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provided by Elsevier - Publisher Connector Cell, Vol. 105, 721–732, June 15, 2001, Copyright 2001 by Cell Press Crystal Structure of Synthase 3␤: Structural Basis for -Primed Substrate Specificity and Autoinhibition

Rana Dajani,1 Elizabeth Fraser,2 S. Mark Roe,1 of proliferative genes such as c-Myc, which are regu- Neville Young,2 Valerie Good,1 Trevor C. Dale,2 lated by ␤-catenin-dependent Tcf/Lef transcription fac- and Laurence H. Pearl1,3 tors (He et al., 1998). 1Section of Structural Biology Despite their common GSK3␤ component, the Wnt 2 CRC Centre for Cell and Molecular Biology and insulin signaling pathways are insulated from each Institute of Cancer Research other by incorporation of Wnt-signaling GSK3␤ into a Chester Beatty Laboratories multiprotein complex involving Axin/Conductin, and the 237 Fulham Road adenomatous polyposis coli-associated protein (APC) London SW3 6JB (reviewed in Polakis, 2000). The Axin-APC complex also United Kingdom binds ␤-catenin, which is efficiently phosphorylated by GSK3␤ in the presence of the Axin-APC scaffold, but not in its absence (Ikeda et al., 1998). The Axin-APC Summary complex may serve a dual role of enhancing access of GSK3␤ to ␤-catenin, while restricting its access to non- kinase 3␤ (GSK3␤) plays a key role Wnt substrates. The requirement of an active Axin-APC- in insulin and Wnt signaling, phosphorylating down- GSK3␤ complex for and consequent stream targets by default, and becoming inhibited fol- degradation of ␤-catenin confers a tumor suppression lowing the extracellular signaling event. The crystal role on this complex. Defects in APC which prevent its structure of human GSK3␤ shows a catalytically active formation are associated with the most common form of conformation in the absence of activation-segment hereditary colorectal cancer (Groden et al., 1991; Kinz- phosphorylation, with the sulphonate of a buffer mole- ler et al., 1991), and somatic loss of functional APC is a cule bridging the activation-segment and N-terminal frequent occurrence in spontaneous colorectal tumors. domain in the same way as the phosphate group of the Phosphorylation of glycogen synthase (GS) by GSK3␤ activation-segment phospho-Ser/Thr in other . is unusual in requiring a prior phosphorylation by an- The location of this oxyanion in the sub- other protein kinase. Thus, GSK3␤ only phosphorylates strate binding cleft indicates direct coupling of Pϩ4 GS once Ser 656 of GS has been phosphorylated by phosphate-primed substrate binding and catalytic ac- casein kinase II (CK2). With GS thus “primed,” GSK3␤ tivation, explains the ability of GSK3␤ to processively will sequentially phosphorylate Ser 652, Ser 648, Ser hyperphosphorylate substrates with Ser/Thr pentad- 644, and Ser 640 in a carboxy-terminal to amino-terminal repeats, and suggests a mechanism for autoinhibition direction, with each subsequent phosphorylation de- ϩ in which the phosphorylated N terminus binds as a pendent on prior phosphorylation of the P 4 position competitive pseudosubstrate with phospho-Ser 9 oc- (Fiol et al., 1988, 1990; Wang and Roach, 1993). A similar cupying the Pϩ4 site. process of sequentially primed phosphorylation is sug- gested by the periodicity of the phosphorylation sites Introduction (Ser 45, Thr 41, Ser 37, Ser 33, and Ser 29) in the main Wnt target ␤-catenin (Polakis, 1999). The kinase provid- ing the priming phosphorylation at Ser 45 is unknown. Glycogen synthase kinase 3␤ (GSK3␤) is a Ser/Thr pro- Efficient phosphorylation of Wnt targets is different from tein kinase with key roles in transduction of regulatory phosphorylation of insulin targets in requiring formation and proliferative signals arising at the cell membrane. of a ␤-catenin–APC–Axin–GSK3␤ complex (Hart et al., Unlike many protein kinases involved in signal transduc- 1998; Ikeda et al., 1998, 2000), and is sensitive to inhibi- tion, GSK3␤ is active in the absence of the external tion by GBP/FRAT proteins and fragments thereof (Farr signal and becomes inhibited in response to signals et al., 2000; Li et al., 1999; Thomas et al., 1999; Yost et from the membrane-bound receptor. In insulin signaling, al., 1998). A requirement for priming phosphorylation of GSK3␤ phosphorylates (and thereby inhibits) glycogen substrates has also been reported for casein kinase I synthase, the translation initiation factor eIF2B, and the (CK1) (Flotow et al., 1990), but here the priming position ␣ C/EBP transcription factor (Plyte et al., 1992; Ross et is located three residues to the N-terminal side of the al., 1999). Insulin binding to the extracellular domains target Ser/Thr. We have now determined the crystal of the receptor activates a phosphorylation cascade structure of human GSK3␤, expressed in insect cells, (Cohen, 1999), resulting in inhibitory phosphorylation of at 2.8 A˚ . The structure is devoid of activation segment ␤ GSK3 by PKB/Akt (Cross et al., 1995) with consequent phosphorylation, but is nonetheless in an activated con- hypophosphorylation and activation of the downstream formation stabilized by the sulphonate group of a buffer ␤ ␤ targets. In Wnt signaling, GSK3 phosphorylates -cat- molecule bound in the substrate binding cleft. Analysis enin, promoting its ubiquitination and subsequent de- of the structure and comparison with other protein ki- struction (Aberle et al., 1997) so that ␤-catenin levels are nase structures suggests a mechanism of catalytic acti- minimized. Binding of Wnt glycopeptide to the Frizzled vation coupled to binding of phosphorylated substrates, receptor inhibits GSK3␤ via Dishevelled, allowing in- serendipitously mimicked by the bound buffer. This ex- creased ␤-catenin levels with consequent up-regulation plains the requirement of GSK3␤ for phosphorylation “priming” of substrates and its ability to processively 3 Correspondence: [email protected] hyperphosphorylate substrates such as glycogen syn- Cell 722

