaces of PKR and PERK are composed almost exclu-sively by the N-lobe (Figure 4A, red box insert), with contactresidues highly conserved across the eIF2a kinase family(Figure 4B, left box). Most notable among the dimer inter-actions are the Asp289–Tyr323 hydrogen bond and theArg262–Asp266 salt-bridge (residue numbering fromhuman PKR), both of which cause a loss of catalytic functionwhen perturbed. Complementation studies centered on theArg262–Asp266 salt bridge cemented a critical role for back-to-back dimerization in enzyme biological function [52,53]:single site mutations disrupting the salt bridge interactionabolished PKR catalytic function both in vitro and in vivo,but charge reversal of this amino-acid pair rescued biologicalfunction. Conservation of the dimer interface residues sug-gests the back-to-back dimer conformation is generallyrelevant for all eIF2a family members. Supporting thisnotion, the corresponding charge-reversal experiments per-formed on PERK and GCN2 [49] revealed the same com-pensatory effects. Interestingly, the streptococcal proteinkinase PknB/PknE, possibly originating from an eukaryotichost by horizontal gene transfer, employs a near identicalmode of dimerization for catalytic activation, albeit using adivergent set of dimer contact residues [55,56] (Figure 5).Not surprisingly, the IRE1 and RNase L family of proteinkinases/pseudo-kinases, which lie adjacent to the eIF2a

kinase family on the kinome tree (Figure 2), also employthe same back-to-back dimer conformation for their regula-tion (Figure 5); however, rather than regulating proteinkinase function, dimerization of the kinase domain servesto regulate the activity of an RNase domain fused directly tothe kinase C-lobe [31,32,57,58].

Although crystal structures of the monomeric, inactivestate of PKR and PERK have been elusive, we speculate thatthey too will display a misposition and/or distortion of helixaC as this appears to be the case for both the related IRE1/RNaseL protein kinase family and the unrelated bacterialPknB kinases. Monomeric structures of PknB displayunwinding and translational shifts to helix aC that kinkthe regulatory spine and disrupt the essential Lys–Glusalt-bridge required for productive ATP coordination(Figure 5A,B). Similarly, inactive IRE1 displays a brokenregulatory spine and a disruption of the Lys–Glu salt-bridge(Figure 5A,C). A twist on the monomer to dimer switchingmechanism is displayed by the two eIF2a kinases GCN2 andHRI, with both enzymes appearing to exist as constitutivedimers (Figure 4A,C). The switching of GCN2 and HRI,therefore, likely involves a transition between alternatedimer states with the active state corresponding to the same

481

H

Helix αCHelix αC

D266

C-spine R-spineDimer Interface C-spine R-spineDimer Interface

R262

1

2

1

2

1

2

Viral dsRNA

dsRNAbindingdomains

NN

CC

PP

N

C

N

C

Bip

NN

CC

PP

NN

CC

PP

N-terminal domain

Regulatory domain

tRNA

C-terminal domain

HisRS-like domain

NN

CC

NN

CC

PP

H

H

Hemedeficiency

H H

?–

PKR GCN2

N

C

N

C

N-lobe

C-lobe

PKR protomer 1

GCN2 protomer 2

N-lobe

C-lobe

GCN2 protomer 1GCN2 protomer 1

PKR protomer 1

ER Lumen

Unfolded protein

Cytosol

Inac�ve monomer Inac�ve dimerAc�ve dimer

PKR

PERK

GCN

2HR

IPKR RDHIDKYYEVADVHYNCDLHRI RENLDQYTLVKQPGYHAILGCN2 RENLDCYIKVTHERYYAILPERK RDNVDCYLMVKEPRYFALL

262

266

286

288

289

291

293

300

306

309

313

316

318

322

323

324

326

328

362

(A)

(C)

(B)

