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Threonine Binding Domains
Structure, Vol. 9, R33–R38, March, 2001, 2001 Elsevier Science Ltd. All rights reserved.
PhosphoSerine/Threonine Binding Domains: You Can’t pSERious? Michael B. Yaffe,*‡ and Stephen J. Smerdon†‡ * Center for Cancer Research Massachusetts Institute of Technology 77 Massachusetts Avenue, E18-580 Cambridge, Massachusetts 02139 † Division of Protein Structure National Institute for Medical Research The Ridgeway Mill Hill London NW7 1AA United Kingdom
Summary The fundamental biological importance of protein phosphorylation is underlined by the existence of more than 500 protein kinase genes within the human genome. In many cases, phosphorylation on serine, threonine, and tyrosine residues creates binding surfaces for a variety of phospho-amino acid binding proteins/modules. Here, we review the insights into serine/threonine phosphorylation-dependent signal transduction processes provided by structures of several of these proteins and their complexes. Introduction Signal transduction in eukaryotic cells involves the assembly of various protein and lipid kinases and phosphatases along with other enzymes, adaptors, and regulatory molecules into large protein complexes. These multicomponent signaling machines integrate and transmit information to control ion fluxes, cytoskeletal rearrangements, patterns of gene expression, cell cycle progression, and programmed cell death. The dynamic nature of signaling events requires rapid assembly and disassembly of signaling complexes, events that are generally regulated through protein phosphorylation. Historically, attention has focused on the assembly of multiprotein complexes that were regulated by tyrosine phosphorylation of transmembrane receptors to generate binding sites for proteins with modular SH2 and PTB domains. In contrast, phosphorylation of proteins on serine and threonine residues was thought to regulate protein function largely through allosteric modifications rather than by directly mediating protein–protein interactions. Within the last few years, however, a variety of signaling molecules and modular domains have been identified that specifically bind to short pSer/Thr-containing motifs, recruiting these substrates into protein– protein signaling complexes in response to phosphorylation by Ser/Thr kinases. In this review, we focus on the identification of these new pSer/Thr binding modules, including 14-3-3 proteins, WW domains, and FHA domains; their physiological functions and the structural basis for their pSer/Thr motif recognition. ‡ To whom correspondence should be addressed (e-mail: myaffe@ mit.edu, [email protected]).
PII S0969-2126(01)00580-9
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14-3-3 Proteins The term “14-3-3” denotes a family of dimeric ␣-helical pSer/Thr binding proteins present in high abundance in all eukaryotic cells [1]. 14-3-3 proteins were the first molecules to be recognized as distinct pSer/Thr binding proteins, forming tight complexes with phosphorylated ligands containing either of two sequence motifs, R[S/ Ar]XpSXP and RX[Ar/S]XpSXP, respectively, where pS denotes both phosphoserine and phosphothreonine, and Ar denotes aromatic residues [2, 3]. In addition, a few 14-3-3 binding ligands have been identified whose sequences deviate significantly from these motifs or do not require phosphorylation for binding. Over 50 distinct substrates have been identified that bind to 14-3-3, many of which play critical roles in regulating progression through the cell cycle, activation of the Erk1/2 subfamily of MAP kinases, initiation of apoptosis, and control of gene transcription and chromosome remodeling. Both the maintenance of cells in interphase [4], for example, and the G2/M cell cycle arrest observed in rapidly dividing cells following DNA damage [5] involve the binding of 14-3-3 proteins to a specific phosphorylated form of the mitotic regulatory tyrosine phosphatase Cdc25. The resulting 14-3-3:Cdc25 complex localizes to the cytoplasm [6–9], blocking Cdc25’s access to its nuclear substrate, Cdc2-CyclinB, and thereby regulating mitotic entry. The binding of 14-3-3 dimers to phosphorylated forms of A-Raf, B-Raf, and c-Raf-1 plays a complex and incompletely understood role in Raf activation and MAP kinase signaling, probably through a combination of stabilizing the active form of Raf, facilitating the interaction of Raf with downstream effectors, and maintaining inactive Raf in a reactivatable conformation [10–12]. Several 14-3-3-regulated ligands are physiological effectors of apoptosis, including phosphorylated forms of the kinase ASK1 [13], the Forkhead transcription factor FKHRL1 [14], and the Bcl-2 family member BAD [15]. Other 14-3-3 binding molecules include several transcriptional activators and coactivators [16], human telomerase [17], and histone deacetylases 4 and 5 [18]. Through what feat of structural gymnastics do 14-3-3 proteins accomplish their phosphospecific recognition while binding to such a diverse array of ligands? Some insight into this dilemma has emerged through studies of 14-3-3:phosphopeptide complexes. Each monomer of the dimeric 14-3-3 molecule contains nine ␣ helices, with the individual monomeric subunits related to each other by C2 symmetry [19, 20] (Figure 1). The three N-terminal helices of each monomer interact extensively with those of the opposing monomer through a combination of salt bridges, hydrogen bonds, and van der Waals interactions, forming a 35 ⫻ 35 ⫻ 20 A˚ wide central channel suitable for binding to peptide and protein ligands. The residues lining this central channel show extensive sequence conservation among all seven 14-3-3 isotypes found in mammalian cells [19]. Three basic residues, Lys49, Arg56, and Arg127, together with Tyr128, emerge from helices C
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Figure 1. 14-3-3 Proteins Bind Phosphoserine Peptides within a Large Central Channel Each monomeric subunit of the dimer, shaded blue and red, respectively, is composed of nine ␣ helices (␣A–␣I), with each monomer simultaneously binding a phosphopeptide. A single protein ligand, when phosphorylated on two sites, probably binds in a similar manner to both monomers of the 14-3-3 dimer. Alternatively, two proteins with single phosphorylation sites may bind to each monomer individually, allowing 14-3-3 to function as a molecular adaptor. The phosphoserine phosphate is coordinated by a basic pocket formed from the indicated residues emerging from two of the ␣ helices (bottom). A loosely similar arrangement of basic and hydroxyl-containing side chains coordinates the peptide phosphotyrosine phosphate in SH2 and some PTB domain complexes.
and E to form a positively charged pocket located toward one end of each edge of the central channel in what is otherwise a largely negatively charged molecule. Each of the residues in this basic pocket interacts directly with the peptide phosphate group, forming five ionic bonds and a hydrogen bond to three of the phosphate oxygens (Figure 1) [2, 3]. This strong network of interactions, like the fingers of a hand balancing a squash ball, forms the critical anchor that orients the remainder of the peptide, arguing for the importance of ligand phosphorylation in high-affinity 14-3-3 binding. The phosphopeptide main chain is stabilized in an extended conformation by a series of hydrogen bonds between the peptide backbone NH and CO groups with the side chains of residues in helices E, G, and I. However, a sharp alteration in peptide chain direction occurs at a position that is two residues C-terminal to the pSer. Most physiological 14-3-3 binding proteins as well as optimal 14-3-3 binding peptides contain a Pro residue at this position. The major effect of this kink is to redirect the chain direction away from 14-3-3 and back into the central channel. This type of four-fingered arrangement for “gripping”
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 highaffinity phospho-amino acid binding. WW Domains WW domains constitute one of the smallest modules found widely distributed in signaling proteins, often in association with HECT family ubiqitin ligase domains, proline isomerase domains, PTB domains, and PDZ domains. Comprised of ⵑ40 amino acids folded into three antiparallel  strands, WW domains bind to short proline-rich sequences containing PPXY, PPLP, or PPR motifs [25, 26] (Figure 2). Two proteins, the proline isomerase Pin1/Ess1 and the ubiquitin ligase Nedd4/Rsp5p, however, contain WW domains that can specifically recognize pSer-Pro and pThr-Pro motifs [27]. Intriguingly, 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 FKBP and cyclophilin families [28]. Since many Pin1 substrates are mitotic phosphoproteins, the conformational changes induced upon proline isomerization have been proposed to regulate mitotic entry and exit. For two of Pin1’s phosphorylated substrates, Cdc25C and Tau, proline isomerization facilitates their subsequent dephosphorylation by the Ser/Thr phosphatase PP2A [29], an event which requires the pSer-Pro bond to be in trans. Curiously, the Pin1 WW domain only binds to pSer-Pro peptides in the trans conformation [30], suggesting that one function of the WW domain may be to stabilize the trans-isomerase product in preparation for dephosphorylation. The Saccharomyces cerevisiae Pin1 homolog, Ess1, regulates transcription and mitosis through binding the hyperphosphorylated C-terminal domain of RNA polymerase II in a region that contains 26–52 repeats of the motif YSPTSPS [31]. The structure of Pin1 complexed to the serine-phosphorylated YpSPTpSPS peptide has provided a structural basis for understanding the specificity displayed by some WW domains toward phosphorylated ligands [30]. Overall, the number of specific contacts between the ligand’s phosphoserine phosphate and the WW domain’s side
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Figure 2. The Pin1 WW Domain Binds Phosphoserine-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 143-3 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 proteins (Figure 2). All of the phosphate contacts are made between the peptide’s second pSer and two residues in the 1–2 loop (Ser16 and Arg17) along with one in the 2 strand (Tyr23). The Arg guanidino group participates in a bidentate interaction with two of the phosphate oxygens along with a hydrogen bond from the Arg17 main chain NH, while the Ser residue forms a hydrogen bond with a third. Moreover, there is an additional water-mediated hydrogen bond between Tyr23 and one of the phosphate oxygens. Residues functionally equivalent to Ser16 and Arg17 are absent in the vast majority of WW domains, suggesting that the pSer/pThr binding function may be limited to a small subset of WW domains. FHA Domains Forkhead-associated (FHA) domains are found in a variety of transcriptional control proteins, DNA damage– activated protein kinases, cell cycle checkpoint proteins, phosphatases, kinesin motors, and regulators of small G proteins in organisms ranging from mycobacteria and yeast to higher plants and humans. In humans, many FHA domain–containing proteins appear to be involved in processes protecting against cancer, since mutations in, or inactivation of, several FHA proteins, including Chk2, p95/Nbs1, and Chfr, have been postulated to play a role in tumor formation. Originally identified as 55–75 amino acid modules by sequence profiling [32], a variety of biophysical studies have now demonstrated that FHA domains are considerably larger, encompassing 130–140 amino acids. Several independent lines of evidence led to the recognition that FHA domains were phosphothreonine binding modules, including work showing that FHA domains played critical roles in the interaction between the protein phosphatase KAPP and phosphorylated receptor-like kinases in Arabidopsis [33, 34] and between the cell cycle checkpoint kinase Rad53p and the phosphorylated DNA damage control protein Rad9p in S. cerevesiae [35, 36]. Nevertheless, the work of Tsai and coworkers [37] has shown that Rad53 FHA2 is able to bind peptides containing phosphotyrosine [37, 38]. Although the biological significance of pTyr-dependent interactions in budding yeast remains to be demonstrated, these data indicate that FHA domains may constitute a rather promiscuous class of phospho-dependent modules that may indeed partic-
ipate in phosphotyrosine signaling processes in higher eukaryotes. The in vivo binding partners for most FHA domain proteins are unknown. Data regarding the specificity of different FHA domains for phosphothreonine-based sequence motifs, however, emerged from peptide library experiments [39]. FHA domains were found to recognize short sequence motifs bracketing the central phosphothreonine residue, with selection extending from the pT⫺4 to the pT⫹3 position. In many cases, the selection was strongest in the pT⫹3 position, allowing a tentative grouping of FHA domains into discrete classes on the basis of pT⫹3 specificity. All FHA domains reported to date are pThr-specific, as pSer substitution eliminates peptide binding [39]. NMR analysis of the C-terminal FHA domain (FHA2) from S. cerevisiae Rad53 revealed the overall fold and identified a likely interacting surface for phosphopeptide binding [37]. The high-resolution crystal structure of FHA1 from Rad53p in complex with a phosphothreonine-containing peptide ligand (SLEVpTEADATFAKK) derived from Rad9p revealed the detailed architecture of this class of signaling module and the unusual features of phospho-dependent binding [39]. Both structures show the FHA domain to consist of an 11-stranded  sandwich that has an identical  strand topology to the MH2 domains of Smad tumor suppressor molecules (Figure 3). In both structures, the FHA homology region encompasses only six of the  strands that largely form one side of the  sandwich. The N- and C-terminal  strands lie adjacent to one another on the nonconserved face of the domain. Indeed, the proximity of the N and C termini is consistent with the modular nature of the FHA domain as an independently folded binding module. The Rad53 FHA1:phosphopeptide complex reveals that ligand binding occurs at one end of the molecule, where it interacts with residues from the loops between 3/4, 4/5, and 6/7 (Figure 3). The conserved residues within the FHA region either interact directly with the peptide or form the structural framework upon which the peptide binding loops are presented. Of the six most highly conserved residues in the FHA family, three make interactions with the peptide and, of these, Arg70 and Ser85 bind directly to the pThr residue itself. Most of the phospho-independent contacts are formed between
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Figure 3. Structure of the Rad53 FHA1:Rad9 Phosphopeptide Complex The fold topology of the FHA domain, an 11stranded  sandwich, and the phosphopeptide binding loops are indicated in the left panel. Two critical Arg residues (green) interact with the phosphothreonine phosphate and the pT⫹3 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 peptide. In this way, the peptide chain is held in an extended conformation similar to that observed in SH2, 14-3-3, and WW domain complexes. The phosphothreonine side chain makes a total of five hydrogen bonds with main and side chain atoms from Thr106 and Asn86 and two of the highly conserved residues, Arg70 and Ser85. Arg70 forms the side of a basic pocket and makes a charged hydrogen bond to the OG1 atom of the phosphothreonine The importance of this interaction is demonstrated by mutagenesis of Arg70 to alanine, which considerably lowers the binding affinity [39]. These observations are largely consistent with the recent NMR structure of Rad53 FHA2 in complex with a phosphotyrosine peptide [38], although in this case, the phosphate moiety appears to be somewhat displaced from the observed position in the pThr complex, presumably due to differences in the size of the phosphorylated side chain. At present, the bulk of available biological and biochemical data support the notions that in most cases, FHA domains function by specifically recognizing phosphorylated threonine residues in target proteins, and substitution of pSer for pThr in model peptides abrogates or severely compromises binding. It appears that the pThr rotamer observed in the Rad53 FHA1 is determined by steric clashes and by hydrogen bonding between the main chain N and a phosphate O. The threonine ␥-methyl group sits in a cavity formed by side chain and main chains atoms from Ser85, the side chains of Thr 106 and Asn 107, and backbone atoms encompassing Ser82–Ile84, forming extensive van der Waals interactions that would not be possible with a pSer substitution (Figure 3). As mentioned, many FHA domains show the strongest amino acid discrimination in the pT⫹3 position in oriented peptide screening experiments. In the case of Rad53 FHA1, these methods select strongly for aspartic acid in the ⫹3 position [39, 40]. Again, the X-ray structure nicely explains this observation, since the carboxylate
oxygens of Asp⫹3 form salt bridging interactions with N⑀ and N atoms of Arg83 from the 4/5 loop. Intriguingly, the type of residue selected at this position varies considerably in other FHA domains [39], but the basis for selection at the ⫹3 position in these cases awaits further structural and biochemical analyses of FHA domain complexes. WD40 and LRR Domains of F-Box Proteins Two other domains may function, in some circumstances, as pSer/Thr binding modules: the WD40 domains and leucine-rich regions present within F-box proteins. F-box proteins recognize substrates of SCF (Skp1Cdc53/Cullin-F-box) ubiquitin ligases, targeting them for phosphorylation-dependent ubiquitination and proteasomal degradation [41, 42]. In addition to the F-boxes from which F-box proteins derive their names, most also contain a WD40 or LRR (leucine-rich repeat) domain that may be responsible for phosphosubstrate recognition. However, unlike 14-3-3, WW, and FHA domains, structural proof for direct binding of pSer/Thr