Figure 1. Structure of Human GSK3␤ (A) Stereo pair secondary structure cartoon of human glycogen synthase kinase 3␤ colored blue-red from the visible N terminus at residue 35 to the visible C terminus at residue 384. The orthogonal ␤ barrel formed by the N-terminal domain is on the left. All molecular graphics were generated using Robert Esnouf’s adaptation (Bobscript) of Molscript (Kraulis, 1991) and Raster3D (Merrit and Murphy, 1994), except for Figures 3A and 6B, which were generated with GRASP (Nicholls et al., 1993). (B) As (A), but with the view rotated by 90Њ around the horizontal. thase and ␤-catenin, and suggests a mechanism for (94–104) topologically equivalent to the C helix originally phosphorylation-dependent autoinhibition of GSK3␤ defined in the structure of cAMP-dependent protein ki- following phosphorylation by PKB/Akt. nase (Knighton et al., 1991). This helix is shorter than in other kinases, consisting of only two turns. The N-terminal Results and Discussion domain connects to the remainder of the protein via an ␣ helix (138–149) extending from the end of the 7th strand. The structure of GSK3␤ conforms to the consensus ob- The core of the ␣-helical domain (152–342) has a similar served for “activation-segment” protein kinases, con- topology to the equivalent region in the mitogen acti- sisting of an amino-terminal ␤-sheet domain, coupled to vated protein (MAP) kinases such as ERK2 and p38 a carboxy-terminal ␣-helical domain. Although crystals (Wang et al., 1997; Zhang et al., 1994). The major differ- were grown from full-length human GSK3␤ (420 residue- ences are the absence of the second helix in the hairpin sϩprotease-cleavable His-tag), clear electron density is segment from 276–293, where GSK3␤ more closely re- only evident for the 351 residues from Lys 35 to Ser sembles the cyclin-dependent kinase fold (DeBondt et 386, and the segments of polypeptide preceding 35 and al., 1993). Beyond residue 342, GSK3␤ departs substan- following 386 are disordered in these crystals. The visi- tially from the MAPK fold, with residues 342–386 forming ble N-terminal domain (35–134) of GSK3␤ consists of a a series of short helices and loops packed against the seven-stranded ␤ sheet curved over on itself to form a long helix (155–175) in the large domain, rather than closed orthogonal ␤ barrel (Figure 1). This is different snaking back to rejoin the N terminus as in the MAPKs. to the equivalent domains in related Ser/Thr kinases, At the C-terminal extremity of the visible polypeptide, and more closely resembles the N termini of protein the two molecules in the asymmetric unit make different tyrosine kinases such as insulin receptor kinase (Hub- lattice contacts and the structures diverge, with the seg- bard et al., 1994) and members of the Src-kinase family ment from Pro379 to Arg383 forming a turn of helix in (Sicheri et al., 1997; Xu et al., 1997). The 5th and 6th one molecule, while having an extended conformation strands of the barrel are connected by a short ␣ helix in the other. Structure of GSK3␤ 723

