How Do Protein Kinases Take a Selfie (Autophosphorylate)?

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Review

How Do Protein Kinases Take a Selfie (Autophosphorylate)? Jonah Beenstock,1 Navit Mooshayef,1,3 and David Engelberg1,2,3,* Eukaryotic protein kinases (EPKs) control most biological processes and play central roles in many human diseases. To become catalytically active, EPKs undergo conversion from an inactive to an active conformation, an event that depends upon phosphorylation of their activation loop. Intriguingly, EPKs can use their own catalytic activity to achieve this critical phosphorylation. In other words, paradoxically, EPKs catalyze autophosphorylation when supposedly in their inactive state. This indicates the existence of another important conformation that specifically permits autophosphorylation at the activation loop, which in turn imposes adoption of the active conformation. This can be considered a prone-to-autophosphorylate conformation. Recent findings suggest that in prone-to-autophosphorylate conformations catalytic motifs are aligned allosterically, by dimerization or by regulators, and support autophosphorylation in cis or trans.

Trends A profound step in the activation of eukaryotic protein kinases (EPKs) is self-activation by autophosphorylation of their activation loop. This autophosphorylation is believed now to be shared by almost all EPKs. Activation-loop phosphorylation induces conversion from an inactive to an active conformation. Intriguingly, EPKs catalyze this reaction when not in their active state. The autophosphorylation reaction must share structural features with the substrate phosphorylation reaction. It is not clear how it utilizes the ATP-binding and catalytic sites.

Autophosphorylation Is a Fundamental Reaction in Eukaryotic Cells Phosphorylation is probably the most common post-translational modification of proteins in eukaryotic cells [1]. Phosphorylation influences the activity of a large portion of the proteome, therefore eukaryotic protein kinases (EPKs; see Glossary), the family of enzymes that catalyze the phosphorylation reaction, are tightly regulated. Although EPKs are regulated in a variety of ways, a major regulatory mode is by phosphorylation, the same modification they catalyze. One critical regulatory phosphorylation event is common to most EPKs, phosphorylation that occurs at kinases activation loop, which is part of the activation segment (Box 1) [2,3]. Phosphorylation of the activation-loop phosphorylation site is a major mechanism that induces the dynamic assembly of the regulatory spine (for elaboration on the catalytic and regulatory spines of EPKs, see [4–8]). Assembly of the regulatory spine is part of the activation process and is accompanied with well-understood structural changes. These include a conformational change of the DFG motif (DFG ‘in’; Box 1), rotation of the /C helix (/C ‘in’) that enables the formation of a Glu–Lys salt bridge, and a domain closure between the N and C lobes that compose the canonical kinase fold. These events collectively stabilize the active conformation that catalyzes the phosphotransfer reaction of a g-phosphate of an ATP molecule to the phosphoacceptor site of the substrate [3,5,9]. Activation-loop phosphorylation is therefore crucial because it is required for the interconversion from an inactive to an active conformation. Although for some EPKs additional steps are required for full activity and in some cases the dependence on activation-loop phosphorylation for activation can be bypassed by other mechanisms, in most EPKs, the absence of activationloop phosphorylation leaves EPKs catalytically impotent. Remarkably, activation-loop phosphorylation is frequently generated by autophosphorylation, implying that EPKs are not only activated by the modification they catalyze but are also selfactivating enzymes. As EPKs are involved in controlling all physiological and pathological processes of life, their autoactivation via autophosphorylation at the activation loop is a fundamental

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The prone-to-autophosphorylate conformation is stabilized allosterically by dimerization or via association with auxiliary proteins. The dimers’ organization and the reaction mechanism (cis or trans) are kinase specific. In mitogen-activated protein kinases (MAPKs), autophosphorylation regulation is very tight and occurs via unique mechanisms and structural motifs, specific to each MAPK.

1

Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel 2 CREATE-NUS-HUJ Cellular & Molecular Mechanisms of Inflammation Program, National University of Singapore, 1 CREATE WAY, Innovation Wing, Singapore, Singapore 3 Department of Microbiology, Yong loo lin School of Medicine, National University of Singapore, Singapore, Singapore

http://dx.doi.org/10.1016/j.tibs.2016.08.006 © 2016 Elsevier Ltd. All rights reserved.

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Box 1. Functional Elements of Eukaryotic Protein Kinases (i) Kinase fold: A structurally conserved core of approximately 250 residues found in all eukaryotic protein kinases (EPKs). Composed of a smaller N lobe and a larger C lobe connected by a flexible hinge. The lobes form a deep cleft that accommodates the ATP adenine base (Figure I). (ii) Activation segment: A 20–30-amino acid long region (the length depends on the EPK) located between the N and C lobes. Bordered by two conserved tripeptide motifs: DFG and APE. This segment is composed of several conserved functional elements:  Asp–Phe–Gly (DFG) tripeptide motif: The N-terminal border of the activation segment. The Asp residue chelates Mg2+ and is required for binding the phosphates of ATP. Undergoes a DFG out/DFG in conversion upon activation, dynamically reorienting the Phe into the regulatory spine.  Activation loop: A flexible region that is mostly disordered in inactive kinases. Most kinases harbor at least one phosphorylation site at the activation loop. When this site is phosphorylated the activation loop is stabilized and imposes a conformational change on the entire protein that promotes activity.  P + 1 loop: A key contact site between the substrate and the kinase. Accommodates the residue located one position C terminal to the phosphorylation site (the P + 1 residue) of the substrate. A major determinant of selectivity.  Alanine–proline–glutamic acid (APE) motif: The C-terminal border of the activation segment and an anchor of this segment to the kinase domain.