Residue numbering from human PKR

TiBS

Figure 4. Dimerization and activation of the eukaryotic initiation factor 2a (eIF2a) family kinases. (A) In the resting state, eIF2a kinases are predicted to exist as monomers,

but their structures have yet to be solved [double-stranded RNA-dependent protein kinase (PKR), PKR-like endoplasmic reticulum kinase (PERK); broken box]. Inactive

dimers of general control non-derepressible 2 kinase (GCN2) have also been observed (GCN2; PDB: 1ZYC; red inlet). Inactive dimers are also suspected to participate in the

Heme regulated inhibitor kinase (HRI) activation process (bottom right, broken box). Upon activation by stress signals, PERK, PKR, GCN2 and HRI form specific back-to-back

dimers (PKR; PDB: 2A1A; blue inlet). Stimuli affecting each family member are: unfolded proteins for PERK, viral dsRNA for PKR, uncharged tRNA for GCN2, or heme

deficiency for HRI. Upon binding of stimulating cues the sensor domains of each kinase dictate a precise productive orientation of the kinase domain that leads to back-to-

back dimer formation and catalytic switching. (B) Sequence alignment of the residues participating in the back-to-back dimer interface of eIF2a family kinases. Identical

residues are shaded in dark grey while similar residues are in pale grey. (C) The dimerization interface and the hydrophobic spines of eIF2a kinases in the active state (PKR;

PDB: 2A1A; left; blue box) and inactive state (GCN2; PDB: 1ZYC; right; red box). Back-to-back dimer interface residues R262 and D266 are shown in yellow on PKR

dimerization surface. The dimer surface buried underneath the stripped out protomer is colored blue (left) or yellow (right).

Review Trends in Biochemical Sciences October 2014, Vol. 39, No. 10

482

N-lobe

C-lobe

IRE1 protomer 1

IRE1 protomer 2

KEN domain

Extracellular

CytosolNN

CC

P PN

C

P

PASTAdomains

Pep�doglycan

Inac�ve monomer

N-lobe

C-lobe

PknB protomer 1 PknB protomer 2

PknB

Ac�ve dimer

N-lobe

C-lobe

PknB monomer

N N

CP

NN

CCP

N

C

Bip

NN

CCPP

Regulatory domain

NN

CC

P

ER Lumen

Unfolded protein

Cytosol

Inac�ve monomer Ac�ve dimer Ac�ve oligomer

IRE1

P

KENdomain

N

C

ANK domain

NN

CC

Ribonucleasedomain

2-5A

RNas

e L

PknB dimer PknB monomer

Helix αC

C-spine R-spine C-spine R-spine

(B)

(A)

(C)

N-lobe

C-lobe

RNase L protomer 1

RNase L protomer 2

RNase domain

ANK domain

IRE1 dimer IRE1 monomer

C-spine R-spineC-spine R-spine

IRE1 monomer

N-lobe

C-lobe

KEN Domain

Helix αC Helix αC Helix αC

TiBS

Figure 5. Dimerization and activation of the eukaryotic initiation factor 2a (eIF2a) kinase family relatives protein kinase B (PknB), IRE1, and ribonuclease L (RNaseL). (A) In

the resting state, bacterial PknB, IRE1, or RNaseL exist as monomers (red inlets; PknB PDB: 3ORM, IRE1 PDB: 3P23). The monomeric state of RNAseL is unresolved (broken

box). Upon binding to regulatory second messengers back-to-back dimerization leads to catalytic activation of the kinase domain (PknB) or activation of a fused

ribonuclease domain (IRE1 and RNaseL) (blue inlets; PknB PDB: 2A19, IRE1 PDB: 2RIO, RNase L PDB: 4O1P). PknB is activated by peptidoglycan, IRE1 by unfolded proteins

while RNaseL binds the small viral-induced ligand 20,50-oligoadenylate (2–5A). Additionally, in the case of IRE1, higher-order oligomers drive maximal activation of RNase

function (top right). (B) Depiction of the hydrophobic spines of PknB in monomeric and active dimeric states. The monomer state displays a kinked regulatory spine not

conducive to catalysis. (C) The hydrophobic spines of IRE1 in its monomeric and dimeric configurations. The monomer state displays a moderately misaligned regulatory

spine.