Conformation of Activation Loop phosphorylation of Thr 183 into activation. Remarkably, and Catalytic Groups in GSK3␤, the same interaction and consequent align- The catalytic activity of protein kinases depends upon ment of the catalytic apparatus is achieved by an exoge- the correct juxtaposition of the catalytic groups contrib- nous anion, occupying an equivalent position to the uting to the transfer of the ␥-phosphate group from ATP covalently attached phosphate of activated ERK2, to a , threonine, or tyrosine side chain of the pro- which cross-links the activation segment to the basic tein substrate. A second factor is the accessibility and cluster by nonbonded polar interactions. The shape and correct positioning of the groups forming the substrate size of the electron density and the nature of the proxi- peptide binding site, which provide affinity and specific- mal groups suggest that the bound anion is a phosphate ity for the substrate (Johnson et al., 1996). In many ki- or sulfate. However, neither of these ions was explicitly nases, these two requirements are simultaneously con- present in the buffers used to purify and crystallize hu- tingent upon the conformation of an “activation segment” man GSK3␤ (see Experimental Procedures). HEPES which contains residues that are themselves subject ([4-(2-hydroxyethyl)-1-piperazineethane sulphonate) to phosphorylation. In the dephosphorylated state, the was used in buffers throughout the purification and conformation of the activation segment disfavors kinase would be Ϸ10 mM (with a protein concentration of Ϸ activity. Phosphorylation promotes a conformation of 0.1 mM) in the crystallization experiments. HEPES pos- the activation segment in which the catalytic and sub- sesses a sulphonate group whose chemistry is consis- strate binding sites are correctly formed, leading to a tent with the requirements of the site. Furthermore, a substantial increase (Ͼ1000-fold for MAP kinases) in second strong electron density feature is present adja- activity. Thus, phosphorylation of the activation seg- cent to the oxyanion site, which corresponds well in size ment provides a means by which the activity of one and position to the piperazineethane moiety of a HEPES protein kinase can be regulated by a second protein molecule bound to the (Figure 2A). kinase, allowing the familiar cas- cades. A Substrate-Coupled Activation Mechanism In the MAP kinases, two sites of phosphorylation exist In MAP kinases, cAMP-dependent kinases, and cyclin- within the activation segment, Thr 183 and Tyr 185 in the dependent kinases, a significant component of activa- sequence 180-GFLTEYVATR-188 (Robbins et al., 1993). tion comes from the interaction of basic residues in the The residue corresponding to Tyr 185 in the activation N-terminal domain and catalytic loop with a phosphate segment sequence in GSK3␤ (210-GEPNVSYICSR-220), covalently attached to the activation segment (Canaga- Tyr 216, has been found to be phosphorylated in GSK3␤ rajah et al., 1997; Knighton et al., 1991; Russo et al., (Hughes et al., 1993). The residue corresponding to Thr 1996). In phosphorylase kinase (PhK), which is not acti- 183 of ERK2 is Val 214 in GSK3␤ and although the adja- vated by phosphorylation (Cox and Johnson, 1992), the cent residue (215) is serine, its phosphorylation has not equivalent of Thr 183 in ERK2 is glutamate, whose nega- been reported. Within the GSK3␤ crystals themselves, tive charge provides a constitutive activating interaction there is no indication of the strong electron density fea- with the basic cluster (Owen et al., 1995). In some (but ture that would be expected if Tyr 216 were phosphory- not all) crystal structures of (CK1) (Long- lated (Figure 2A). Despite the absence of any phosphory- necker et al., 1996; Xu et al., 1995), oxyanions were lation, the GSK3␤ activation segment has a very similar bound close to the activation segment in a similar fash- conformation to the activation segment in the doubly ion to the oxyanion we observe in GSK3␤, interacting phosphorylated active form of ERK2 (Canagarajah et with two basic residues from the underlying structure. al., 1997), but is significantly different from that in the However, CK1 is not activated by phosphorylation and unphosphorylated ERK2 structure (Zhang et al., 1994) (Figure 2B). Furthermore, the arrangement of residues only two basic residues, both from the C-terminal do- ␤ in GSK3␤ implicated in the catalytic mechanism of phos- main, interact with the anion. In contrast, in GSK3 (as phoryl-transfer is superimposable on the arrangement in ERK2 and cAPK/PKA), three basic residues, including ␤ of the equivalent residues in other structures of acti- one from the C helix in the N-terminal domain, con- vated Ser/Thr protein kinases (Canagarajah et al., 1997; verge at this site. In the absence of an anion, the intense Lowe et al., 1997) (Figure 2C). Taken together, these data positive potential generated by this cluster of basic side suggest that unlike MAP kinase or cAMP-dependent chains could not be adequately neutralized, and most kinases, GSK3␤ can achieve a catalytically active con- significantly, the interaction with Arg 96 from the N-terminal formation in the absence of activation segment phos- domain, which positions the catalytic residues in their phorylation. active conformation, would be hard to maintain (Figure While there is no electron density for co- 3). The crystal structure of Chk1, a Ser/Thr kinase in- valently attached to the activation segment in GSK3␤, volved in cell-cycle checkpoint regulation (Chen et al., we observed a very strong electron density feature in 2000), also contained a phosphate ion bound near to difference Fourier maps (Ͼ 6␴) within hydrogen bonding the activation segment, but this does not interact with distance of the peptide main chain at Asn 213 to Val the N-terminal domain and plays no role in activation 214. This feature is also close to the positively charged segment conformation, which was unchanged in crys- side chain head groups of Arg 96, Arg180, and Lys 205, tals grown in the absence of oxyanion. which converge at this site, suggesting that this feature In vivo, the site occupied by the sulphonate of HEPES represents an anionic species. In activated ERK2, the could be occupied by inorganic phosphate present in equivalent residues (Arg 68, Arg 146, and Arg 170) form the cell so that, other regulatory factors aside, GSK3␤ the binding site for the phosphate of pThr 183 and via would be constitutively present in a catalytically compe- that interaction, translate the signal represented by the tent state. However, the unusual substrate preference Cell 724

Figure 2. Activation Loop and Conformation (A) Stereo pair showing electron density for the activation segment from residues 212–219 and the bound HEPES buffer (HEP). Gold “chicken- wire” is from a 2FoϪFc Fourier map contoured at 1.0␴; blue chicken-wire is from an FoϪFc map contoured at 3.0␴. In both cases, calculated amplitudes and phases are from the refined structure of the protein only. No electron density consistent with phosphorylation of Tyr 216 is visible in either map. (B) Comparison of the activation-segment conformation of GSK3␤ (blue) with the equivalent region of unphosphorylated inactive ERK2 (yellow, right) and diphosphorylated active ERK2 (red, left). (C) Stereo pair showing superposition of the side chains of the catalytic residues of activated ERK2 (yellow), phosphorylase kinase (magenta), and GSK3␤ (CPK colors). Only the secondary structure of GSK3␤ is shown. of GSK3␤ suggests an alternative and much more inter- (Lowe et al., 1997) identifies those GSK3␤ residues that esting role for this anion binding site. Analysis of sites constitute the binding subsites for the phosphorylatable phosphorylated by GSK3␤ (see Table 2) suggests a pref- Ser/Thr in a substrate peptide (site P 0), and for adjacent erence for sequences of the type: Ser/Thr–X–X–X–Ser/ substrate residues N-terminal (sites PϪn) and C-termi- Thr, where the C-terminal serine or threonine is already nal (sites Pϩn) to that. As in PhK, the PϪ3, PϪ1, Pϩ1, phosphorylated (Fiol et al., 1988, 1990; Wang and and Pϩ3 substrate residues would be bound to exposed Roach, 1993), so that prior phosphorylation of the nϩ4th surface sites on GSK3␤, and would exert relatively little residue facilitates phosphorylation of the nth residue. sequence specificity. The PϪ2 binding site in GSK3␤ Superposition of GSK3␤ with the PhK substrate complex (formed by Ser 219, Arg220, and Tyr 221) is more Structure of GSK3␤ 725

Figure 3. Oxyanion Binding Site (A) Stereo pair of solvent-accessible surface of GSK3␤ colored according to the electro- static potential: red, negative; blue, positive. The intense positive patch generated by the basic side chains of Arg 96, Arg 180, and Lys 205 is indicated, as is the location of the catalytic aspartate 181 and Arg 220, which could interact with a phosphorylated Tyr 216. (B) Position of the bound HEPES molecule relative to the overall structure of GSK3␤. The negatively charged sulphonate group of HEPES is close to the positive patch (see [A]) generated by Arg 180 and Lys 205 from the large domain and Arg 96 on the C helix of the N-terminal domain (blue side chains). The side chains of the catalytic residues Asp 181 and Asp 200 are shown in red. (C) Detail of the hydrogen bonding network involving the sulphonate of HEPES, the side chains of Arg 96, Arg 180, and Lys 205, and the main chain of Asn 213 and Val 214 in the activation segment.