N lobe

(iii) The /C helix Glu and the b3-sheet Lys salt bridge: The /C helix and b3 sheet are two structural elements of kinases’ N lobe. The b3 sheet contains an invariant Lys residue that binds the ATP phosphates. This Lys is positioned by the invariant /C-helix Glu site in a favorable conformation for phosphate binding. The conformation of the /C-helix Glu is determined by rotation of the /C helix to a favorable position. Such rotations are induced by activation-loop phosphorylation or by binding of allosteric activators to this helix. (iv) Hydrophobic spines: Two ensembles of hydrophobic residues that traverse the kinase domain. The catalytic spine is mostly assembled even in inactive kinases and is completed with the adenine ring of the bound ATP molecule and positions g-phosphate for the phosphotransfer reaction. The regulatory spine is dynamically assembled in kinases adopting their active conformation and discontinuous in the inactive conformation. (v) Catalytic loop: A structural element that contains the catalytic Asp residue, required for activating the hydroxyl of the phosphoacceptor residue of the substrate by making it a nucleophile for the phosphotransfer reaction. In kinases that are regulated by activation-loop phosphorylation, this Asp is frequently preceded by an Arg residue (RD kinases). (vi) Kinase active conformation: Once phosphorylated, the activation-loop site forms a network of interactions with positive residues of the N-terminal part of the /C helix and the catalytic loop, coordinating conformational changes of the DFG motif (‘DFG in’), the /C helix (‘/C in’), the formation of the Glu–Lys salt bridge, and a domain closure, enabling catalytic activity.

Gly-rich loop

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DFG mof

DFG mof

pThr

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Acvaon loop

ATP

2+

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Catalyc Asp RD mof

Acvaon loop Substrate phosphorylaon site

Figure I. Activation-Loop Phosphorylation Stabilizes the Active Conformation of Protein Kinases. Activationloop phosphorylation aligns the functional elements of protein kinases required for their activity. These are shown in the structure of phosphorylated and active p38/ (PDB 3PY3). The kinases fold is composed of a bilobal core with the active site and the ATP-binding pocket located in a cleft between these lobes. The phosphorylated activation-loop site forms interactions with multiple regions, including with an Arg located in the catalytic loop and with N-terminal residues of the /C helix. This aligns the active-site DFG motif[8_TD$IF] and the Glu–Lys salt bridge in conformations that support catalysis.

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*Correspondence: [email protected], [email protected] (D. Engelberg).

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and essential reaction. Moreover, recent studies have suggested that activation-loop autophosphorylation is far more common in EPKs than previously appreciated. EPKs such as mitogenactivated protein kinases (MAPKs), which until recently were not considered to be activated by autophosphorylation, have been found to do so efficiently [10–15]. In other words, although evolution provided some kinases with specific upstream kinases that phosphorylate their activation-loop sites, many of these kinases preserved inherent autophosphorylation capability that seems physiologically relevant. Thus, autoactivation appears to be a capability that was preserved in many EPKs, and hence must be critical for their functionality. It was further realized recently that autoactivation targets kinases to particular physiological functions so that this mode of activation is directly associated with cancer, drug resistance, ischemia, and immune responses [16–22]. It is noteworthy that autophosphorylation of other sites, outside the activation loop, is also highly prevalent in kinases. While important, these autophosphorylations do not determine activity and are mechanistically different from activation-loop autophosphorylation (see in the ‘The Enigma of Autophosphorylation’ section). In this review, we discuss only autophosphorylation of activationloop sites, which is crucial for stabilizing the catalytically active conformation. Thus, we henceforth use the term ‘autophosphorylation’ in a narrower sense to refer only to this more specific reaction. Particularly, this review focuses on particular mechanisms through which an ‘inactive’ kinase can autophosphorylate and autoactivate. We also provide a comprehensive view of the kinases within the kinome that autophosphorylate and address the physiological relevance of their autophosphorylation (Figure 1, Table S1).

Autophosphorylation Is a Highly Prevalent Activity amongst EPKs Although activation-loop phosphorylation is a clear hallmark of the active conformation of kinases [3] and although it is well accepted that many EPKs undergo autophosphorylation, the extent of the phenomenon has not been assessed so far. To estimate how many EPKs are regulated by activation-loop phosphorylation, we annotated the human kinome for kinases that contain an arginine–aspartic acid (RD) motif within their catalytic loop (Box 1), using the ProKinO Web server [23]. The presence of an Arg residue before the catalytic Asp (RD) indicates, in most cases, that a kinase depends on activation-loop phosphorylation, because the positively charged side chain of this Arg serves to stabilize the phosphorylated activation-loop site [9,24]. This analysis revealed that 374 kinases, approximately 72% of the kinome, harbor an RD motif (Figure 1A,B). Remarkably, a systematic survey of the literature revealed that 238 of the 374 RD kinases have an intrinsic and unconditioned autophosphorylation activity, that is, an autophosphorylation activity that is independent of an upstream kinase (Figure 1A,C, Table S1). As much as 167 of those have been shown to autophosphorylate on their activation-loop regulatory site (Figure 1A,C, Table S1). Importantly, activation-loop phosphorylation of 94 kinases, including the stress-response eukaryotic translational initiation factor 2/ (eIF2/) kinases, the p21-activated kinases (PAKs), and many tyrosine kinases, seems to depend exclusively on autophosphorylation as no relevant upstream kinases have been identified. Thus, according to our current understanding, approximately 45% of the kinome can autophosphorylate their activation loop, and for approximately 18% of the kinome, activation-loop phosphorylation depends solely on autophosphorylation. Because for 87 RD kinases autophosphorylation has not yet been experimentally tested (Table S1) and for 71 autophosphorylating RD kinases the sites have not been mapped, it is likely that the number of EPKs capable of autophosphorylation is in fact higher. In addition, in some cases, autophosphorylation is not spontaneous and occurs only under specific conditions [see the example of p38 mitogen-activated protein kinase / (p38/) in the ‘Cis-Autophosphorylation of a Monomeric Kinase’ section]. Although a global analysis of EPKs autophosphorylation is not complete and for a number of EPKs the physiological relevance of autophosphorylation as an activating mechanism has