Review Trends in Biochemical Sciences October 2014, Vol. 39, No. 10

483

Review Trends in Biochemical Sciences October 2014, Vol. 39, No. 10

dimer configuration sampled by PERK and PKR. This isreminiscent of the KSR structure representing an inactivedimer state, while RAF structures represent the activedimer state. In support of the non-productive dimer toproductive dimer transition model, the structure of thekinase domain of GCN2 revealed an altogether novel inac-tive dimer configuration [59]. Although the GCN2 dimer-ization surface displayed 80% overlap with the active statedimerization surface of PERK and PKR, the inactive GCN2protomers were positioned in an anti-parallel orientationversus the parallel orientation (rotated by approximately1808 relative to the contacting surface) observed for activePKR/PERK protomers. Consistent with an inactive stateconformation, the activation segment of the GCN2 structurewas not phosphorylated and the regulatory spine was bro-ken due to a non-productive position of helix aC that dis-rupted the Lys–Glu ion pair required for ATP coordination.In addition, the essential salt-bridge between R594 andD598 (residue numbering for yeast GCN2) on the dimerinterface (analogous to PKR R262 and D266) was not acces-sible due to a large 13 A separation of side chains. Interest-ingly, GCN2 also contains a pseudokinase domain Nterminal to its protein kinase domain and this domainhas been implicated in the allosteric regulation of kinasecatalytic function in yeast [60]. Whether the pseudokinasedomain can directly engage the functional eIF2a kinasedomain in cis or in trans in an analogous manner proposedfor KSR, remains to be explored.

Stimuli influencing eIF2a kinase dimerization

Under stress conditions, the eIF2a kinases are activated invivo by stimulatory factors that bind to their regulatorydomains, which then promote the active state dimer config-uration of their catalytic kinase domains (Figure 4A). ForPKR, viral infection produces double stranded RNA(dsRNA) that simultaneously binds to the dsRNA bindingdomains of minimally two PKR protomers, thereby promot-ing dimerization concomitant with activation of kinasefunction, inhibition of translation and ultimately the atten-uation of viral propagation. Dimerization of the ER residenttransmembrane receptor PERK, by contrast, is driven bythe accumulation of misfolded proteins in the endoplasmicreticulum (ER). Under normal conditions, binding immuno-globulin protein (BiP)-like chaperones prevent PERK di-merization by binding to the IRE1-like ER luminal domain.The accumulation of unfolded proteins is thought to promoteluminal domain dimerization by direct engagement or byindirect titration of BiP from PERK [61]. This, in turn,promotes dimerization and activation of the cytoplasmickinase domain to retard protein translation and reducethe protein synthetic load on the ER. IRE1, which as noted,shares a similar luminal domain as PERK and functions inparallel to PERK to regulate the unfolded protein response(UPR), is regulated by a common mechanism [57]. In thecase of IRE1 however, dimerization-induced activation ofits RNase activity enables the non-conventional splicing ofthe transcription factor XBP1, which in turn acts as a mastertranscriptional regulator of the UPR (Figure 5A, right). Inparallel to PKR, a pseudo-kinase paralogue of IRE1 calledRNaseL is activated by 2,050-oligoadenylate (2–5A) second-ary messengers produced during viral replicative stress.

484

2–5A directly binds to the ankyrin (ANK) domain ofRNaseL to drive dimerization [31], and subsequent activa-tion of its RNase activity (Figure 5A, right). ActivatedRNaseL then cleaves messenger and structural RNAsnon-specifically to signal an antiviral response that limitsviral replication [62]. Thus, in a parallel twist of kinasesubfamily co-evolution, PERK and IRE1 function in theUPR while PKR and RNaseL function in innate immunity.

