Protein–Protein Interactions in the Allosteric Regulation of Protein Kinases Patricia Pellicena and John Kuriyan

COSTBI-407; NO OF PAGES 8

Protein–protein interactions in the allosteric regulation of protein kinases Patricia Pellicena and John Kuriyan

Protein–protein interactions involving the catalytic domain of protein interactions can bring about conformational protein kinases are likely to be generally important in the changes in the kinase domain, in this case resulting in regulation of signal transduction pathways, but are rather activation [4]. Since then, much effort has been focused sparsely represented in crystal structures. Recently on filling in structural information for the kinome [5], determined structures of the kinase domains of the mitogen- understanding how intramolecular interactions control activated protein kinase Fus3, the RNA-dependent kinase the kinase domain (e.g. in the Src [6,7] and Abl [8,9] PKR, the epidermal growth factor receptor and Ca2+/ tyrosine kinases), how the conformation of the activation calmodulin-dependent protein kinase II have revealed loop controls kinase activity [10,11], how peptide sub- unexpected and distinct mechanisms by which interactions strates are recognized [12,13] and how the modulation of with the catalytic domain can modulate kinase activity. kinase structure is reflected in the specificity of kinase inhibitors [14]. Addresses Department of Molecular and Cell Biology, Department of Chemistry and Howard Hughes Medical Institute, University of California, and Surprisingly little has been revealed so far at the detailed Division of Physical Biosciences, Lawrence Berkeley National structural level about how protein–protein interactions Laboratory, Berkeley, CA 94720, USA involving the kinase domain result in changes in kinase activity, with one prominent exception being the early Corresponding author: Kuriyan, John ([email protected]) analysis of the Cdk–cyclin interaction [4]. This is now changing with several recent studies providing new infor- Current Opinion in Structural Biology 2006, 16:1–8 mation on protein–protein interactions involving the catalytic domain of protein kinases. We summarize the This review comes from a themed issue on Catalysis and regulation findings of some of these studies here. Edited by Gourisankar Ghosh and Susan S Taylor A kinase domain acting alone: activation loop phosphorylation in cis 0959-440X/$ – see front matter We begin by digressing somewhat to describe a remark- # 2006 Elsevier Ltd. All rights reserved. able observation concerning the DYR (dual-specificity tyrosine-phosphorylation-regulated) kinases. The most DOI 10.1016/j.sbi.2006.10.007 obvious protein–protein interaction involving kinase domains is the formation of a dimer that promotes transphosphorylation of the centrally located activation loop Introduction and subsequent activation (Figure 1). The kinase active The clustering of receptor molecules is emerging as a key site appears to be so constructed that phosphorylation feature of intracellular signal transduction. Such cluster- cannot occur in cis. This restriction arises because peptide ing brings signaling molecules into close proximity at high substrates bind to the catalytic domain in an orientation local concentrations and promotes a diverse array of opposite to that of the activation loop backbone [12,15] protein–protein interactions, often through the utilization (Figure 1). Crystal structures of inactive kinases, such as of specialized domains [1]. Within these clusters, protein those of insulin receptor kinase (IRK) [16] and c-Abl [8], kinases, which use ATP to phosphorylate proteins show that the active site is disrupted when the activation on serine/threonine or tyrosine residues, become activated loop folds back so as to present itself as a substrate mimic. by being phosphorylated themselves or by interacting with other proteins or ligands. DYR kinases (DYRKs) phosphorylate their substrates on serine/threonine residues, but are themselves autopho- In the 15 years since the first structure of a protein kinase sphorylated on a critical tyrosine residue in their activa- was determined, that of cAMP-dependent protein kinase tion loop [17–21]. Intriguingly, autophosphorylation of (PKA) [2], much has been learnt at the structural level DYRKs is a result of intramolecular rather than intermo- about how conformational control is exerted on the kinase lecular phosphate transfer, and this phosphorylation domain. The structure of the cyclin-dependent protein occurs before the kinase leaves the ribosome [22]. It kinase Cdk2 was determined shortly after that of PKA and is proposed that this in cis reaction is catalyzed by a revealed an inactive conformation of the kinase domain, translational intermediate of the kinase domain that rather than the active form seen for PKA [3]. The struc- differs from the mature and fully folded enzyme in that ture of a Cdk2–cyclin A complex showed us how protein– the intermediate can phosphorylate the activation loop www.sciencedirect.com Current Opinion in Structural Biology 2006, 16:1–8

Please cite this article in press as: Pellicena P, Kuriyan J, Protein–protein interactions in the allosteric regulation of protein kinases, Curr Opin Struct Biol (2006), doi:10.1016/j.sbi.2006.10.007 COSTBI-407; NO OF PAGES 8

