Phosphoserine/Threonine Binding Minireview Domains: You Can’T Pserious?

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Michael B. Yaffe,*‡ and Stephen J. Smerdon†‡ 14-3-3 Proteins *Center for Cancer Research The term “14-3-3” denotes a family of dimeric ␣-helical Massachusetts Institute of Technology pSer/Thr binding proteins present in high abundance in 77 Massachusetts Avenue, E18-580 all eukaryotic cells [1]. 14-3-3 proteins were the first Cambridge, Massachusetts 02139 molecules to be recognized as distinct pSer/Thr binding † Division of Protein Structure proteins, forming tight complexes with phosphorylated National Institute for Medical Research ligands containing either of two sequence motifs, R[S/ The Ridgeway Ar]XpSXP and RX[Ar/S]XpSXP, respectively, where pS Mill Hill denotes both phosphoserine and phosphothreonine, London NW7 1AA and Ar denotes aromatic residues [2, 3]. In addition, a United Kingdom few 14-3-3 binding ligands have been identified whose sequences deviate significantly from these motifs or do not require phosphorylation for binding. Summary Over 50 distinct substrates have been identified that bind to 14-3-3, many of which play critical roles in regu- The fundamental biological importance of protein phos- lating progression through the cell cycle, activation of phorylation is underlined by the existence of more than the Erk1/2 subfamily of MAP kinases, initiation of apo- 500 protein kinase genes within the human genome. In ptosis, and control of gene transcription and chromo- many cases, phosphorylation on serine, threonine, and some remodeling. Both the maintenance of cells in in- tyrosine residues creates binding surfaces for a variety terphase [4], for example, and the G2/M cell cycle arrest of phospho-amino acid binding proteins/modules. observed in rapidly dividing cells following DNA damage Here, we review the insights into serine/threonine phos- [5] involve the binding of 14-3-3 proteins to a specific phorylation-dependent signal transduction processes phosphorylated form of the mitotic regulatory tyrosine provided by structures of several of these proteins and phosphatase Cdc25. The resulting 14-3-3:Cdc25 com- their complexes. plex localizes to the cytoplasm [6–9], blocking Cdc25’s access to its nuclear substrate, Cdc2-CyclinB, and Introduction thereby regulating mitotic entry. The binding of 14-3-3 Signal transduction in eukaryotic cells involves the as- dimers to phosphorylated forms of A-Raf, B-Raf, and sembly of various protein and lipid kinases and phos- c-Raf-1 plays a complex and incompletely understood role phatases along with other enzymes, adaptors, and regu- in Raf activation and MAP kinase signaling, probably latory molecules into large protein complexes. These through a combination of stabilizing the active form of multicomponent signaling machines integrate and trans- Raf, facilitating the interaction of Raf with downstream mit information to control ion fluxes, cytoskeletal re- effectors, and maintaining inactive Raf in a reactivatable arrangements, patterns of gene expression, cell cycle conformation [10–12]. Several 14-3-3-regulated ligands progression, and programmed cell death. The dynamic are physiological effectors of apoptosis, including phos- nature of signaling events requires rapid assembly and phorylated forms of the kinase ASK1 [13], the Forkhead disassembly of signaling complexes, events that are transcription factor FKHRL1 [14], and the Bcl-2 family generally regulated through protein phosphorylation. member BAD [15]. Other 14-3-3 binding molecules in- Historically, attention has focused on the assembly of clude several transcriptional activators and coactivators multiprotein complexes that were regulated by tyrosine [16], human telomerase [17], and histone deacetylases phosphorylation of transmembrane receptors to gener- 4 and 5 [18]. ate binding sites for proteins with modular SH2 and PTB Through what feat of structural gymnastics do 14-3-3 domains. In contrast, phosphorylation of proteins on proteins accomplish their phosphospecific recognition serine and threonine residues was thought to regulate while binding to such a diverse array of ligands? Some protein function largely through allosteric modifications insight into this dilemma has emerged through