crowded than the equivalent site in PhK (Thr 186, Pro interactions with a substrate. Immediately beyond the 187, and Ser 188), and would disfavor bulky substrate Pϩ3 binding site in GSK3␤ is the position of the strong side chains. The position and conformation of the phos- electron density feature, which we attribute to a HEPES phorylatable substrate serine in PhK is determined by molecule with its sulphonate group bound to the side a stretch of antiparallel ␤ sheet formed between the chains of Arg 96, Arg180, and Lys 205, and to the peptide substrate from P0 to Pϩ3 and the activation segment NH of Val 214 in the activation segment. The Pϩ4 residue of the enzyme from Val 183 to Thr 186. In GSK3␤, the of a substrate peptide, bound to GSK3␤ in a similar conformation of the corresponding part of the activation conformation to that seen in PhK, could be readily ac- segment, Tyr 216 to Ser 219, would facilitate similar commodated at this position such that the phosphate Cell 726

Table 1. Crystallographic Statistics activity regardless of the state of Thr 183 (Robbins et al., 1993; Zhang et al., 1995). Phosphorylation of Tyr 216 Data Collection All data (outer shell) in GSK3␤ has been described (Hughes et al., 1993), and Rmerge 0.074 (0.323) a requirement for tyrosine phosphorylation observed in ␴ I/ (I) 8.4 (2.3) vivo (Murai et al., 1996). However, unlike MAP kinases, Completeness (%) 99.9 (99.9) ␤ Multiplicity 5.6 (5.6) GSK3 devoid of Tyr 216 phosphorylation is capable No. Unique Reflections 33,985 (4,883) of achieving an activated configuration of its catalytic residues and a conformation of the activation segment Structure Refinement receptive to substrate binding, suggesting that phos- No. of Atoms (Protein) 5,567 phorylation of Tyr 216 is not essential for activity. Con- No. of Atoms (All) 5,786 sistent with this, mutation of Tyr 216 to phenylalanine Resolution Range 35–2.8 A˚ did not impair GSK3␤ activity in Wnt signaling, nor signif- Rcryst 0.220 icantly alter the kinase activity of GSK3␤ on myelin basic Rfree 0.254 protein (Itoh et al., 1995). Furthermore, saturating tyro- sine phosphorylation in vitro by ZAK1, the only specific GSK3␤-tyrosine kinase so far identified (Kim et al., 1999) group of a phosphoserine or phosphothreonine could only doubled GSK3␤ kinase activity. This low level of bind in the same site as the sulphonate of HEPES (Fig- activation compared to MAP kinases suggests that the ure 4A). ␤ Although we have not observed the structure of effect of Tyr 216 phosphorylation on GSK3 is subtle. ␤ GSK3␤ in an inactive conformation, it seems likely that, In the conformation observed in the GSK3 structure, as in ERK2 or cAPK, the convergence of three basic the unphosphorylated Tyr 216 side-chain has a gauche residues required for the active conformation can only conformation, directed down toward the bottom of the be achieved with the counterbalancing negative charge peptide binding cleft. In this orientation, it crowds the provided by an oxyanion. Thus, the the side chains of substrate binding site and impedes access to the puta- ϩ Arg 96, Arg180, Lys 205, and the peptide NH of Val tive P 4 phosphate binding site. At the equivalent posi- 214 in GSK3␤ provide a specific binding site for the tion in activated ERK2, the side chain of pTyr 185 has phosphate groups of Pϩ4 phosphate-primed sub- an anti conformation which directs it out of the substrate strates, whose binding then stabilizes the active confor- binding site so that the phosphate group interacts with mation of the enzyme, directly coupling substrate speci- Arg 189 and Arg 192. A comparable conformation for a ficity to enzymatic activity. pTyr 216 would be possible in GSK3␤, with the tyrosyl phosphate group interacting with Arg 220 and Arg 223, A Role for Tyrosine Phosphorylation the topological equivalent of 189 and 192 in ERK2, and in Modulating GSK3␤ Activity allowing full access to the Pϩ4 binding site (Figure 4B). One important difference between GSK3␤ and PhK oc- While phosphorylation would force the “up” conforma- curs in the vicinity of the Pϩ3 substrate binding site, tion for Tyr 216, there is no inherent barrier to this confor- which is provided by the side chain and main chain of mation in its absence, so that although Tyr 216 phos- Val 183 in PhK. The corresponding residues in GSK3␤ phorylation would facilitate substrate binding, it may not and in ERK2 are tyrosines (Tyr 216 and Tyr 185), whose be an absolute requirement in situations where other phosphorylation is implicated in kinase activation. In factors serve to enhance the affinity of GSK3␤ for a ERK2, phosphorylation of Tyr 185 is essential for kinase substrate.