2 Catalysis and regulation

Figure 1 Figure 2

Anatomy of a protein kinase catalytic domain. Ribbon diagram representing the main features of a prototypical kinase catalytic domain based on the structure of IRK (PDB code 1IR3) [12]. The N-lobe is shown in green, the C-lobe is shown in purple and the activation loop is colored yellow. The substrate peptide, docked onto the activation loop, and an ATP analog are shown in black. Helix-C, which contains residues critical for catalysis, and helix-G, which is involved in docking protein substrates, are labeled. tyrosine whereas the mature form cannot. Autophosphor- ylation by a folding intermediate has so far only been described for this family of kinases, but it raises the possibility that such translational intermediates might provide alternative targets for inhibitor development [22].

Scaffolds as allosteric regulators of kinases By bringing signaling components into close proximity, the yeast scaffold protein Ste5 contributes to the speed and speciﬁcity of the mating pheromone signaling pathway. In addition to assembling components of the mitogen-activated protein (MAP) kinase pathway, it now appears that Ste5 regulates at least one of them allosterically [23]. A peptide containing a 29-residue minimal region of Ste5 stimulates the rate of Fus3 autophosphor- Comparison of Fus3 in complex with a peptide derived from the ylation by 50-fold. The co-crystal structure of Fus3 in scaffolding protein Ste5 and PKA. (a) The Ste5 peptide, shown in complex with the Ste5 peptide shows a bipartite mode of dark blue, docks onto Fus3 (PDB code 2F49) [23], shown in white. (b) The Ste5 peptide follows a path similar to that of the C-terminal binding, with both N- and C-lobes of the kinase domain tail extension (dark blue) of PKA (PDB code 2CPK) [2]. engaged by the peptide.

Superposition of peptide-bound and unbound structures of Fus3 shows a relative displacement between the N- reminiscent of the way in which a C-terminal extension and C-lobes, suggesting that Ste5 modulates Fus3 activity of PKA folds back onto the distal face of the kinase [2]. by coordinating an optimal orientation between the two The C-terminal extension of PKA is thought to stabilize lobes of the kinase (Figure 2). In going from the C-lobe to the active state and a similar mechanism may be one the N-lobe through a path that is distal to the active site, component of the activating effect of the Ste5 peptide on the topology of the Ste5 peptide bound to Fus3 is Fus3 [24] (Figure 2).

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Allosteric regulation of protein kinases Pellicena and Kuriyan 3