Glossary Activation-loop phosphorylation site: a key regulatory phosphorylation site found in most eukaryotic protein kinases (EPKs) within a region termed the activation loop. Activation-loop phosphorylation has a crucial role in stabilizing the active conformation of many protein kinases and is considered a biochemical marker for activity. Depending on the EPK, the activation-loop site can be phosphorylated either by autophosphorylation, by a regulating upstream kinase, or by both mechanisms. Autophosphorylation: phosphotransfer activity of eukaryotic protein kinases (EPKs), in which the receiving residue resides within the catalyzing EPK itself or within another twin molecule. Autophosphorylation can be an intermolecular (trans) or an intramolecular (cis) reaction. Eukaryotic protein kinases (EPKs): a family of enzymes that catalyze phosphotransfer of the gphosphate group of ATP to serine, threonine, or tyrosine residues of target proteins (substrates). EPKs share 12 conserved subdomains and a conserved 3D fold of the catalytic core, termed the kinase domain. Most EPKs contain additional domains or structural elements, tethered to the kinase domain, which have important roles in their functionality. Kinase dead: a eukaryotic protein kinase engineered to lack catalytic activity but to maintain the overall canonical fold of the kinase domain. This is achieved by mutagenesis of key sites that serve for the phosphotransfer reaction. Kinome: the complement of protein kinases encoded by a genome of an organism. The human kinome, for example, is composed of 518 eukaryotic protein kinases. The kinome can be cataloged into subgroups based on features found within or outside the kinase domain. Pseudokinase: traditionally defined as a eukaryotic protein kinase that naturally lacks at least one of the functional elements that enable catalytic activity. Recent experimental evidence has shown in a number of cases, for example, the predicted pseudokinases WNK and kinase suppressor of Ras, that kinases missing some canonical catalytic residues do manifest some activity.

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not been established, recent advances have identified autophosphorylation as an activating mechanism for a growing number of EPKs. How these EPKs autophosphorylate and the physiological context of their autophosphorylation will be discussed in this review. Based on these cases and the global survey of the literature (Figure 1 and Table S1), it is clear that autophosphorylation is prevalent amongst EPKs and is a reaction with tremendous physiological importance.

The Enigma of Autophosphorylation Although autophosphorylation and substrate phosphorylation are both phosphotransfer reactions catalyzed by kinase molecules, significant differences between them are evident (Figure 2, Key Figure). Numerous studies have compared the biochemical and structural traits of kinases when phosphorylated or not phosphorylated at their activation loops [9], leading to the currently accepted model that activation-loop phosphorylation coordinates the active conformation. Paradoxically, however, the supposedly inert nonphosphorylated molecules are the ones that manage to catalyze the phosphotransfer reaction on their own activation-loop sites (Figure 2). The phosphotransfer activity of kinases in their nonphosphorylated state is probably exclusive to the innate activation-loop site. Other substrates are not phosphorylated by kinases in this state. In addition, the autophosphorylation reaction, unlike standard substrate phosphorylation, takes place just once in the cycle of activity of the kinase (Figure 2). Once the single substrate (the activation-loop site) has been loaded with a phosphate group, the reaction, obviously, cannot be repeated and the kinase adopts the active conformation. This implies that autophosphorylation cannot be explained by models that describe how phosphorylation of common substrates occurs. Thus, although both the autophosphorylation reaction and the substrate phosphorylation reaction use the single ATP-binding site and the single catalytic site of the kinase molecule, they nevertheless must have distinct structural and mechanistic properties. Two major and obvious obstacles hinder catalysis of autophosphorylation by the inactive conformation. First, the active-site residues are not correctly aligned in the absence of one of their major stabilizing elements. Second, kinases have a clear selectivity toward phosphoacceptors within a consensus sequence (Box 2) [25]. The phosphoacceptor site at the heart of the autophosphorylation reaction, commonly the activation-loop Thr (or Tyr in tyrosine kinases), usually does not reside within such a consensus sequence and is not oriented toward the catalytic Asp. These structural obstructions have to be overcome by a prone-to-autophosphorylate conformation, which is by definition distinct from the two hitherto known major conformations of kinases: the active and inactive conformations [9]. Theoretically, autophosphorylation can occur via several mechanisms (Figure 3). A straightforward mechanism would involve the formation of a homodimer in which one monomer is phosphorylated at its activation loop and is therefore catalytically active, and uses the other monomer (which resides in an inactive conformation) as a substrate. However attractive, this mechanism is not supported experimentally and cannot explain how de novo autoactivation is achieved. Rather, most of the experimentally supported models involve a multistage process that includes dimerization of nonactive monomers, leading, allosterically, to stabilization of a unique conformation that specifically supports trans- or cis-autophosphorylation (Figure 2). In some kinases, similar allosteric effects are imposed by specific proteins and not by another monomer. Finally, some EPKs show cis-autophosphorylation as monomers. Thus, unlike the active conformation, which is similar in most kinases [9], there seems to be a variety of prone-to-autophosphorylate conformations. These are described in detail in the next section.

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Upstream kinase: some eukaryotic protein kinases (EPKs) have been developed in evolution to function as activators of other EPKs, by phosphorylating their activation-loop phosphorylation sites. Therefore, they function ‘upstream’ to their substrate EPKs. This organizes some EPKs in cells into signaling pathways that are composed of cascades of tiered kinases, in which some serve as upstream kinases for others in a hierarchical manner. Frequently, upstream kinases serve as dedicated kinases to their downstream EPKs and have no other substrates. Examples for this are the mitogenactivated protein kinase kinases that activate mitogen-activated protein kinase family members, phosphoinositide-dependent kinase 1 that serves as an upstream kinase for the AGC kinase family, and liver kinase B1 that serves as a master regulator of the AMP-activated protein kinase-related kinases.

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(A)

(B)

RD kinases (374)

Intrinsic and uncondional autophosphorylaon (238)

Proven to occur at AL (167)

AL autophosphorylaon is obligatory for acvaon (94)

STE

70% Mixed (73)

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AGC

No autophosphorylaon Non mapped autophosphorylaon Autophosphorylaon dependent

CAMK

Mixed regulated

Figure 1. Self-Activation Is a Prevalent Phenomenon in Eukaryotic Protein Kinases. (A) The human kinome [77] was divided using the ProKinO Web server [23] into arginine–aspartic acid (RD) and non-RD kinases. The RD kinases were further annotated following a literature curation as follows: light blue, experimentally shown to possess intrinsic and unconditional autophosphorylation activity; blue, have not been shown to autophosphorylate; pale green, autophosphorylation was mapped to activation-loop sites; green, autophosphorylation activity was not mapped to specific sites; red, activation-loop phosphorylation is dependent on autophosphorylation; orange, mixed dependence, namely, dependent on either autophosphorylation or an upstream kinase for activation-loop phosphorylation. (A full list of the distributions of the RD kinases and links to the relevant studies can be found in Table S1.) (B) RD kinases (red dots) are superimposed on the human kinome [77]. (C) A global map of autophosphorylation activity of the RD kinases is superimposed on the human kinome [77]. Categories are color coded as in (A). The illustrations were reproduced courtesy of Cell Signaling Technology, Inc. (www.cellsignal.com). AL, activation loop.