Activation of GCN2 is achieved by binding to unchargedtRNAs through its HisRS domain C terminal to the kinasedomain (Figure 4A). How precisely this occurs at a struc-tural level awaits further investigation. For its part, HRI isactivated by a shortage of heme, which normally seques-ters the kinase domain in an auto-inhibited state(Figure 4A). By analogy to GCN2, a shortage of hememay cause a transition of HRI protomers from a non-productive dimer to a productive dimer configuration.

Finally, PknB, a structural homologue of PKR and amember of the eukaryotic-like protein serine/threonine ki-nase (PSTK) family of proteins in bacteria, likely dimerize inthe plane of the plasma membrane in response to binding topeptidoglycans via their penicillin-binding protein and bacte-rial serine/threonine kinase associated (PASTA) sensordomains [63] (Figure 5A, left). Therefore, regulated dimeriza-tion is a conserved phenomenon that can be traced evolution-arily to some of the earliest protein kinases in prokaryotes.

eIF2a kinases are emerging as targets for therapeuticintervention in cancer, neurodegeneration, and diabetes[64–66]. Given that dimerization is allosterically linked tothe kinase active site, the design of small molecules as ATPcompetitive inhibitors may require careful examinationfor unanticipated trans-activation effects that afflictRAF family kinase inhibitors. However, unlike the RAFfamily kinases, there is no report that eIF2a kinases formheterotypic interactions that can signal in vivo.

Concluding remarksDimerization is a recurring theme in the regulation ofprotein kinase catalytic function across the kinome(Figure 2A). Interestingly, despite the diversity of mecha-nisms and interfaces employed to modulate kinase func-tion, a common feature appears to involve the use of asignal-detecting domain controlling an allosteric dimeriza-tion surface embedded within the kinase domain: theremodeling of sensor domains upon interaction with stim-ulating cues triggers the precise configuration of kinasedomain dimers yielding productive conformations of thekinase domain (Table 1) to achieve catalytic activation.Evolutionarily, this convergent approach to detect andtransmit incoming signals suggests a shared principleguiding the exploitation and adaptation of protein kinasesas biological sensors. As seen for RAF and eIF2a familykinases, high-resolution structural data suggests tremen-dous conformational variation in the ability of the dimer-ization interface to allosterically regulate the kinase activesite, primarily through the repositioning of helix aC into aproductive conformation conducive to the alignment of thehydrophobic spines and kinase domain closure. It wouldnot be surprising if additional kinase families exploit asimilar, evolutionarily conserved mechanism for regulat-ing their catalytic outputs.

Review Trends in Biochemical Sciences October 2014, Vol. 39, No. 10

An understanding of allosteric regulation through di-merization has also brought a new dimension to the engi-neering of kinase inhibitors. Because dimerization isallosterically linked to the catalytic properties of the kinaseactive site, it is not unexpected that the converse would betrue, and hence that ATP competitive kinase inhibitorscould promote dimerization and thus cause unanticipatedtransactivation of the kinase dimerization partner. As aresult, future efforts at drug design for dimer-regulatedkinases should heed the lessons learned from the RAFfamily kinases in an attempt to prevent dimerization-in-duced effects. The recent observation that Janus kinases(JAK) undergo homo- and heterodimer formation [67],which in turn appear to modulate the cellular resistanceto JAK ATP-competitive inhibitors, echoes this warning[68]. To surmount the pitfalls of unintended pathwayactivation and resistance mechanisms, it may proveuseful to target binding sites remote from the kinase cata-lytic cleft. Peptide inhibitors targeting the dimerizationinterface recently provided an elegant proof-of-conceptalong this line of inquiry [46]. Alternatively, it may bepossible to engineer small-molecule inhibitors thatexploit and stabilize inactive, monomeric conformationsof the kinase domain and thereby prevent dimerization.

References1 Miele, A.E. et al. (2013) Hemoglobin allostery: new views on old players.