Table 2. Sites Phosphorylated by GSK3␤ Protein Sequence Residues

Glycogen synthase ASVPPS 639–644 Glycogen synthase PSPSLS 643–648 Glycogen synthase LSRHSS 647–652 Glycogen synthase SSPHQS 651–656 Elongation initiation factor 2B ⑀ subunit DSRAGS 539–544 cAMP-responsive element binding protein (CREB) LSRRPS 128–133 CCAAT/enhancer binding protein ␣ (C/EBP␣)PTPPPT 225–230 CCAAT/enhancer binding protein ␣ (C/EBP␣)PTPVPS 229–234 Microtubule associated protein Tau KSPVVS 395–400 Microtubule associated protein Tau VSGDTS 399–404 ␤-catenin QSYLDS 28–34 ␤-catenin DSGIHS 32–37 ␤-catenin HSGATT 36–41 ␤-catenin TTTAPS 40–45 Axin TSANDS 321–326 Axin DSEQQS 325–330 Dd-STATa SSPLPS 130–135 Dd-STATa PSSPLS 125–130 Cyclin D1 CTPTDV 285–290 The site to be phosphorylated is in bold, while the Pϩ4 residue whose phosphorylation “primes” GSK3␤ activity is underlined. All sequence numbers are for the mammalian protein except Dd-STATa which are for the Dictystelium protein. Structure of GSK3␤ 727

Figure 4. Phospho-Substrate Binding Model and Tyr 216 Conformation (A) Model of substrate binding to GSK3␤ based on structural alignment with the structure of a phosphorylase kinase–peptide substrate complex (PDB code 2PHK). The modeled substrate corresponds to residues 642–648 of human glycogen synthase (-642-PPSPSLS-648), whose phosphorylation at Ser 644 by GSK3␤ requires a “priming” phosphoserine at 648. The position of the serine to be phosphorylated is indicated at P(0) and that of the priming phospho-serine at P(ϩ4). The phosphate at P(ϩ4) occupies the same position as the sulphonate of the bound HEPES. The rotamer of Tyr 216 was changed to an anti conformation to eliminate steric clashes. (B) Comparison of the conformations of the activation segment tyrosine in GSK3␤ (left) and the phosphorylated equivalent tyrosine in activated ERK2. Simple rotation of the side chain of Tyr 216 in GSK3␤ around the C␣-C␤ bond brings it into the same position as the pTyr 185 in ERK2, and, if phosphorylated, would be stabilized in that conformation by interaction with Arg 220 and Arg 223.

Mechanism of Inhibition by SER 9 Phosphorylation (Figure 5A); however, clear dose-dependent inhibition Insulin signaling promotes phosphorylation of Ser 9 in of GSK3␤ kinase activity was observed with the identical GSK3␤ by PKB/Akt (Cross et al., 1995), with consequent peptide when Ser 9 was phosphorylated (Figure 5B). An inhibition of GSK3␤. The structural basis for this has not unrelated peptide containing a phosphoserine had no been defined, but could involve one of two mechanisms. effect on GSK3␤ activity at comparable concentrations Phosphorylation at Ser 9 could be an allosteric effect, (Figure 5C), showing that the inhibition was not simply promoting a catalytically inactive conformation, in an mediated by a phosphate group, but dependent on the inverse version of the more commonly observed phos- sequence context of the phospho-serine and therefore phorylation-dependent activation found in many protein specific. Detailed kinetic analysis (Figure 5D) clearly kinases. Alternatively, the phosphorylated N-terminal re- showed competitive inhibition by the phosphorylated N gion might be directly autoinhibitory, blocking access (3–12) peptide, with a Ki of Ϸ 700 ␮M. While this inhibi- to the active site and/or substrate binding cleft, but only tion, although specific, is relatively weak in these inter- when phosphorylated. To distinguish between these molecular in vitro assays, in vivo, the peptide is cova- mechanisms, we analyzed GSK3␤ activity in the pres- lently attached to the enzyme so that binding is an ence of a peptide based on its N-terminal segment intramolecular reaction with a very large effective con- (3–12), including the phosphorylatable Ser 9. No inhibi- centration. tion was observed with the unphosphorylated peptide The requirement for phosphorylation and competition Cell 728

Figure 5. Competitive Inhibition by Phosphorylated N Terminus (A) Phosphorylation of GSM peptide (Km Ϸ 70 ␮M) by GSK3␤ in vitro in the presence of a peptide derived from residues 3–12 of GSK3␤. The activity is undiminished even in the presence of 5-fold excess of GSK3␤-N(3-12) peptide. All points in (A), (B), and (C) are averages of two measurements. (B) As (A), but with varying concentrations of GSK3␤-N(3-12) peptide in which Ser 9 is phosphorylated, resulting in clear inhibition of substrate phosphorylation. (C) As (A) but with varying concentrations of an unrelated peptide containing a phosphoserine. As in (A), no inhibition of GSM phosphorylation by GSK3␤ is observed. (D) Double-reciprocal (Lineweaver-Burk) plot of reaction velocity versus substrate concentration in the presence of increasing concentrations of the Ser 9 phosphorylated GSK3␤-N(3-12) peptide. All points are means of three measurements. The progressive increase in slope indicates competitive inhibition, with a Ki for the peptide of Ϸ 700 ␮M. (E) Inhibition of GSK3␤ activity via direct competitive autoinhibition by the N terminus binding in the substrate binding site with the phosphory- lated Ser 9 occupying the P(ϩ4) phospho-priming binding site. The segment connecting the bound N terminus and the visible N terminus of GSK3␤ in the present crystal is shown as a dotted magenta line, whose detailed path is purely hypothetical. Structure of GSK3␤ 729