peptides to purified WD40 or LRR domains is lacking. LRR domains form multiple repeats of single ␣ helix/ strand units that align to form a large surface area for interaction with substrates [43]. How such a structure might recognize short pSer/Thr peptides is unclear. In contrast, WD40 domains adopt a  propeller structure, which has precedence for binding linear peptide motifs within grooves formed by adjacent propeller blades [44]. Whether or not contributions from other components within the ubiquitination complex play important roles in phosphospecific binding is currently unknown. Concluding Remarks The identification of several families of pSer/Thr binding modules within the last four years presents new paradigms for how cell signaling events can be regulated by Ser/Thr phosphorylation. The remarkable diversity of protein folds displayed by these domains, together with differences in how the ionized phosphate group is rec-
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ognized, suggests that their phospho binding functions evolved independently through a process of convergent evolution. Challenges for the future include the identification of additional pSer/Thr binding modules, the development of a deeper molecular understanding into their phosphospecificity along with the design of specific molecular inhibitors, and the elucidation of their physiological role within signaling pathways relevant to human health and disease. Acknowledgments Andrew E. H. Elia contributed to the section on WD40/LRR modules. We thank Drs. Mark Verdecia and Joseph Noel for providing the Pin1 WW domain:phosphopeptide coordinates used in Figure 2 and Dr. Ming-Daw Tsai for discussion on FHA domains. We apologize to the many investigators whose work was not cited due to space limitations. References 1. Fu, H., Subramanian, R.R., and Masters, S.C. (2000). 14-3-3 proteins: structure, function, and regulation. Annu. Rev. Pharmacol. Toxicol. 40, 617–647. 2. Yaffe, M.B., et al., and Cantley, L.C. (1997). The structural basis for 14-3-3:phosphopeptide binding specificity. Cell 91, 961–971. 3. Rittinger, K., et al., and Yaffe, M.B. (1999). Structural analysis of 14-3-3 phosphopeptide complexes identifies a dual role for the nuclear export signal of 14-3-3 in ligand binding. Mol. Cell 4, 153–166. 4. Dalal, S.N., Schweitzer, C.M., Gan, J., and DeCaprio, J.A. (1999). Cytoplasmic localization of human cdc25C during interphase requires an intact 14-3-3 binding site. Mol. Cell. Biol. 19, 4465– 4479. 5. Peng, C.Y., Graves, P.R., Thoma, R.S., Wu, Z., Shaw, A.S., and Piwnica-Worms, H. (1997). Mitotic and G2 checkpoint control: regulation of 14-3-3 protein binding by phosphorylation of Cdc25C on serine-216. Science 277, 1501–1505. 6. Zeng, Y., and Piwnica-Worms, H. (1999). DNA damage and replication checkpoints in fission yeast require nuclear exclusion of the Cdc25 phosphatase via 14-3-3 binding. Mol. Cell. Biol. 19, 7410–7419. 7. Yang, J., Winkler, K., Yoshida, M., and Kornbluth, S. (1999). Maintenance of G2 arrest in the Xenopus oocyte: a role for 143-3-mediated inhibition of Cdc25 nuclear import. EMBO J. 18, 2174–2183. 8. Kumagai, A., and Dunphy, W.G. (1999). Binding of 14-3-3 proteins and nuclear export control the intracellular localization of the mitotic inducer Cdc25. Genes Dev. 13, 1067–1072. 9. Lopez-Girona, A., Furnari, B., Mondesert, O., and Russell, P. (1999). Nuclear localization of Cdc25 is regulated by DNA damage and a 14-3-3 protein. Nature 397, 172–175. 10. Roy, S., et al., and Hancock, J.F. (1998). 14-3-3 facilitates Rasdependent Raf-1 activation in vitro and in vivo. Mol. Cell. Biol. 18, 3947–3955. 11. Tzivion, G., Luo, Z., and Avruch, J. (1998). A dimeric 14-3-3 protein is an essential cofactor for Raf kinase activity. Nature 394, 88–92. 12. Van Der Hoeven, P.C., Van Der Wal, J.C., Ruurs, P., Van Dijk, M.C., and Van Blitterswijk, J. (2000). 14-3-3 isotypes facilitate coupling of protein kinase C-zeta to Raf-1: negative regulation by 14-3-3 phosphorylation. Biochem. J. 345 Pt 2, 297–306. 13. Zhang, L., Chen, J., and Fu, H. (1999). Suppression of apoptosis signal-regulating kinase 1-induced cell death by 14-3-3 proteins. Proc. Natl. Acad. Sci. USA 96, 8511–8515. 14. Brunet, A., et al., and Greenberg, M.E. (1999). Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell 96, 857–868. 15. Zha, J., Harada, H., Yang, E., Jockel, J., and Korsmeyer, S.J. (1996). Serine phosphorylation of death agonist BAD in response to survival factor results in binding to 14-3-3 not BCLX(L). Cell 87, 619–628.