Multiple Mechanisms Generate Prone-to-Autophosphorylate Conformations in Different EPKs Trans-Autophosphorylation in ‘Face-to-Face’ Dimers via Activation-Loop Swapping An appealing structural solution for fulfilling the requirements for autophosphorylation was first described for the DNA-damage response kinase checkpoint kinase 2(Chk2) [26], and later for its yeast ortholog Rad53 [27]. Double-strand breaks in the DNA lead to the phosphorylation of Chk2 on a residue outside the kinase domain in an ataxia telangiectasia mutated (ATM)-dependent

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Key Figure

The Autoactivation Route of Protein Kinases

The autoacvaon route of protein kinases Prone to autophosphorylate conformaon (commonly a dimer)

n o va )  ac on ric iza e t er os All (dim

(si

a ng utop Cis\ l e - h o Tr h i t s p an ph ho sos ryl ph a or on yla o n)

Nonacve conformaon

Acve conformaon

Inacvaon by phosphatases

Substrate phosphorylaon (mulple cycles of phosphorylaon)

Figure 2. For adopting their active conformation (green) many kinases must become phosphorylated at the activation loop. Activation-loop phosphorylation is catalyzed in many cases by nonphosphorylated kinases, although nonphosphorylated kinases are considered to be unactive (red). This implies that an active conformation that is specialized in catalyzing a single autophosphorylation reaction (prone-to-autophosphorylate; yellow) must exist. This conformation, in many cases induced by dimerization, performs only one cycle of activity and is distinct from the bona fide active conformation. Importantly, kinases can also be activated by a heterologous upstream kinase and bypass the prone-to-autophosphorylate conformation to achieve activity (not shown). Unlike the single-hit autophosphorylation reaction catalyzed by the prone-to-autophosphorylate conformation, kinases in the active conformation perform multiple cycles of substrate phosphorylation.

manner (Box 3) [28]. This phosphorylation enhances dimerization and autophosphorylation of Chk2 [26,28]. The 3D structure of Chk2 revealed why dimerization is important for activation. Chk2 kinases crystallized as completely symmetric ‘face-to-face’ homodimers (Figure 4A), in which each monomer exchanges its activation loop with the other, a phenomenon known as ‘3D domain swapping’ [29]. A striking feature of these symmetric dimers is that both monomers gain characteristics of the active conformation, including a Glu–Lys salt bridge, a characteristic of active kinases (Box 1). The active sites within these dimers have only one available substrate, the activation-loop phosphoacceptor of the other monomer. Notably, within the crystal structure, this substrate does not reside in a favorable position for autophosphorylation, namely, the activationloop site of one monomer is not accessible for activation by the catalytic Asp of the other monomer (Figure 4A). However, biophysical analysis showed that Chk2/Rad53 dimerization

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occurs in solution [26,27]. Biochemical analysis using a mixture of kinase dead and catalytically competent Chk2/Rad53 showed that trans-autophosphorylation occurs in solution [26,27] (although one study claims that the autophosphorylation of Chk2 occurs in cis [30]). The physiological role of Chk2 autophosphorylation is emphasized by a disease-causing mutation found in Chk2 in Li–Fraumeni syndrome patients that impairs Chk2 dimerization and activity [28]. These observations combined, plus the fact that to date no Chk2-activating kinase has been identified (Table S1) [28], suggest that Chk2 kinases within the dimer are in a prone-to-autophosphorylate conformation, providing an example for how stabilization of the catalytic and ATPbinding sites can be achieved without activation-loop phosphorylation. The catalytic domains of many other kinases, including STE20-like kinase, lymphocyte-oriented kinase, death-associated protein kinase 3, TANK binding kinase 1 (TBK1), I-kappaB kinase beta, and mammalian STE20like protein kinase 4 [1_TD$IF](MST[12_TD$IF]4), have since been crystallized in a similar dimeric form with exchanged activation loops [31–36]. For some of these kinases, such as TBK1, autophosphorylation seems to be the sole mechanism for activation (Table S1), emphasizing the physiological relevance of this autophosphorylation-promoting conformation. Importantly, dimers with swapped activation loops were also reported for oxidative stress responsive kinase 1, Ste20-related proline alanine-rich kinase, p70S6 Kinase 1 (p70S6K1), and p38/ [37–40], but these structures did not show hallmarks of active conformations. For example, the catalytic loop of p38/ within this dimer is disrupted and does not seem to be in a favorable position for catalysis [37]. Furthermore, p70S6K1 has not been documented to autophosphorylate (Table S1), indicating that [13_TD$IF]swapping [14_TD$IF]of activation loops might[15_TD$IF] also have other roles than promoting autophosphorylation. Trans-Autophosphorylation within ‘Face-to-Face’ Asymmetric Dimers PAK1 also forms face-to-face homodimers, but in these cases they are asymmetric [41]. PAK1 activity controls multiple cellular processes, including the dynamics of the cytoskeleton proteins and the activation of MAPK pathways, and has an important role in human disease [42]. PAK1 is Box 2. How Is Phosphorylation of Nonconsensus Sites Achieved? Kinases in their active conformation show a clear preference to phosphoacceptor sites within linear consensus motifs [84]. Although in some kinases, such as Aurora A (Figure IA), the activation-loop site is within its consensus sequence, in many cases the activation-loop sites, either partially or completely, do not reside within such motifs. Active protein kinase A (PKA), for example, shows a strong selectivity to phosphorylation of substrates with basic amino acids at positions –2 and –3 (Figure [9_TD$IF]IB). Thr197, the activation-loop phosphorylation site, only partially meets these requirements, and has an Arg at position –3. Mutation of this Arg to Ala abolishes autophosphorylation but has no effect on phosphoinositidedependent kinase 1 phosphorylation, showing that this mutation did not disrupt the activation-loop structure completely [85]. In addition, Gly and Thr residues at the P + 3 and P + 4 positions were required for autophosphorylation but do not seem to be required for substrate phosphorylation. Similarly, active p38/ has a clear preference for a Pro residue at the P + 1 position, while its activation-loop autophosphorylation site is followed by a Gly residue, but can be autophosphorylated (Figure [9_TD$IF]IC). Why do kinases frequently have such different specificities in their active conformation and their prone-to-autophosphorylate conformations? While there is currently no full answer to this, a possible resolution to this paradox is that in the prone-to-autophosphorylate conformations, the P + 1 pocket, a major determinant of kinase specificity [3,5,9], acquires specificity toward the activation-loop site. Of note, the conformation of the P + 1 pocket is, in many cases, determined by the phosphorylation of the activation loop (in mitogen-activated protein kinases and PKA, for example, [71,86]). In the prone-to-autophosphorylate conformation, activation-loop phosphorylation is absent and it is likely, therefore, that this pocket will have a different conformation than in the active conformation. Another important difference between substrate and activation-loop phosphorylation, especially evident in cis-autophosphorylating eukaryotic protein kinases, is the availability of the substrate activation-loop site. Because the activation loop is tethered to the kinase, there is a proximity effect that undoubtedly increases the probability for autophosphorylation when the kinase fluctuates between the active and inactive conformations (see the ‘Cis-Autophosphorylation of a Monomeric Kinase’ section). It can be postulated that during the early stages of kinase evolution, the original use of the g-phosphate of ATP was applied for autophosphorylation.