studies rather than by directly mediating protein–protein inter- of 14-3-3:phosphopeptide complexes. actions. Within the last few years, however, a variety of Each monomer of the dimeric 14-3-3 molecule con- signaling molecules and modular domains have been tains nine ␣ helices, with the individual monomeric sub- identified that specifically bind to short pSer/Thr-con- units related to each other by C2 symmetry [19, 20] taining motifs, recruiting these substrates into protein– (Figure 1). The three N-terminal helices of each monomer protein signaling complexes in response to phosphory- interact extensively with those of the opposing mono- lation by Ser/Thr kinases. In this review, we focus on the mer through a combination of salt bridges, hydrogen identification of these new pSer/Thr binding modules, bonds, and van der Waals interactions, forming a 35 ϫ including 14-3-3 proteins, WW domains, and FHA do- 35 ϫ 20 A˚ wide central channel suitable for binding to mains; their physiological functions and the structural peptide and protein ligands. The residues lining this basis for their pSer/Thr motif recognition. central channel show extensive sequence conservation among all seven 14-3-3 isotypes found in mammalian ‡ To whom correspondence should be addressed (e-mail: myaffe@ cells [19]. Three basic residues, Lys49, Arg56, and mit.edu, [email protected]). Arg127, together with Tyr128, emerge from helices C Structure R34

phosphates is not unique to the all-helical 14-3-3 proteins. The ␤ sheet–based SH2 and PTB domains bind to all or part of their tyrosine-phosphorylated ligands in an extended conformation and use a similar pattern of basic and hydroxyl-containing side chains to form multiple ionic and hydrogen bonds with the phosphotyrosine phosphate. Two Arg residues in SH2 domains make important contributions: there is a bidentate ionic interaction between two of the guanidino nitrogens in a buried Arg residue, ␤B5, and two of the phosphate oxygens and a combined salt bridge and amino-aromatic interaction between guanidino nitrogens in Arg ␣A2 and the Tyr phosphate and ring system, respectively [21, 22]. In addition, there are interactions involving the amino side chain of Lys ␤D6, the hydroxyl of Ser ␤B7, the main chain NH of Glu BC1, and the hydroxyl of Thr/ Ser BC2 in the Src and Lck SH2 domain structures [22, 23]. Likewise, in the Shc PTB domain, the phosphotyrosine interaction involves two Arg residues (Arg175 and Arg67), the amino group on a Lys side chain (Lys169), and the hydroxyl side chain of Ser151 [24]. Thus, it would appear that 14-3-3 proteins, most SH2 domains, and a number of PTB domains have independently evolved a similar arrangement of amino acid side chains for high- affinity phospho-amino acid binding. WW Domains Figure 1. 14-3-3 Proteins Bind Phosphoserine Peptides within a Large Central Channel WW domains constitute one of the smallest modules Each monomeric subunit of the dimer, shaded blue and red, respec- found widely distributed in signaling proteins, often in tively, is composed of nine ␣ helices (␣A–␣I), with each monomer association with HECT family ubiqitin ligase domains, simultaneously binding a phosphopeptide. A single protein ligand, proline isomerase domains, PTB domains, and PDZ do- amino acids folded into three 40ف when phosphorylated on two sites, probably binds in a similar man- mains. Comprised of ner to both monomers of the 14-3-3 dimer. Alternatively, two pro- antiparallel ␤ strands, WW domains bind to short proteins with single phosphorylation sites may bind to each monomer line-rich sequences containing PPXY, PPLP, or PPR individually, allowing 14-3-3 to function as a molecular adaptor. The phosphoserine phosphate is coordinated by a basic pocket formed motifs [25, 26] (Figure 2). Two proteins, the proline isom- from the indicated residues emerging from two of the ␣ helices erase Pin1/Ess1 and the ubiquitin ligase Nedd4/Rsp5p, (bottom). A loosely similar arrangement of basic and hydroxyl-con- however, contain WW domains that can specifically rec- taining side chains coordinates the peptide phosphotyrosine phos- ognize pSer-Pro and pThr-Pro motifs [27]. Intriguingly, phate in SH2 and some PTB domain complexes. the proline isomerase domain of Pin1 catalyzes the specific cis-trans isomerization