J. Mol. Biol. 425, 1515–15262 Perutz, M.F. (1970) Stereochemistry of cooperative effects in

haemoglobin. Nature 228, 726–7393 Changeux, J-P. (2012) Allostery and the Monod–Wyman–Changeux

model after 50 years. Annu. Rev. Biophys. 41, 103–1334 Manning, G. et al. (2002) Evolution of protein kinase signaling from

yeast to man. Trends Biochem. Sci. 27, 514–5205 Hunter, T. (1991) Protein kinase classification. Meth. Enzymol. 200, 3–376 Nolen, B. et al. (2004) Regulation of protein kinases controlling activity

through activation segment conformation. Mol. Cell 15, 661–6757 Kornev, A.P. and Taylor, S.S. (2010) Defining the conserved internal

architecture of a protein kinase. Biochim. Biophys. Acta 1804, 440–4448 Eyck and Ten, L.F. et al. (2008) Conserved spatial patterns across the

protein kinase family. Biochim. Biophys. Acta 1784, 238–2439 De Bondt, H.L. et al. (1993) Crystal structure of cyclin-dependent

kinase 2. Nature 363, 595–60210 Jeffrey, P.D. et al. (1995) Mechanism of CDK activation revealed by the

structure of a cyclinA–CDK2 complex. Nature 376, 313–32011 Endicott, J.A. et al. (2012) The structural basis for control of eukaryotic

protein kinases. Annu. Rev. Biochem. 81, 587–61312 Sicheri, F. et al. (1997) Crystal structure of the Src family tyrosine

kinase Hck. Nature 385, 602–60913 Xu, W. et al. (1997) Three-dimensional structure of the tyrosine kinase

c-Src. Nature 385, 595–60214 Filippakopoulos, P. et al. (2008) Structural coupling of SH2-kinase

domains links Fes and Abl substrate recognition and kinase activation.Cell 134, 793–803

15 Yan, Q. et al. (2013) Structural basis for activation of ZAP-70 byphosphorylation of the SH2-kinase linker. Mol. Cell. Biol. 33, 2188–2201

16 Pellicena, P. and Kuriyan, J. (2006) Protein–protein interactions in theallosteric regulation of protein kinases. Curr. Opin. Struct. Biol. 16,702–709

17 Endres, N.F. et al. (2011) Regulation of the catalytic activity of the EGFreceptor. Curr. Opin. Struct. Biol. 21, 777–784

18 Jura, N. et al. (2011) Catalytic control in the EGF receptor and itsconnection to general kinase regulatory mechanisms. Mol. Cell 42, 9–22

19 Udell, C.M. et al. (2010) Mechanistic principles of RAF kinasesignaling. Cell. Mol. Life Sci. 68, 553–565

20 Baljuls, A. et al. (2013) It takes two to tango – signalling by dimeric Rafkinases. Mol. Biosyst. 9, 551–558

21 Forbes, S.A. et al. (2011) COSMIC: mining complete cancer genomes inthe catalogue of somatic mutations in cancer. Nucleic Acids Res. 39,D945–D950

22 Roskoski, R. (2012) ERK1/2 MAP kinases: structure, function, andregulation. Pharmacol. Res. http://dx.doi.org/10.1016/j.phrs.2012.04.005

23 Sundaram, M. and Han, M. (1995) The C. elegans ksr-1 gene encodes anovel Raf-related kinase involved in Ras-mediated signal transduction.Cell 83, 889–901

24 Douziech, M. et al. (2006) A KSR/CNK complex mediated by HYP, anovel SAM domain-containing protein, regulates RAS-dependent RAFactivation in Drosophila. Genes Dev. 20, 807–819

25 Rajakulendran, T. et al. (2009) A dimerization-dependent mechanismdrives RAF catalytic activation. Nature 461, 542–545

26 Cheng, S. and Niv, M.Y. (2010) Molecular dynamics simulations andelastic network analysis of protein kinase B (Akt/PKB) inactivation.J. Chem. Inf. Model. 50, 1602–1610