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AurA

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... 172RHTDDEMTGYVATRW188 ... Figure [9_TD$IF]I. Autophosphorylation Can Occur in Nonconsensus Motifs. Consensus phosphoacceptor motifs in substrates compared with the activation-loop sequence that includes the autophosphorylated site (green) of (A) Aurora A, (B) protein kinase A (PKA), and (C) p38/. Consensus phosphoacceptor motifs were illustrated using WebLogo software and adapted from the PhosphoSite Web server (www.phosphosite.org). Activation-loop phosphorylation sites are marked in green.

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Trans-autophosphorylaon between an acve and nonacve kinase (ii)

Symmetric

(iii)

Asymmetric

Dimerizaon-induced Dimerizaon-independent cis-autophosphorylaon cis-autophosphorylaon (iv)

(v)

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Trans-autophosphorylaon in a dimer of nonacve kinases

Nonacve Acve kinase kinase (‘enzyme’) (‘substrate’)

Examples: ?

Scaffold protein, translaonal intermediate

Each monomer is an Allosterically Allosteric ‘enzyme’, a ‘substrate’ acvated acvator and an kinase kinase ‘allosteric acvator’ (‘enzyme’) (‘substrate’)

LOK, Chk2, SLK, DAPK3, TBK1

Aurora A, IRAK4, PAK1

Allosteric Allosterically acvator acvated kinase kinase (‘enzyme’+ ‘substrate’) RAF, PKR

Allosterically acvated kinase (‘enzyme’+ ‘substrate’) GSK3β, DYRK, p38α

Figure 3. Possible Models for the Mechanism of Autophosphorylation. Autophosphorylation can be mediated through a number of possible mechanisms – these are shown with schematic representations of kinase monomers and the direction of the phosphate group (yellow) transfer is shown with arrows. Examples of kinases shown to follow each mechanism are indicated beneath the schemes. From left to right: (i) Trans-autophosphorylation between a phosphorylated and active kinase and a nonactive kinase that functions as a substrate. No experimental evidence points to this mechanism. (ii) Symmetric trans-autophosphorylation. Each monomer phosphorylates and activates the other. Examples: LOK (PDB 2J7T), Chk2 (PDB 2CN5), SLK (PDB 2J51), [7_TD$IF]DAPK3[1_TD$IF] (PDB 2J90), and TBK1 (PDB 4EUT). (iii) Asymmetric trans-autophosphorylation. One monomer serves as an ‘enzyme’ and phosphorylates the other. The substrate monomer extends its activation loop into the active site of the ‘enzyme’ monomer. This induces an active conformation on the ‘enzyme’ monomer. Examples: Aurora A (PDB 4C3P), IRAK4 (PDB 4U9A), and PAK1 (PDB 3Q4Z). (iv) Dimerization-induced cis-autophosphorylation. Protein kinase dimers with active sites not facing the dimeric interface; each monomer induces cisautophosphorylation of the other monomer in a mechanism that can either be simultaneous (symmetric) or nonsimultaneous (asymmetric). Examples: RAF (PDB IUWH) and PKR (PDB 2A1A). (v) Dimerization-independent cis-autophosphorylation. Autophosphorylation occurs in cis. This is either a property of a translational intermediate of a kinase (GSK3b and DYRK1A, for example) or as a result of an induction by interaction with another protein (p38/ and PDB 4LOQ, for example). Chk2, checkpoint kinase 2; DYRK1A, dual-specificity tyrosine-regulated kinase 1A; GSK3b, glycogen synthase kinase 3b; IRAK4, interleukin-1 receptor-associated kinase 4; LOK, lymphocyte-oriented kinase; PAK1, p21-activated kinase 1; PKR, protein kinase R; SLK, STE20-like kinase; TBK1, TANK binding kinase 1.

regulated by a multitude of mechanisms (Box 3), and requires activation-loop phosphorylation to achieve full activity [42]. Because no upstream kinase has been identified for PAK1 (Table S1), autophosphorylation is crucial for achieving its active conformation and is central for many biological processes. PAK1 is crystallized as a dimer, and although the monomers face each other, each one displays a very different conformation [41] (Figure 4B). Specifically, one monomer exhibits a closed activation-loop conformation that resembles an active kinase. It is likely, therefore, that this monomer serves as an ‘enzyme’ within this dimer. By contrast, the activation loop of the other monomer is extended and protrudes into the active site of the opposite monomer. The activation-loop Thr residue of the ‘substrate’ monomer forms a hydrogen bond with the catalytic Asp of the other monomer, positioning this Thr in a favorable position for phosphorylation [41,43]. This monomer serves, most probably, simultaneously as a substrate and as an allosteric activator of its partner. The kinase domains of Aurora A and interleukin-1 receptor-associated kinase 4 (IRAK4) have also been found to crystallize as asymmetric ‘face-to-face’ dimers that seem to facilitate autophosphorylation [44,45]. In the IRAK4 dimer, for example, the putative ‘enzyme’ monomer resembles the conformation of phosphorylated and active IRAK4 [44,46], while the ‘substrate’ monomer has an extended activation-loop conformation and its Thr phosphorylation site engages the active site of the other