of pSer/Thr-Pro bonds, in contrast to the conventional proline isomerases in the and E to form a positively charged pocket located to- FKBP and cyclophilin families [28]. Since many Pin1 ward one end of each edge of the central channel in substrates are mitotic phosphoproteins, the conforma- what is otherwise a largely negatively charged molecule. tional changes induced upon proline isomerization have Each of the residues in this basic pocket interacts di- been proposed to regulate mitotic entry and exit. For rectly with the peptide phosphate group, forming five two of Pin1’s phosphorylated substrates, Cdc25C and ionic bonds and a hydrogen bond to three of the phos- Tau, proline isomerization facilitates their subsequent phate oxygens (Figure 1) [2, 3]. This strong network of dephosphorylation by the Ser/Thr phosphatase PP2A interactions, like the fingers of a hand balancing a [29], an event which requires the pSer-Pro bond to be squash ball, forms the critical anchor that orients the in trans. Curiously, the Pin1 WW domain only binds to remainder of the peptide, arguing for the importance of pSer-Pro peptides in the trans conformation [30], sug- ligand phosphorylation in high-affinity 14-3-3 binding. gesting that one function of the WW domain may be The phosphopeptide main chain is stabilized in an ex- to stabilize the trans-isomerase product in preparation tended conformation by a series of hydrogen bonds for dephosphorylation. The Saccharomyces cerevisiae between the peptide backbone NH and CO groups with Pin1 homolog, Ess1, regulates transcription and mitosis the side chains of residues in helices E, G, and I. How- through binding the hyperphosphorylated C-terminal ever, a sharp alteration in peptide chain direction occurs domain of RNA polymerase II in a region that contains at a position that is two residues C-terminal to the pSer. 26–52 repeats of the motif YSPTSPS [31]. The struc- Most physiological 14-3-3 binding proteins as well as ture of Pin1 complexed to the serine-phosphorylated optimal 14-3-3 binding peptides contain a Pro residue YpSPTpSPS peptide has provided a structural basis for at this position. The major effect of this kink is to redirect understanding the specificity displayed by some WW the chain direction away from 14-3-3 and back into the domains toward phosphorylated ligands [30]. Overall, central channel. the number of specific contacts between the ligand’s This type of four-fingered arrangement for “gripping” phosphoserine phosphate and the WW domain’s side Minireview R35

Figure 2. The Pin1 WW Domain Binds Phos- phoserine-Proline Motifs The left panel shows the two Trp residues, for which these three-stranded ␤ sheet structures are named, along with the trans conformation of the pSer-Pro bonds in the bound phosphopeptide from RNA polymerase II (shaded purple). The right panel shows that oxygen atoms from only one of the two pSer phosphates interact directly with side chains from residues in the ␤1/2 loop. This pattern of phosphoserine coordination, together with a water-mediated hydrogen bond from a Tyr residue within ␤2, is less than that seen in 14- 3-3 and SH2 domain structures, consistent with the finding that only a subset of WW domains show phospho-specific binding. chains was reduced compared to that seen in 14-3-3 ipate in phosphotyrosine signaling processes in higher proteins (Figure 2). All of the phosphate contacts are eukaryotes. made between the peptide’s second pSer and two resi- The in vivo binding partners for most FHA domain dues in the ␤1–␤2 loop (Ser16 and Arg17) along with proteins are unknown. Data regarding the specificity one in the ␤2 strand (Tyr23). The Arg guanidino group of different FHA domains for phosphothreonine-based participates in a bidentate interaction with two of the sequence motifs, however, emerged from peptide li- phosphate oxygens along with a hydrogen bond from brary experiments [39]. FHA domains were found to rec- the Arg17 main chain NH, while the Ser residue forms ognize short sequence motifs bracketing the central a hydrogen bond with a third. Moreover, there is an phosphothreonine residue, with selection extending from additional water-mediated hydrogen bond between Tyr23 the pTϪ4 to the pTϩ3 position. In many cases, the selec- and one of the