27 Brennan, D.F. et al. (2011) A Raf-induced allosteric transition of KSRstimulates phosphorylation of MEK. Nature 472, 366–369

28 Zeqiraj, E. and van Aalten, D.M.F. (2010) Pseudokinases-remnants ofevolution or key allosteric regulators? Curr. Opin. Struct. Biol. 20, 772–781

29 Lavoie, H. et al. (2013) Inhibitors that stabilize a closed RAF kinasedomain conformation induce dimerization. Nat. Chem. Biol. 9, 428–436

30 Baselga, J. and Swain, S.M. (2009) Novel anticancer targets: revisitingERBB2 and discovering ERBB3. Nat. Rev. Cancer 9, 463–475

31 Huang, H. et al. (2014) Dimeric structure of pseudokinase RNase Lbound to 2-5A reveals a basis for interferon-induced antiviral activity.Mol. Cell 53, 221–234

32 Han, Y. et al. (2014) Structure of human RNase L reveals the basis forregulated RNA decay in the IFN response. Science 343, 1244–1248

33 Mineo, C. et al. (1997) Physical association with ras enhancesactivation of membrane-bound raf (RafCAAX). J. Biol. Chem. 272,10345–10348

34 Chong, H. and Guan, K-L. (2003) Regulation of Raf throughphosphorylation and N terminus–C terminus interaction. J. Biol.Chem. 278, 36269–36276

35 Tran, N.H. et al. (2005) B-Raf and Raf-1 are regulated by distinctautoregulatory mechanisms. J. Biol. Chem. 280, 16244–16253

36 Plowman, S.J. et al. (2005) H-ras, K-ras, and inner plasma membraneraft proteins operate in nanoclusters with differential dependence onthe actin cytoskeleton. Proc. Natl. Acad. Sci. U.S.A. 102, 15500–15505

37 Dementiev, A. (2012) K-Ras4B lipoprotein synthesis: biochemicalcharacterization, functional properties, and dimer formation. ProteinExpr. Purif. http://dx.doi.org/10.1016/j.pep.2012.04.021

38 Gu ldenhaupt, J. et al. (2012) N-Ras forms dimers at POPC membranes.Biophys. J. 103, 1585–1593

39 Lin, W-C. et al. (2014) H-Ras forms dimers on membrane surfaces via aprotein–protein interface. Proc. Natl. Acad. Sci. U.S.A. 111, 2996–3001

40 Baska, F. et al. (2014) Pharmacophore and binding analysis of knownand novel B-RAF kinase inhibitors. Curr. Med. Chem. 21, 1938–1955

41 Hatzivassiliou, G. et al. (2010) RAF inhibitors prime wild-type RAF toactivate the MAPK pathway and enhance growth. Nature 464, 431–435

42 Poulikakos, P.I. et al. (2010) RAF inhibitors transactivate RAF dimersand ERK signalling in cells with wild-type BRAF. Nature 464, 427–430

43 Gibney, G.T. et al. (2013) Paradoxical oncogenesis – the long-termeffects of BRAF inhibition in melanoma. Nat. Rev. Clin. Oncol. 10,390–399

44 Holderfield, M. et al. (2014) Targeting RAF kinases for cancer therapy:BRAF-mutated melanoma and beyond. Nat. Rev. Cancer 14, 455–467

45 Macdonald-Obermann, J.L. et al. (2013) Dynamic analysis of theepidermal growth factor (EGF) receptor–ErbB2–ErbB3 proteinnetwork by luciferase fragment complementation imaging. J. Biol.Chem. 288, 30773–30784

46 Freeman, A.K. et al. (2013) Effects of Raf dimerization and itsinhibition on normal and disease-associated Raf signaling. Mol. Cell49, 751–758