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Box 3. How Is the Autophosphorylation Reaction Regulated? With the exception of only a handful of eukaryotic protein kinases (EPKs; e.g., mitogen-activated protein kinases), when tested as purified proteins in vitro, most kinase domains self-activate spontaneously via autophosphorylation. In vivo, however, autophosphorylation is tightly controlled by a number of distinct mechanisms that enable this to occur in the correct physiological context. Because dimerization is an important event in the allosteric control of the prone-toautophosphorylate conformations, the local concentration of EPKs is frequently tightly controlled, influencing the probability of dimerization and self-activation. Chk2 kinases, for example, are composed of three functional domains, the SCD, FHA, and kinase domains. The kinase domain of Chk2 has a low affinity for dimerization. This is sufficient for dimerization and autophosphorylation when expressed in Escherichia coli or overexpressed in cell culture [28]. Upon DNA damage, Chk2 is phosphorylated at Thr68, which is located within the SCD domain in an ataxia telangiectasia mutated-dependent manner [28]. Phospho-Thr68 binds to the FHA domain of another monomer, increasing the avidity of Chk2 kinases to dimerize and subsequently to autophosphorylate [28]. In a similar manner, the ability of RAF kinases to dimerize and autoactivate is enhanced by recruitment to GTP-bound Ras through an N-terminal Ras-binding domain. Interleukin-1 receptor-associated kinase 4 dimerization is controlled by Myd88 oligomerization at the myddosome [44]. The kinase domains of protein kinase R [59] and receptor tyrosine kinases [47] are conjugated to receptor domains that dimerize upon ligand binding. Autophosphorylation can also be regulated by inhibitory elements that prevent it, which can be either inherent elements associated with the kinase domain or regulatory proteins that, upon dissociation from the kinase, enable autophosphorylation. For example, p21-activated kinase 1–3 (PAK1–3), which are considered group 1 PAKs [42], contain an inhibitory domain that prevents autophosphorylation [42,87]. Partial proteolysis of the inhibitory domain or binding of small GTPases, such as Rac1, to a specialized binding domain releases autoinhibition [87] and enables activation-loop autophosphorylation [41] as well as additional autophosphorylations that collectively stabilize the active conformation [87]. The autophosphorylation of p38/ is inhibited by a C-terminal extension termed the L16/L16 helix. This element is tethered to the kinase domain by a core of hydrophobic residues [79]. Disruption of this core, either by mutations [75,80] or by phosphorylation of Tyr323 [14], which is part of this core, induces autophosphorylation most probably in cis [74]. Chk2, checkpoint kinase 2.

monomer. The structures of PAK1, Aurora A, and IRAK4 are most probably the best representation of a bona fide prone-to-autophosphorylate conformation as the ‘enzyme’ monomer shows clear characteristics of the active conformation and the phosphorylation site of the ‘substrate’ monomer engages its active site (for exact measurements of distances between the phosphorylation site and catalytic Asp in autophosphorylation complexes, please see [43]). Kinases of the epidermal growth factor receptor (EGFR) family also employ autoactivation imposed by allosteric interactions between two monomers that form asymmetric dimers [47]. The kinase domains of these receptors form head-to-tail dimers in which the C lobe of the ‘substrate’ monomer associates with the N lobe of the ‘enzyme’ monomer and stabilizes its active conformation. Stabilization of the active conformation of EGFR via dimerization and allostery is efficient enough to render the activation-loop phosphorylation dispensable [47]. Autophosphorylation within Dimers That Are Not ‘Face-to-Face’ The role of dimerization in autophosphorylation mechanisms involving trans-autophosphorylation can be understood intuitively. Intriguingly, dimerization also frequently plays a role when autophosphorylation occurs in cis. Important examples are the proto-oncoprotein B-RAF and the stress-response eIF2/ kinase protein kinase R (PKR). B-RAF is part of the Ras-MAPK pathway, critical for mitogenic growth-factor signaling. B-RAF is frequently mutated in human cancers, mainly in melanoma, and has been shown to self-activate via homodimerization or heterodimerization with other RAF proteins or with the closely related kinase suppressor of Ras pseudokinases [18,20,21,48–51]. B-RAF homodimers are best described as ‘side-by-side’ dimers, in which the activation loop of each monomer faces a different way (Figure 4C). B-RAF dimers, which form also depending on regions that are not part of the putative kinase domain (Box 3) [48,52], involve an elaborate interaction between the N lobes of the kinases (Figure 4C) [18,21,52]. Most notably, this interface includes many regions in the vicinity of the /C helix, inducing its conformational change [21,53,54]. Conformational

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(A)

Monomer A

Monomer B

Monomer B (‘substrate’)

(B) Monomer A (‘enzyme’)

KE salt bridge ATP Catalyc D

Thr phos’ site

AL monomer B

AL monomer B Thr phos’ site

(C) Monomer B

Monomer A AL phos’ site

Catalyc D

Figure 4. Dimerization-Induced Autophosphorylation Can Occur via Several Mechanisms. (A) Autophosphorylation mediated by activation-loop swapping within a symmetric dimer. Example: a [7_TD$IF]DAPK3[1_TD$IF] dimer (PDB 2J90). The activation loop (dark blue) of monomer B (pale blue) protrudes into the active site of monomer A (wheat). Correspondingly, the activation loop of monomer A (dark red) symmetrically protrudes into the active site of monomer B. Monomer A is considered active due to the Lys–Glu salt bridge (colored black). The activation-loop Thr site of monomer B (colored black) is shown to be in the proximity of the catalytic Asp, although not positioned well for catalysis. (B) Autophosphorylation mediated by a face-to-face asymmetric dimer. Example: an asymmetric PAK1 dimer (PDB 3Q4Z). The activation loop (dark blue) of monomer B (pale blue) protrudes into the active site of this monomer A (wheat). The activation loop of monomer A (dark red) does not protrude the active site of monomer B in the same manner. Notably, due to point mutations, the catalytic Asp and the Lys–Glu salt bridge are not shown. (C) Cis-autophosphorylation mediated by a side-to-side asymmetric dimer. Example: an asymmetric B-RAF dimer (PDB IUWH) in which the activation loop (dark blue) of monomer B (pale blue) does not face the active site of monomer A (wheat). Note the proximity of the activation-loop phosphorylation sites to the catalytic Asp. AL, activation loop; PAK1, p21-activated kinase 1.