In another study concerning a MAP kinase cascade an intact protein substrate. eIF2 docks onto a region of the enzyme, TAK1 (a MAP kinase kinase kinase), the inter- kinase domain that is adjacent to the active site, next to actions between the kinase domain of TAK1 and a pep- helix-G in the C-lobe. The structure suggests an allosteric tide segment of TAB1 (TAK1-binding protein 1) have coupling mechanism between substrate docking to helix- been deﬁned [25]. The structure shows that a 36-resi- G and the conformation of the activation loop. Helix-G due TAB1 fragment docks onto a hydrophobic patch in has also been implicated recently as a docking site for the the C-lobe. Just how this interaction increases the cata- mutual recognition of two kinase domains during trans- lytic activity of TAK1 is unclear at present, but it is phosphorylation of the activation loop of p21-activated intriguing that the a helix formed by the TAK1-interact- kinases (PAKs) [40,41]. The coupling of kinase activa- ing segment of TAB1 inserts hydrophobic sidechains into tion to substrate recognition has also been recognized in a cavity at the base of the C-lobe of the kinase domain other protein kinase families, such as Src [42] and Cdks [25], in a similar position to where a myristoyl group is [43]. bound to the structure of autoinhibited c-Abl [9]. EGF receptor: a case of molecular A symmetrical kinase domain dimer: PKR plagiarism RNA-dependent protein kinase (PKR) shuts down pro- Members of the epidermal growth factor (EGF) receptor tein translation by phosphorylating the translation initia- family, important in many cancers [44], transduce a tion factor eIF2 [26]. The upstream signal to this event is ligand-binding event outside the cell into an increase the binding of double-stranded RNA to PKR. As for the in kinase activity inside the cell. EGF receptor is a majority of protein kinases, activation of PKR involves transmembrane protein possessing ligand-binding and autophosphorylation of the activation loop [27]. Muta- kinase domains that are separated from each other by tions in PKR that impair RNA binding also affect the the plasma membrane. The C-terminal tail of EGF ability of the kinase molecules to dimerize. These muta- receptor contains autophosphorylation sites that recruit tions also result in reduced PKR autophosphorylation and signaling molecules when phosphorylated. catalytic activity [28–31], suggesting a link between dimerization, autophosphorylation and substrate phos- Receptor dimerization, or the formation of higher order phorylation. Indeed, a recent study uncovered several oligomers, results in the activation of EGF receptor [45– mutations in the kinase domain that result in elevated 47]. Unlike most protein kinases, the EGF receptor does kinase activity by promoting dimerization [32]. not require activation loop phosphorylation [48–51] and thus the mechanism for the increase in activity upon The dimerization mechanism of PKR has been revealed by ligand-induced dimerization has remained elusive. crystallizing the kinase domain of PKR in complex with its Recently, a re-examination of previously determined protein substrate, eIF2 [33]. The PKR kinase domains in crystal structures [51,52], as well as the analysis of new these structures form a symmetrical dimer with back-to- ones, led to the discovery that an interface between two back interactions between the N-lobes of both kinases kinase domains that is seen in all crystal structures of (Figure 3). Although the mutational analysis makes it clear active EGF receptor kinase domains provides an expla- that the formation of this dimer is important for the activa- nation for the mechanism of receptor activation by dimer- tion of PKR, the structure does not completely explain why ization [53]. this is the case. In this regard, it is most intriguing that a very similar mode of dimerization has been observed in the In the active kinase crystal structures [51,53], the crystal crystal structures of a group of protein kinases that are packing between kinase molecules includes an asym- unrelated to PKR, the Mycobacterium tuberculosis kinases metric C-lobe to N-lobe interaction between two kinase PknB and PknE [34,35,36]. Again, the consequences of domains. The crystals also contain a symmetric interface dimerization for the catalytic activity of the kinase domain that has been analyzed extensively [54], but does not are not entirely clear from these studies [34,35], but the seem to be critical for the activation process [53]. The close similarity of the dimer interfaces of PKR and the M. intriguing feature of the asymmetric arrangement is that it tuberculosis kinases emphasizes the functional importance resembles the interactions seen between Cdk2 and its of this interaction. A key aspect of the interaction between activator, cyclin A [4] (Figure 4). Sequence alignments kinases in these dimers is the engagement by one kinase show that residues at this interface are invariant among domain of the region abutting helix-C in the N-lobe of the the three catalytically active EGF receptor family mem- other kinase domain. Interactions in this region are critical bers, EGF receptor, HER2 and HER4, but are not con- for the regulation of many protein kinases, for example, the served in the N-lobe face of the catalytically inactive Src kinases [37,38] and G-protein-coupled receptor kinases ErbB3 [53]. The formation of the cyclin-like N-lobe to (GRKs) [39] (Figure 3). C-lobe interface explains how ErbB3, despite being catalytically dead, can still activate other kinase domains The PKR structure is also very important because it by heterodimerization [55]. Disruption of this interface provides the ﬁrst view of a kinase domain recognizing by mutagenesis results in the adoption by the kinase www.sciencedirect.com Current Opinion in Structural Biology 2006, 16:1–8

4 Catalysis and regulation

Figure 3

Protein kinases subjected to regulation via N-lobe interactions. In many protein kinases, regulation of activity occurs via a region that couples helix-C to regulatory domains, as in GRK2 (PDB code 2BCJ) [39] and the Src family kinase Hck (PDB code 1QCF) [6,7]. In the protein kinases PknE (PDB code 2H34) [34] and PKR (PDB code 2A1A) [33], a similar back-to-back dimer is observed. In this arrangement, the kinase catalytic domains act as their own regulatory regions via the same N-lobe interface that is seen in GRK2 and the Src kinases. domain of an inactive structure that closely resembles Dimers within supramolecular assemblies: those of Cdk2 and the Src kinases [53]. This inactive CaMK-II Src/Cdk-like structure is also seen in the crystal structure Ca2+/calmodulin-dependent protein kinase II (CaMK-II) of the EGF receptor kinase domain bound to the cancer is a key player in processes underlying learning and drug tykerb [52]. memory, and also in other functions throughout the body [61]. The most remarkable property of this enzyme is its Src/Cdk-like inactive conformations have recently been apparent ability to respond not only to the amplitude of observed in several kinases, including GCN2 [56], intracellular Ca2+ spikes that occur during neuronal sti- c-Abl [57] and Snf1 [58,59], a member of the AMP- mulation but also to their frequency [61,62]. CaMK-II is activated kinase family [60]. Snf1 also forms a dimer, unique among protein kinases in that it forms stable but, in contrast to the EGF receptor, the Snf1 dimer dodecamers. Although it is not clear how CaMK-II can stabilizes the inactive rather than the active conformation decode Ca2+ spike frequencies, the answer may lie in the [59]. supramolecular organization of the enzyme.