Figure 6. GSK3␤ Dimer Interactions (A) Secondary structure cartoon of GSK3␤ crystal dimer, viewed approximately down the noncrystallographic diad axis. Monomers are color- ramped blue-red from N to C terminus on the left and by chain on the right. (B) Stereo pair of solvent accessible surface for the two monomers calculated separately and colored by chain. (C) Comparison between the dimer interactions in the vicinity of the activation segment from one monomer (color ramped), with Asp 260, Ser 261, and Val 262 from the other monomer (LEFT), and the same region in the substrate-complex modeled on the structure of the phosphorylase kinase–substrate complex 2PHK (right). The side chain of Ser 261 in the dimer occupies approximately the same site as the phosphorylatable serine in the substrate peptide, close to the side chains of the catalytic residues Asp 181 and Asp 200 (in red). with substrate peptide suggests a mechanism for autoin- inhibition could occur in cis, with 24 residues available hibition in which the phosphorylated N-terminal segment to bridge the Ϸ 42 A˚ between the visible N terminus in binds in the substrate binding cleft as a pseudo-primed the present GSK3␤ structure (Ser 35), and pSer9 at the substrate, with the phosphorylated Ser occupying the C terminus of the bound inhibitory peptide (Figure 5E). Pϩ4 phospho-Ser/Thr binding site. With the N-terminal Residues 1–34 lie outside the canonical protein kinase segment thus positioned, the P0 binding site, in which fold, and are completely disordered in this present the phospho-accepting serine or threonine of a real sub- GSK3␤ crystal structure, so that this model for autoin- strate would be located, would be occupied by Pro 5. hibition remains to be verified structurally. A proline at this position would be readily accommo- dated, as its side chain has a similar volume to serine or Dimer Structure threonine, and can conform to the main chain substrate GSK3␤ crystallized as a dimer, with the monomers re- conformation observed at this position in other Ser/Thr lated by a noncrystallographic diad (Figures 6A and 6B). kinase-peptide complexes. With an inhibitory Ser 9-phos- Dimers have been observed in crystals of other protein phorylated N-terminal segment bound in that way, auto- kinases, including ERK2 (Canagarajah et al., 1997) and Cell 730

may have functional significance (Cobb and Goldsmith, strates with Pϩ4 priming phosphorylation. Together 2000). However, the GSK3 dimer more closely resembles with our observation that peptides derived from the N the head-to-tail interaction observed in phosphorylase terminus of GSK3␤ competitively inhibit its kinase activ- kinase (Lowe et al., 1997). The main sites of dimeric ity, this suggests a model for the mechanism of inhibition interaction are residues Asp 260 to Val 263 at the N of GSK3␤ following phosphorylation of Ser 9 by PKB/Akt terminus of an ␣ helix (262–273) from one monomer, in response to the insulin signal. At least one apparent which interacts with Tyr 216 to Arg 220 at the end of the GSK3␤ substrate, Cyclin D1, is phosphorylated at a loca- activation segment in the other. Direct polar interactions tion in which the Pϩ4 residue is valine (Diehl et al., 1998), are limited to an ion pair between Asp 260 and Arg 220, suggesting that unprimed phosphorylation can occur and main chain to main chain hydrogen bonds between under some circumstances. GSK3␤ is specifically inhib- the peptide carbonyls of Asp 260 and Val 263 with the ited by lithium ions (Stambolic et al., 1996), and this is peptide nitrogens of Arg 220 and Tyr 216, respectively. a source of considerable medical interest, as lithium is The hydrophobic face of the side chain of Tyr 216 inter- a valuable agent in the treatment of depressive disease acts with a hydrophobic patch formed by Ile 228, Phe (Williams and Harwood, 2000). Even at much higher res- 229, Gly 262, Val 263, and Leu 266 from the other mono- olution than we have so far obtained, it will be very mer. Despite the overall shape complementarity at the difficult to identify the lithium binding site as it is virtually interface, there are few other direct contacts, although invisible to X-rays. several solvent-bridged interactions are evident. Over- The major questions remaining in the molecular biol- all, dimer formation buries Ϸ 2358 A˚ 2 of accessible sur- ogy of GSK3␤ relate to its ability to operate in two dis- face, which is slightly more than in the PhK dimer (Lowe tinct signaling pathways with no apparent cross-talk. et al., 1997). The positions occupied in one monomer Insulation of the Wnt and insulin signals is achieved by the segment Asp 260–Ser 261–Val 262 from the other by recruitment of GSK3␤ into a multiprotein complex coincides approximately with the expected location of containing Axin and APC, which facilitates phosphoryla- the PϪ1, P0, and Pϩ1 residues of a substrate peptide. tion of ␤-catenin, and allows GSK3␤ activity to be regu- Productive binding of even a small peptide substrate is lated by Dishevelled in response to Wnt binding to the thus precluded by the observed dimer. However, within Frizzled receptor. Structural studies of the specific inter- the dimer, both the position and conformation of Ser actions between GSK3␤ and the components of the 261 are close to those expected for the phosphorylata- Wnt-signalosome hold the key to a full understanding ble serine of a productively bound substrate peptide of this system. (Figure 6C). This raises the possibility that these dimer interactions are on the path to a productive trans-phos- phorylation of Ser 261, although no such phosphoryla- Experimental Procedures tion has been reported so far. Cloning, Expression, and Purification The presence of an intimate dimer prompts consider- Human GSK3␤ was expressed in the Bac-to-Bac baculovirus ex- ation of its biological relevance. With GSK3␤, we can pression system (Life Technologies). The hGSK3␤ gene was ampli- detect dimer formation in solution by cross-linking, at fied by PCR from an existing clone, and inserted as an NcoI/XhoI concentrations as low as 5 ␮M, suggesting that dimer- fragment into the donor plasmid, pFASTBAC HTa. This construct ization is not an artifact of crystallization. While the pres- prepends a His6-affinity purification tag and a cleavage site for the ent data does not allow a definitive assessment of its TEV protease to the N terminus of the GSK3␤ peptide sequence. functional relevance, the structure of the GSK3␤ dimer The construct was sequenced to ensure no errors had been intro- duced. An apparent His-Leu mutation was found at codon 350 in at least allows us to predict several properties that would the PCR product, but was also found to be leucine in the original bear upon a putative biological role. First, the substrate template cDNA. In sequences from other species, this position is a binding site is obstructed by dimerization, so that the conserved leucine/hydrophobic residue, and is buried in the crystal dimer would be inactive in heterophosphorylation, but structure. We believe that the original assignment of histidine for potentially active in homophosphorylation in trans.As this codon in the deposited peptide sequence (Swissprot a corollary, N-terminal autoinhibition in the manner we KG3B_HUMAN) is wrong and that leucine is correct. suggest and dimerization would be mutually exclusive. The recombinant pFASTBAC HTa plasmid was transformed into DH10Bac cells for transposition of the GSK3␤ gene into the bacmid Second, as the side chain of Tyr 216 in the “down” according to manufacturer’s instructions, and transposition was conformation participates in the dimer interface, its confrmed by PCR. Bacmids were transfected into Sf9 cells (2.0 ϫ phosphorylation and consequent rotation to the up con- 105 to 2.0 ϫ 106 cells/ml) in SF900II media supplemented with penicil- formation would be antagonistic, at least in part, to di- lin and streptomycin, and grown in shaker flasks at 27ЊC, 150 rpm., merization. Dimerization of GSK3␤ in vivo could be regu- according to manufacturer’s protocols (Life Technologies). Virus lated by interaction with other proteins in the APC-Axin was amplified using a multiplicity of infection (MOI) of 0.1, a cell density of 2.0 ϫ 106 and a 3 day incubation. Expression was with scaffolded Wnt-regulated complex. In support of that ϫ 6 ␤ an MOI of 2, a cell density of 2.0 10 and a 3 day incubation. Cells idea, we have observed disruption of GSK3 dimeriza- were harvested by centrifugation at 1000 ϫ g for 5 min, and cell tion in cross-linking experiments, on addition of other pellets were stored at Ϫ80ЊC. Cells were lysed by thawing, hand components of the Wnt signaling complex (in prepara- homogenizing in 50 mM HEPES-NaOH and 50 mM NaCl (pH 7.2) tion), but the biological significance of this remains to supplemented with protease inhibitors, and sonicating for 1 min. The be determined. clarified supernatant was applied to an SP-Sepharose (Pharmacia) column (2.6 ϫ 15 cm), and the protein was eluted using a gradient of 0–1 M NaCl in 50mM HEPES-NaOH (pH 7.2). Fractions were Conclusions (and applied to a Talon (Clontech ,7.6ف pooled, pH adjusted to column (1.6 ϫ 2.5 cm), which was washed with 50 mM HEPES- The structure of GSK3␤ presented here provides a satis- NaOH, 300 mM NaCl and 5 mM Im-HCl (pH 7.6), and the protein factory explanation for the preference of GSK3␤ for sub- eluted with 50 mM HEPES-NaOH, 300 mM NaCl, and 200 mM Im-HCl Structure of GSK3␤ 731