changes of the /C helix are important steps in the activation of protein kinases [5,8]. The /C helix of monomeric nonactive B-RAF is held by a short helix found in the activation segment in an ‘open’ conformation that does not support ATP binding and the integrity of the regulatory spine [54]. The inhibitory activation-segment helix is destabilized upon dimerization, enabling a rotation of the /C helix and acquisition of a prone-to-autophosphorylate conformation, as indicated by the integrity of the regulatory spine [48,54]. A bona fide active conformation is stabilized only after activation-loop cis-autophosphorylation [54]. Interestingly, B-RAF can induce c-RAF cis-autophosphorylation via heterodimerization, even as a kinase-dead protein [48]. These observations, combined with recent reports of pseudokinases that induce an active conformation with their kinase-binding partners [55–57], raise a new and interesting role of kinases that has so far been overlooked, as allosteric activators of other kinases [56]. Thus, reassessment of the autophosphorylation mechanism of kinases that were assumed to autophosphorylate in trans by the virtue of their dimerization is needed. Finally, a recent study has shown that mice knocked-in with a BRAF mutant that cannot be phosphorylated at the activation loop display just mild developmental defects compared with the B-RAF knockout mice that are not viable [58], indicating that

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perhaps, like the EGFRs [47], for B-RAF dimerization by itself is sufficient for executing most of its physiological activity [58]. PKR, an eIF2/ kinase activated by double-stranded RNA and an important mediator of the cellular response to viral infection, is also activated by sequential dimerization and cisautophosphorylation[3_TD$IF] [59,60]. The geometry of the PKR dimer shows a ‘back-to-back’ organization. Thus, it could be assumed that the interface formed between the monomers induces an allosteric effect that positions the catalytic residues in the correct orientation for autoactivation. Biochemical analysis has shown that other eIF2/ kinases [general control nonderepressible 2 kinase, protein kinase RNA-like endoplasmic reticulum kinase (PERK), and hemeregulated eIF2/ kinase] self-activate by a similar dimerization mode, demonstrating that this mechanism has been conserved in the course of evolution [61]. ‘Back-to-back’ dimers of Nek7 [62] have also been proposed to promote trans-autophosphorylation in a similar way. Interestingly, a ‘back-to-back’ dimeric assembly of the endoplasmic reticulum stress-activated EPKs, namely, PERK and Ire1, has been proposed to render activation by trans-autophosphorylation rather than cis, suggested to occur via oligomerization between dimerized kinases [63–65]. Of note, both Ire1 and PERK have also been shown to dimerize ‘face-to-face’ [64,66]. However, physiologically relevant active conformation of these kinases seems to be multimeric [64,65]. Cis-Autophosphorylation of a Monomeric Kinase EPKs autophosphorylation can also be dimerization independent. Most curiously, two Ser/Thr kinases, glycogen synthase kinase 3b (GSK3b) and dual-specificity tyrosine-regulated kinase (DYRK), self-activate via cis-autophosphorylation of a tyrosine residue at their activation loops. DYRK was found to be phosphorylated at an activation-loop Tyr residue when purified from Escherichia coli cells, which lack tyrosine kinases. However, purified DYRK proteins could not autophosphorylate on tyrosines [67]. This paradox was resolved when it was found that in the course of its translation, DYRK folds into a stable intermediate structure that is catalytically active as a cis-autophosphorylating enzyme [67]. This prone-to-autophosphorylate conformation and its catalytic characteristics are completely different from those of the mature full-length DYRK protein and even inhibited by different small molecules [68]. Similar to DYRK, a translational intermediate of GSK3b was also found to manifest cis-autophosphorylation activity on a tyrosine residue [69]. This activity is sensitive to Hsp90 inhibitors, indicating that this chaperone assists in stabilizing the intermediate prone-to-autophosphorylate conformation. p38/ is a member of the MAPK family, which are known to lack significant autoactivation capacity and must be phosphorylated at their activation loop by dedicated MAPK kinases (MEKs). p38/ is activated by this canonical mechanism, through the MEKs MKK3 and MKK6, but can also be induced to autoactivate by autophosphorylation (Box 3). For example, p38/ autoactivates following association with the scaffold protein tumor growth factor-b-activated kinase 1/MAP3K7-binding protein 1 (TAB1) [12]. TAB-1-dependent p38/ autophosphorylation is physiologically and pathologically relevant in cardiomyocytes after ischemia reperfusion and in endothelial cells in response to thrombin [11,70]. Crystal structures of the TAB1–p38/ complex [11] indicate that TAB1 binding induces characteristics that were observed in the structure of phosphorylated and active MAPKs [71,72], including a domain closure and formation of the Glu–Lys salt bridge required for ATP binding. Most importantly, TAB1 binding leads to a dramatic conformational change of the activation-loop Thr, bringing it close (10 Å) to the catalytic Asp. In this structure, there is no bound ATP, but it seems likely that in the presence of an ATP, the activation-loop Thr would be in an even more favorable position for serving as a phosphoacceptor [11]. TAB1-dependent activation of p38/ and enhancement of Fus3 activation by Ste5 [73] may suggest that other MAPKs are also activated by specific allosteric activators.