16. Kanai, F., et al., and Yaffe, M.B. (2000). TAZ: a novel transcriptional co-activator regulated by interactions with 14-3-3 and PDZ domain proteins. EMBO J. 19, 6778–6791. 17. Seimiya, H., et al., and Tsuruo, T. (2000). Involvement of 14-3-3 proteins in nuclear localization of telomerase. EMBO J. 19, 2652–2661. 18. Grozinger, C.M., and Schreiber, S.L. (2000). Regulation of histone deacetylase 4 and 5 and transcriptional activity by 14-33-dependent cellular localization. Proc. Natl. Acad. Sci. USA 97, 7835–7840. 19. Xiao, B., et al., and Gamblin, S.J. (1995). Structure of a 14-3-3 protein and implications for coordination of multiple signalling pathways. Nature 376, 188–191. 20. Liu, D., Bienkowska, J., Petosa, C., Collier, R.J., Fu, H., and Liddington, R. (1995). Crystal structure of the zeta isoform of the 14-3-3 protein. Nature 376, 191–194. 21. Waksman, G., et al., and Overduin, M. (1992). Crystal structure of the phosphotyrosine recognition domain Sh2 of V Src complexed with tyrosine phosphorylated peptides. Nature 358, 646–653. 22. Waksman, G., Shoelson, S.E., Pant, N., Cowburn, D., and Kuriyan, J. (1993). Binding of a high-affinity phosphotyrosyl peptide to the Src SH2 domain crystal structures of the complexed and peptide-free forms. Cell 72, 779–790. 23. Eck, M.J., Shoelson, S.E., and Harrison, S.C. (1993). Recognition of a high-affinity phosphotyrosyl peptide by the Src homology-2 domain of p56lck. Nature 362, 87–91. 24. Zhou, M.M., et al., and Fesik, S.W. (1995). Structure and ligand recognition of the phosphotyrosine binding domain of Shc. Nature 378, 584–592. 25. Sudol, M., and Hunter, T. (2000). NeW wrinkles for an old domain. Cell 103, 1001–1004. 26. Zarrinpar, A., and Lim, W.A. (2000). Converging on proline: the mechanism of WW domain peptide recognition. Nat. Struct. Biol. 7, 611–613. 27. Lu, P.J., Zhou, X.Z., Shen, M., and Lu, K.P. (1999). Function of WW domains as phosphoserine- or phosphothreonine-binding modules. Science 283, 1325–1328. 28. Yaffe, M.B., et al., and Lu, K.P. (1997). Sequence-specific and phosphorylation-dependent proline isomerization: a potential mitotic regulatory mechanism. Science 278, 1957–1960. 29. Xiao, Z.Z., et al., Lu, K.P. (2000). Pin1-dependent prolyl isomerization regulates dephosphorylation of Cdc25C and Tau proteins. Mol. Cell 6, 873–883. 30. Verdecia, M.A., Bowman, M.E., Lu, K.P., Hunter, T., and Noel, J.P. (2000). Structural basis for phosphoserine-proline recognition by group IV WW domains. Nat. Struct. Biol. 7, 639–643. 31. Morris, D.P., Phatnani, H.P., and Greenleaf, A.L. (1999). Phospho-carboxyl-terminal domain binding and the role of a prolyl isomerase in pre-mRNA 3⬘-end formation. J. Biol. Chem. 274, 31583–31587. 32. Hofmann, K., and Bucher, P. (1995). The FHA domain: a putative nuclear signalling domain found in protein kinases and transcription factors. Trends Biochem. Sci. 20, 347–349. 33. Li, J., Smith, G.P., and Walker, J.C. (1999). Kinase interaction domain of kinase-associated protein phosphatase, a phosphoprotein-binding domain. Proc. Natl. Acad. Sci. USA 96, 7821– 7826. 34. Stone, J.M., Collinge, M.A., Smith, R.D., Horn, M.A., and Walker, J.C. (1994). Interaction of a protein phosphatase with an Arabidopsis serine-threonine receptor kinase. Science 266, 793–795. 35. Sun, Z., Hsiao, J., Fay, D.S., and Stern, D.F. (1998). Rad53 FHA domain associated with phosphorylated Rad9 in the DNA damage checkpoint. Science 281, 272–274. 36. Durocher, D., Henckel, J., Fersht, A.R., and Jackson, S.P. (1999). The FHA domain is a modular phosphopeptide recognition motif. Mol. Cell 4, 387–394. 37. Liao, H., Byeon, I.J., and Tsai, M.D. (1999). Structure and function of a new phosphopeptide-binding domain containing the FHA2 of Rad53. J. Mol. Biol. 294, 1041–1049. 38. Wang, P., et al., and Tsai, M.D. (2000). II. Structure and specificity of the interaction between the FHA2 domain of rad53 and phosphotyrosyl peptides. J. Mol. Biol. 302, 927–940. 39. Durocher, D., et al., and Yaffe, M.B. (2000). The molecular basis