phosphate oxygens. Residues function- tion was strongest in the pTϩ3 position, allowing a tenta- ally equivalent to Ser16 and Arg17 are absent in the vast tive grouping of FHA domains into discrete classes on majority of WW domains, suggesting that the pSer/pThr the basis of pTϩ3 specificity. All FHA domains reported binding function may be limited to a small subset of WW to date are pThr-specific, as pSer substitution eliminates domains. peptide binding [39]. FHA Domains NMR analysis of the C-terminal FHA domain (FHA2) Forkhead-associated (FHA) domains are found in a vari- from S. cerevisiae Rad53 revealed the overall fold and ety of transcriptional control proteins, DNA damage– identified a likely interacting surface for phosphopeptide activated protein kinases, cell cycle checkpoint pro- binding [37]. The high-resolution crystal structure of teins, phosphatases, kinesin motors, and regulators of FHA1 from Rad53p in complex with a phosphothreo- small G proteins in organisms ranging from mycobac- nine-containing peptide ligand (SLEVpTEADATFAKK) teria and yeast to higher plants and humans. In humans, derived from Rad9p revealed the detailed architecture many FHA domain–containing proteins appear to be of this class of signaling module and the unusual fea- involved in processes protecting against cancer, since tures of phospho-dependent binding [39]. Both struc- mutations in, or inactivation of, several FHA proteins, tures show the FHA domain to consist of an 11-stranded including Chk2, p95/Nbs1, and Chfr, have been postu- ␤ sandwich that has an identical ␤ strand topology to lated to play a role in tumor formation. Originally identi- the MH2 domains of Smad tumor suppressor molecules fied as 55–75 amino acid modules by sequence profiling (Figure 3). In both structures, the FHA homology region [32], a variety of biophysical studies have now demon- encompasses only six of the ␤ strands that largely form strated that FHA domains are considerably larger, en- one side of the ␤ sandwich. The N- and C-terminal ␤ compassing 130–140 amino acids. Several independent lines of evidence led to the recognition that FHA do- strands lie adjacent to one another on the nonconserved mains were phosphothreonine binding modules, includ- face of the domain. Indeed, the proximity of the N and ing work showing that FHA domains played critical roles C termini is consistent with the modular nature of the in the interaction between the protein phosphatase FHA domain as an independently folded binding module. KAPP and phosphorylated receptor-like kinases in Ara- The Rad53 FHA1:phosphopeptide complex reveals bidopsis [33, 34] and between the cell cycle checkpoint that ligand binding occurs at one end of the molecule, kinase Rad53p and the phosphorylated DNA damage where it interacts with residues from the loops between control protein Rad9p in S. cerevesiae [35, 36]. Never- ␤3/4, ␤4/5, and ␤6/7 (Figure 3). The conserved residues theless, the work of Tsai and coworkers [37] has shown within the FHA region either interact directly with the that Rad53 FHA2 is able to bind peptides containing peptide or form the structural framework upon which phosphotyrosine [37, 38]. Although the biological signifi- the peptide binding loops are presented. Of the six most cance of pTyr-dependent interactions in budding yeast highly conserved residues in the FHA family, three make remains to be demonstrated, these data indicate that interactions with the peptide and, of these, Arg70 and FHA domains may constitute a rather promiscuous class Ser85 bind directly to the pThr residue itself. Most of of phospho-dependent modules that may indeed partic- the phospho-independent contacts are formed between Structure R36

Figure 3. Structure of the Rad53 FHA1:Rad9 Phosphopeptide Complex The fold topology of the FHA domain, an 11- stranded ␤ sandwich, and the phosphopeptide binding loops are indicated in the left panel. Two critical Arg residues (green) interact with the phosphothreonine phosphate and the pTϩ3 Asp residue in the Rad9 phosphopeptide (purple). Residues participating in phosphothreonine phosphate coordination are shown in the upper right panel. In addition, there is a pocket in the molecular surface of the FHA domain that accepts the threonine ␥-methyl group (lower right).