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In summary, although not completely defined, it seems that different EPKs developed different prone-to-autophosphorylate conformations. Those include several types of dimers, intermediate folds that form during biosynthesis, and conformational changes induced by phosphorylation on other sites or binding to other proteins. Based on structural evidence, all various prone-toautophosphorylate conformations share common features, which are the stabilization of the activation loop, orienting it toward the catalytic Asp and g-phosphate of ATP, and alignment of the catalytic residues. The activation loop is the most flexible element of the activation segment (Box 1) and typically goes through a disordered/ordered transition upon activation. Kinases can certainly fluctuate between the active and nonactive conformations without activation-loop phosphorylation. Although in some kinases the equilibrium allows some degree of activation, in most cases the equilibrium is shifted toward the nonactive conformation without this phosphorylation. Once the intermediate prone-to-autophosphorylate conformation is adopted and orders the activation loop, the equilibrium is shifted toward activating the kinases (Figure 2). Kinases that [16_TD$IF]rarely adopt a prone-to-autophosphorylate conformation will not be skewed toward the active conformation within this equilibrium. [17_TD$IF]The allosterically induced stabilization[18_TD$IF] of the activation loop in the putative prone-to-autophosphorylate conformations provides a rational explanation for how autophosphorylation is catalyzed in the absence of activation-loop phosphorylation.

The Riddle of MAP Kinases: Many Barriers of Autophosphorylation? Unlike most EPKs, MAPKs [extracellular signal-regulated kinases (ERKs), c-Jun NH2-terminal kinases (JNKs), p38s, and big mitogen-activated protein kinases] manifest extremely slow rates of autophosphorylation as purified proteins in vitro [10,13,15,74–76]. In vivo, however, MAPKs are found to self-activate under very specific conditions, indicating that they are capable of such activity and that it is tightly regulated [11,15,19,22]. The overall structure of MAPKs is very similar to that of other EPKs, with the exception of the presence of two regions that exist only in MAPKs: the MAPK insert and C-terminal extension [77]. The active site of MAPKs is stabilized, as in most EPKs, by phosphorylation of the activation loop. It is not clear, therefore, why autophosphorylation is strongly suppressed in MAPKs. Answering this question might explain which domains control the prone-to-autophosphorylate conformation. Recent studies have revealed domains and residues important for regulating autophosphorylation in some MAPKs. Structural insights were provided by the analyses of the p38b isoform and the JNK2 splice variant JNK2/2. These MAPKs are atypical as they possess intrinsic and unconditioned autophosphorylation activity [10,74,78]. These findings are extremely puzzling because p38b and JNK2/2 are almost identical, at the levels of sequence and crystal structure, to other p38s and JNKs, respectively, which do not autophosphorylate. Rigorous studies have revealed short fragments (13-residue long in the case of p38b) responsible for this activity. These have been found to reside within one of the MAPK-unique structural elements, the MAPK insert [10,78]. It is still not clear how these fragments enforce, or rather fail to prevent, a prone-toautophosphorylate conformation. Another structural motif that is strongly associated with autophosphorylation of p38/ (and probably p38b as well) is stabilized by hydrophobic interactions between the /C helix and residues of the C-terminal extension [75,79,80]. Disrupting these interactions, either by mutating the hydrophobic residues [75,80] or by phosphorylation of the C-terminal Tyr323 [22], induces strong autophosphorylation of the activation-loop Thr180. Importantly, Tyr323 is phosphorylated in vivo in T-cell receptor-activated T cells and serves as an MEK-independent mode of p38//b activation [14,22]. For Hog1, the yeast ortholog of p38, it was shown that any modification of the hydrophobic interactions is sufficient to render it capable of autoactivation [75]. It seems, therefore, that the tight association between the /C helix and the C-terminal extension is an inherent structural barrier of autophosphorylation. A series of point mutations were found to render Erk1/2 capable of spontaneous autophosphorylation [13,15,81,82]. A prominent mutation is the change of Arg to Ser within the /C helix

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[13,15]. In Erk1, this mutation (R84S) transforms it to an oncoprotein [15]. It is not known how the mutation leads to autophosphorylation. Another notable mutation in Erk2 is I84A, occurring in the vicinity of the gatekeeper [81]. It seems to induce a prone-to-autophosphorylate conformation via intramolecular connectivity, ultimately affecting the activation loop. All activating mutations in Erk induce autophosphorylation in cis [15,81], an inherent activity that was also identified very weakly in ErkWT[10_TD$IF] proteins [76]. Thus, the effect of the activating mutations in Erks, similar to the case of p38, is to enhance an inherent activity rather than to introduce a novel activity.

Concluding Remarks While a global assessment of the physiological role of autophosphorylation is incomplete, a growing number of studies have shown that this is an important regulatory step in the activation of many EPKs. Surprisingly, this key regulatory reaction is still poorly understood. However, insightful working models are emerging. Autoactivation of EPKs is not only important in health but also strongly associated with clinical problems. Constitutively, autophosphorylating tyrosine kinases, for example, are associated with a variety of cancers. In addition, in many cases of melanoma, induction of dimerization in other RAF isoforms renders the patients resistant to anticancer effects of oncogenic B-RAF inhibitors [83]. Autophosphorylation of p38/ is closely associated with rheumatoid arthritis [17,19]. As described in this review, it seems that a prone-to-autophosphorylate conformation is achieved via allosteric regulation, which is often a consequence of dimerization. Even in kinases that are not controlled by activation-loop phosphorylation, such as the secreted Fam kinases and liver kinase B1, dimerization is a basis for allosteric activation of catalytic activity [55,57]. Thus, dimerization is a highly conserved mean for allosteric induction of kinase activation. It imposes prone-to-autophosphorylate conformations that have a single purpose and a single reaction cycle. Unlike the active conformation, which is similar in most EPKs, there seems to be a number of prone-to-autophosphorylate conformations. Such a conformation holds, unusually, for just a one-time reaction cycle. Once the activation loop has been autophosphorylated, the kinase interconverts to its active conformation. These notions are the starting points for future studies that should achieve an in-depth understanding of the structure–function relationships of the autophosphorylation reaction. This knowledge will enable, for example, the development of specific inhibitors of the autoactivation of each EPK. It will also disclose currently concealed concepts with respect to the evolution of EPKs in particular and enzymes in general (see Outstanding Questions). Acknowledgments We wish to thank Dr Natarajan Kannan and Mr Daniel Mcskimming for their help with the ProKinO Web server. We would also like to thank Dr Ron Diskin, Dr Ze’ev Paroush, and Dr Rony Seger for their critical reading and helpful comments.

[19_TD$IF]Supplemental [20_TD$IF]Information Supplemental [21_TD$IF]information related to this article can be found[5_TD$IF] online[6_TD$IF] at doi:10.1016/j.tibs.2016.08.006.

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