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Allosteric regulation of protein kinases Pellicena and Kuriyan 5

Figure 4

Comparison of the mechanism of EGF receptor activation by dimerization with the Cdk2–cyclin A complex. In the EGF receptor asymmetric dimer, one kinase catalytic domain is activated by another kinase catalytic domain in a C-lobe to N-lobe interaction [53]. This is analogous to the mechanism of activation of Cdk2 by cyclin A (PDB code 1FIN) [4].

The domain structure of CaMK-II resembles that of other tion. Studies using electron microscopy have shown that CaM-dependent kinases in that it has an N-terminal CaMK-II forms two hexameric rings that are stacked one kinase domain followed by a regulatory region of approxi- on top of the other [63–65]. A detailed view of the manner mately 40 residues. Unique to CaMK-II is the C-terminal in which the two stacked rings are assembled has been association domain, which is responsible for oligomeriza- provided by a crystal structure of the association domain of

Figure 5

Structure of the CaMK-II kinase domain and regulatory region. (a) The kinase catalytic domains within the dimer are shown in blue and green. Dimerization occurs through the regulatory regions, which form a coiled-coil strut. The regulatory regions contain the CaM-binding site, shown in red. The location of the major regulatory autophosphorylation site, Thr286, is shown for one of the monomers (PDB code 2BDW) [68]. (b) Hypothetical reconstruction of the CaMK-II holoenzyme, based on the crystal structure and SAXS analysis [68], showing a possible location of the dimer unit within the dodecameric holoenzyme. www.sciencedirect.com Current Opinion in Structural Biology 2006, 16:1–8

6 Catalysis and regulation

CaMK-II [66]. One curious fact is that the association with potential protein–protein interaction surfaces. Cau- domain on its own (without the kinase domain) forms tion must therefore be employed in interpreting the rings that are sevenfold rather than sixfold symmetric [67]. biological significance of crystal packing interfaces [72]. Some of the studies discussed here demonstrate that the The crystal structure of the autoinhibited kinase domain development of hypotheses based on crystal packing and and regulatory region has been described recently [68] confirmed by biochemical and biophysical techniques can (Figure 5). As seen in other CaM-dependent protein greatly contribute to our understanding of protein kinase kinases [69,70], the regulatory region blocks the active regulation. site. 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Manning G, Whyte DB, Martinez R, Hunter T, Sudarsanam S: crucial to prevent background activation by transpho- The protein kinase complement of the human genome. sphorylation. Maintaining the dodecamer in a continu- Science 2002, 298:1912-1934. ously repressed state is not an easy task considering that, in 6. Schindler T, Sicheri F, Pico A, Gazit A, Levitzki A, Kuriyan J: this setting, the local concentration of kinase domains is in Crystal structure of Hck in complex with a Src family- selective tyrosine kinase inhibitor. Mol Cell 1999, the millimolar range [68]. Second, such an arrangement 3:639-648. can help explain the cooperative nature of Ca2+/CaM 7. Xu W, Doshi A, Lei M, Eck MJ, Harrison SC: Crystal structures binding to the holoenzyme [68,71]: if the dimer is the of c-Src reveal features of its autoinhibitory mechanism. basic unit of the holoenzyme assembly, binding of the first Mol Cell 1999, 3:629-638. CaM to one kinase domain would, in principle, free up its 8. 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The interaction between Ste5 and Fus3 bears Mol Cell 2004, 15:661-675. some resemblance to the manner in which extra segments 12. Hubbard SR: Crystal structure of the activated insulin receptor of PKA interact with the kinase domain. The dimeriza- tyrosine kinase in complex with peptide substrate and ATP tion of PKR is strikingly similar to that of bacterial kinases analog. EMBO J 1997, 16:5572-5581. of the PknB family. The activation of the EGF receptor 13. Lowe ED, Noble ME, Skamnaki VT, Oikonomakos NG, Owen DJ, uses the same trick seen in the Cdk–cyclin complex, with Johnson LN: The crystal structure of a phosphorylase kinase peptide substrate complex: kinase substrate recognition. the EGF receptor kinase domain serving as its own EMBO J 1997, 16:6646-6658. ‘cyclin’. It will be interesting to determine if the con- 14. Noble ME, Endicott JA, Johnson LN: Protein kinase inhibitors: served kinase domain fold possesses a limited number of insights into drug design from structure. Science 2004, interfaces from which control of activity can be exerted. 303:1800-1805. 15. Knighton DR, Zheng JH, Ten Eyck LF, Xuong NH, Taylor SS, Sowadski JM: Structure of a peptide inhibitor bound to the Protein molecules in a crystal must necessarily interact catalytic subunit of cyclic adenosine monophosphate- with each other and crystal structures are usually replete dependent protein kinase. Science 1991, 253:414-420.

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