(pH 7.0). One mM EDTA was added, and the protein was concen- on kinase assays, Maggie Yeo for practical assistance, and Adrian trated and applied to a Superdex 200 column (Pharmacia, 26/60) Harwood, David Barford, and Chris Marshall for helpful discussion. developed with 20 mM HEPES-NaOH, 150 mM NaCl, 1 mM EDTA, This work was supported by the Cancer Research Campaign (T.C.D.) and 1 mM DTT (pH 7.2). Fractions were applied to a Mono-S (Phar- and the Institute of Cancer Research Structural Biology Initiative macia) 1 ml column, and eluted with a gradient of 0.15–1 M NaCl (L.H.P.). in 20 mM HEPES-NaOH, and 1 mM DTT (pH 7.2). The protein was ف concentrated to 10 mg/ml using a 0.5 ml Microcon centrifugal Received November 21, 2000; revised May 3, 2001. concentrator (Amicon), and MgCl2 added to a final concentration of 2 mM. Purified GSK3␤ was stored at Ϫ80ЊC. References Crystallization and Data Collection Crystallization trials of hGSK3␤ were conducted at 10 mg/ml using Aberle, H., Bauer, A., Stappert, J., Kispert, A., and Kemler, R. (1997). ␤ Hampton Research Screens I and II, in 2 ␮l microbatch experiments -catenin is a target for the ubiquitin-proteasome pathway. EMBO under mineral oil. Small elongated bipyramids were observed in J. 16, 3797–3804. several experiments, and conditions optimized in hanging drop ex- Brunger, A.T., Adams, P.D., Clore, G.M., DeLano, W.L., Gros, P., periments by mixing 1 ␮l of protein (4 mg/ml in 20 mM HEPES- Grosse-Kunstleve, R.W., Jiang, J.S., Kuszewski, J., Nilges, M.,