FHA domain side chains and main chain atoms of the oxygens of Aspϩ3 form salt bridging interactions with peptide. In this way, the peptide chain is held in an N⑀ and N␩ atoms of Arg83 from the ␤4/5 loop. Intrigu- extended conformation similar to that observed in SH2, ingly, the type of residue selected at this position varies 14-3-3, and WW domain complexes. The phosphothreo- considerably in other FHA domains [39], but the basis nine side chain makes a total of five hydrogen bonds for selection at the ϩ3 position in these cases awaits with main and side chain atoms from Thr106 and Asn86 further structural and biochemical analyses of FHA do- and two of the highly conserved residues, Arg70 and main complexes. Ser85. Arg70 forms the side of a basic pocket and makes WD40 and LRR Domains of F-Box Proteins a charged hydrogen bond to the OG1 atom of the phos- Two other domains may function, in some circum- phothreonine The importance of this interaction is dem- stances, as pSer/Thr binding modules: the WD40 do- onstrated by mutagenesis of Arg70 to alanine, which mains and leucine-rich regions present within F-box pro- considerably lowers the binding affinity [39]. These ob- teins. servations are largely consistent with the recent NMR F-box proteins recognize substrates of SCF (Skp1- structure of Rad53 FHA2 in complex with a phosphotyro- Cdc53/Cullin-F-box) ubiquitin ligases, targeting them for sine peptide [38], although in this case, the phosphate phosphorylation-dependent ubiquitination and protea- moiety appears to be somewhat displaced from the ob- somal degradation [41, 42]. In addition to the F-boxes served position in the pThr complex, presumably due from which F-box proteins derive their names, most also to differences in the size of the phosphorylated side contain a WD40 or LRR (leucine-rich repeat) domain chain. that may be responsible for phosphosubstrate recogni- At present, the bulk of available biological and bio- tion. However, unlike 14-3-3, WW, and FHA domains, chemical data support the notions that in most cases, structural proof for direct binding of pSer/Thr peptides FHA domains function by specifically recognizing phos- to purified WD40 or LRR domains is lacking. LRR do- phorylated threonine residues in target proteins, and mains form multiple repeats of single ␣ helix/␤ strand substitution of pSer for pThr in model peptides abro- units that align to form a large surface area for interaction gates or severely compromises binding. It appears that with substrates [43]. How such a structure might recog- the pThr rotamer observed in the Rad53 FHA1 is deter- nize short pSer/Thr peptides is unclear. In contrast, mined by steric clashes and by hydrogen bonding be- WD40 domains adopt a ␤ propeller structure, which has tween the main chain N and a phosphate O. The threo- precedence for binding linear peptide motifs within nine ␥-methyl group sits in a cavity formed by side chain grooves formed by adjacent propeller blades [44]. and main chains atoms from Ser85, the side chains of Whether or not contributions from other components Thr 106 and Asn 107, and backbone atoms encom- within the ubiquitination complex play important roles passing Ser82–Ile84, forming extensive van der Waals in phosphospecific binding is currently unknown. interactions that would not be possible with a pSer substitution (Figure 3). Concluding Remarks As mentioned, many FHA domains show the strongest The identification of several families of pSer/Thr binding amino acid discrimination in the pTϩ3 position in ori- modules within the last four years presents new para- ented peptide screening experiments. In the case of digms for how cell signaling events can be regulated by Rad53 FHA1, these methods select strongly for aspartic Ser/Thr phosphorylation. The remarkable diversity of acid in the ϩ3 position [39, 40]. Again, the X-ray structure protein folds displayed by these domains, together with nicely explains this observation, since the carboxylate differences in how the ionized phosphate group is rec- Minireview R37

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The molecular basis Structure R38

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