47 Le, K. et al. (2013) Selective RAF inhibitor impairs ERK1/2phosphorylation and growth in mutant NRAS, vemurafenib-resistant melanoma cells. Pigment Cell Melanoma Res. 26, 509–517

48 Wek, R.C. et al. (2006) Coping with stress: eIF2 kinases andtranslational control. Biochem. Soc. Trans. 34, 7–11

49 Dey, M. et al. (2007) Conserved intermolecular salt bridge required foractivation of protein kinases PKR, GCN2, and PERK. J. Biol. Chem.282, 6653–6660

485

Review Trends in Biochemical Sciences October 2014, Vol. 39, No. 10

50 Dever, T.E. (2002) Gene-specific regulation by general translationfactors. Cell 108, 545–556

51 Ung, T.L. et al. (2001) Heterologous dimerization domains functionallysubstitute for the double-stranded RNA binding domains of the kinasePKR. EMBO J. 20, 3728–3737

52 Dar, A.C. et al. (2005) Higher-order substrate recognition ofeIF2alpha by the RNA-dependent protein kinase PKR. Cell 122,887–900

53 Dey, M. et al. (2005) Mechanistic link between PKR dimerization,autophosphorylation, and eIF2alpha substrate recognition. Cell 122,901–913

54 Cui, W. et al. (2011) The structure of the PERK kinase domain suggeststhe mechanism for its activation. Acta Crystallogr. D: Biol. Crystallogr.67, 423–428

55 Ortiz-Lombardıa, M. et al. (2003) Crystal structure of the catalyticdomain of the PknB serine/threonine kinase from Mycobacteriumtuberculosis. J. Biol. Chem. 278, 13094–13100

56 Greenstein, A.E. et al. (2007) Allosteric activation by dimerization ofthe PknD receptor Ser/Thr protein kinase from Mycobacteriumtuberculosis. J. Biol. Chem. 282, 11427–11435

57 Lee, K.P.K. et al. (2008) Structure of the dual enzyme Ire1 reveals thebasis for catalysis and regulation in nonconventional RNA splicing.Cell 132, 89–100

58 Korennykh, A.V. et al. (2009) The unfolded protein response signalsthrough high-order assembly of Ire1. Nature 457, 687–693

486

59 Padyana, A.K. et al. (2005) Structural basis for autoinhibition andmutational activation of eukaryotic initiation factor 2alpha proteinkinase GCN2. J. Biol. Chem. 280, 29289–29299

60 Lageix, S. et al. (2014) Enhanced interaction between pseudokinaseand kinase domains in Gcn2 stimulates eIF2a phosphorylation instarved cells. PLoS Genet. 10, e1004326

61 Wiseman, R.L. et al. (2010) SnapShot: the unfolded protein response.Cell 140, 590–590.e2

62 Chakrabarti, A. et al. (2011) New insights into the role of RNase L ininnate immunity. J. Interferon Cytokine Res. 31, 49–57

63 Barthe, P. et al. (2010) The structure of PknB extracellular PASTAdomain from Mycobacterium tuberculosis suggests a ligand-dependentkinase activation. Structure 18, 606–615

64 Ma, T. et al. (2013) Suppression of eIF2a kinases alleviates Alzheimer’sdisease-relatedplasticityandmemorydeficits.Nat.Neurosci.16,1299–1305

65 Nakamura, T. et al. (2010) Double-stranded RNA-dependent proteinkinase links pathogen sensing with stress and metabolic homeostasis.Cell 140, 338–348

66 Atkins, C. et al. (2013) Characterization of a novel PERK kinaseinhibitor with antitumor and antiangiogenic activity. Cancer Res.73, 1993–2002

67 Brooks, A.J. et al. (2014) Mechanism of activation of protein kinaseJAK2 by the growth hormone receptor. Science 344, 1249783

68 Koppikar, P. et al. (2012) Heterodimeric JAK-STAT activation as amechanism ofpersistence toJAK2 inhibitor therapy. Nature 489, 155–159