NaOH, 500 mM NaCl, 2 mM MgCl2, and 1 mM DTT [pH 7.2]) with Pannu, N.S., et al. (1998). Crystallography & NMR system: a new 1 ␮l precipitant containing 6% PEG 8000 and 100 mM Tris-HCl (pH software suite for macromolecular structure determination. Acta 7.5). Data to 2.8 A˚ were collected from a single crystal at 100K on Crystallogr. D54, 905–921. ␭ϭ ˚ Station 14.2 ( 1.488 A) at the SRS Daresbury Laboratory, and Canagarajah, B.J., Khokhlatchev, A., Cobb, M.H., and Goldsmith, recorded on an ACSD scanner. Images were integrated using E.J. (1997). Activation mechanism of the MAP kinase ERK2 by dual MOSFLM (Leslie, 1995), and reduced and scaled using programs phosphorylation. Cell 90, 859–869. of the CCP4 suite (CCP4, 1994). GSK3␤ crystallized in P2 2 2 with 1 1 1 CCP4 (1994). Programs for protein crystallography. Acta Crystallogr. cell dimensions a ϭ 83.20 A˚ ,bϭ 86.06 A˚ , and c ϭ 178.09 A˚ . The D50, 760–763. crystal volume was consistent with a solvent content of Ϸ 61% by volume, and two molecules in the asymmetric unit. In support of Chen, P., Luo, C., Deng, Y., Ryan, K., Register, J., Margosiak, S., this, a self-rotation function suggested a noncrystallographic diad Tempczyk-Russell, A., Nguyen, B., Myers, P., Lundgren, K., et al. close to the ab face diagonal. Statistics for the data collection are (2000). The 1.7 A˚ crystal structure of human cell cycle checkpoint given in Table 1. kinase Chk1: implications for Chk1 regulation. Cell 100, 681–692. Cobb, M.H., and Goldsmith, E.J. (2000). Dimerization in MAP-kinase Structure Determination and Refinement signalling. Trends Biochem. Sci. 25, 7–9. The structure of hGSK3␤ was determined by molecular replacement Cohen, P. (1999). The Croonian Lecture 1998. Identification of a using AmoRe (Navaza, 1994). Structures of several protein kinases protein kinase cascade of major importance in insulin signal trans- present in the Protein Data Bank were used as search models, duction. Phil. Trans. Roy. Soc. Lond. B354, 485–495. but an inhibitor complex of ERK2 kinase (PDB Code 1PME) gave convincing rotation and translation function solutions at 4 A˚ , with Cox, S., and Johnson, L.N. (1992). Expression of the phosphorylase ␥ good lattice packing. The search model consisted of residues 6–324, kinase subunit catalytic domain in E.coli. Prot. Eng. 5, 811–819. corresponding to Ϸ 75% of the hGSK3␤ sequence with Ϸ 31% Cross, D.A.E., Alessi, D.R., Cohen, P., Andjelkovich, M., and Hem- sequence identity. Two solutions were identified, approximately re- mings, B.A. (1995). Inhibition of glycogen-synthase kinase-3 by insu- lated by the observed noncrystallographic diad, generating an inti- lin-mediated by protein-kinase-B. Nature 378, 785–789. mate dimer. The ERK2 structure was used as a template to construct DeBondt, H.L., Rosenblatt, J., Jancarik, J., Jones, H.D., Morgan, ␤ a starting model of the core of hGSK3 , which was reorientated to D.O., and Kim, S.-H. (1993). Crystal structure of cyclin dependent the molecular replacement solutions to generate the noncrystallo- kinase 2. Nature 363, 592–602. graphic dimer. The N and C domains of each of the monomers were refined against the 2.8 A˚ data as rigid bodies, and then by torsional Diehl, J.A., Cheng, M.G., Roussel, M.F., and Sherr, C.J. (1998). Gly- molecular dynamics simulated annealing in CNS (Brunger et al., cogen synthase kinase 3 regulates cyclin D1 proteolysis and subcel- 1998), with application of noncrystallographic symmetry restraints lular localization. Genes Dev. 12, 3499–3511. relating equivalent atom in the two monomers. Difference maps Farr, G.H., Ferkey, D.M., Yost, C., Pierce, S.B., Weaver, C., and were used to rebuild the initial model in O (Jones et al., 1991), and Kimelman, D. (2000). Interaction among GSK-3, GBP, axin, and APC many cycles of refinement and manual rebuilding were required to in Xenopus axis specification. J. Cell. Biol. 148, 691–701. achieve the current model, which consists of 5567 protein atoms, Fiol, C.J., Haseman, J.H., Wang, Y.H., Roach, P.J., Roeske, R.W., 177 solvent atoms (including two glycerol molecules), and 24 buffer Kowalczuk, M., and Depaoliroach, A.A. (1988). Phosphoserine as a ϭ ϭ atoms, with an R factor 0.220 and an R free 0.254 for 5% of recognition determinant for glycogen-synthase kinase-3 -phosphor- the data omitted from refinement. Coordinates have been deposited ylation of a synthetic peptide based on the G-component of protein in the Protein Data Bank with PDB code1H8F. -1. Arch. Biochem. Biophys. 267, 797–802.

N-Terminal Peptide Inhibition Fiol, C.J., Wang, A.Q., Roeske, R.W., and Roach, P.J. (1990). Ordered Peptides for inhibition assays were prepared by solid-phase synthe- multisite protein-phosphorylation—analysis of glycogen-synthase sis, HPLC purified, and verified by mass-spectrometry. Peptides kinase-3 action using model peptide-substrates. J. Biol. Chem. 265, were—S9: residues 3–12 of GSK3␤ (GRPRTTSFAE); PS9: as S9, but 6061–6065. with phosphoserine at position 9 (GRPRTTS(p)FAE); and RP: peptide Flotow, H., Graves, P.R., Wang, A., Fiol, C.J., Roeske, R.W., and of unrelated sequence to GSK3␤ but also containing a phosphoser- Roach, P.J. (1990). Phosphate groups as substrate determinants ine (QRRAS(p)DDGK). The phospho-primed, glycogen-synthase- for casein kinase I action. J. Biol. Chem. 265, 14264–14269. ␤ derived substrate peptide GSM and GSK3 kinase assay have been Groden, J., Thliveris, A., Samowitz, W., Carlson, M., Gelbert, L., ␤ described previously (Ryves et al., 1998). GSK3 activity was deter- Albertsen, H., Joslyn, G., Stevens, J., Spirio, L., Robertson, M., et ␤ mined in assays containing 2 ng or 50 ng of recombinant GSK3 al. (1991). Identification and characterization of the familial adeno- ␮ ␥32 Њ and 100 M ATP (containing P-ATP), at 30 C, in a buffer containing matous polyposis-coli gene. Cell 66, 589–600. 50 mM HEPES (pH 8.0), 12.5 mM MgCl2, 2 mM DTT, 0.05 mM EDTA, and varying concentrations of GSM and “inhibitory” peptide. Hart, M.J., de los Santos, R., Albert, I.N., Rubinfeld, B., and Polakis, P. (1998). Downregulation of ␤-catenin by human Axin and its associ- ␤ Acknowledgments ation with the APC tumor suppressor, -catenin and GSK3 . Curr. Biol. 8, 573–581. We are grateful to Pawel Dokurno for assistance with data collection, He, T.-C., Sparks, A.B., Rago, C., Hermeking, H., Zawel, L., da Costa, Jackie Metcalfe for synthesis of peptides, Jonathan Ryves for advice L.T., Morin, P.J., Vogelstein, B., and Kinzler, K.W. (1998). Identifica- Cell 732

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