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PhosphoSerine/Threonine Binding Domains: You Can’t pSERious? Michael B. Yaffe,*‡ and Stephen J. Smerdon†‡ * Center for Cancer Research Massachusetts Institute of Technology 77 Massachusetts Avenue, E18-580 Cambridge, Massachusetts 02139 † Division of Protein Structure National Institute for Medical Research The Ridgeway Mill Hill London NW7 1AA United Kingdom
Summary The fundamental biological importance of protein phosphorylation is underlined by the existence of more than 500 protein kinase genes within the human genome. In many cases, phosphorylation on serine, threonine, and tyrosine residues creates binding surfaces for a variety of phospho-amino acid binding proteins/modules. Here, we review the insights into serine/threonine phosphorylation-dependent signal transduction processes provided by structures of several of these proteins and their complexes. Introduction Signal transduction in eukaryotic cells involves the assembly of various protein and lipid kinases and phosphatases along with other enzymes, adaptors, and regulatory molecules into large protein complexes. These multicomponent signaling machines integrate and transmit information to control ion fluxes, cytoskeletal rearrangements, patterns of gene expression, cell cycle progression, and programmed cell death. The dynamic nature of signaling events requires rapid assembly and disassembly of signaling complexes, events that are generally regulated through protein phosphorylation. Historically, attention has focused on the assembly of multiprotein complexes that were regulated by tyrosine phosphorylation of transmembrane receptors to generate binding sites for proteins with modular SH2 and PTB domains. In contrast, phosphorylation of proteins on serine and threonine residues was thought to regulate protein function largely through allosteric modifications rather than by directly mediating protein–protein interactions. Within the last few years, however, a variety of signaling molecules and modular domains have been identified that specifically bind to short pSer/Thr-containing motifs, recruiting these substrates into protein– protein signaling complexes in response to phosphorylation by Ser/Thr kinases. In this review, we focus on the identification of these new pSer/Thr binding modules, including 14-3-3 proteins, WW domains, and FHA domains; their physiological functions and the structural basis for their pSer/Thr motif recognition. ‡ To whom correspondence should be addressed (e-mail: myaffe@ mit.edu, [email protected]).
PII S0969-2126(01)00580-9
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14-3-3 Proteins The term “14-3-3” denotes a family of dimeric ␣-helical pSer/Thr binding proteins present in high abundance in all eukaryotic cells [1]. 14-3-3 proteins were the first molecules to be recognized as distinct pSer/Thr binding proteins, forming tight complexes with phosphorylated ligands containing either of two sequence motifs, R[S/ Ar]XpSXP and RX[Ar/S]XpSXP, respectively, where pS denotes both phosphoserine and phosphothreonine, and Ar denotes aromatic residues [2, 3]. In addition, a few 14-3-3 binding ligands have been identified whose sequences deviate significantly from these motifs or do not require phosphorylation for binding. Over 50 distinct substrates have been identified that bind to 14-3-3, many of which play critical roles in regulating progression through the cell cycle, activation of the Erk1/2 subfamily of MAP kinases, initiation of apoptosis, and control of gene transcription and chromosome remodeling. Both the maintenance of cells in interphase [4], for example, and the G2/M cell cycle arrest observed in rapidly dividing cells following DNA damage [5] involve the binding of 14-3-3 proteins to a specific phosphorylated form of the mitotic regulatory tyrosine phosphatase Cdc25. The resulting 14-3-3:Cdc25 complex localizes to the cytoplasm [6–9], blocking Cdc25’s access to its nuclear substrate, Cdc2-CyclinB, and thereby regulating mitotic entry. The binding of 14-3-3 dimers to phosphorylated forms of A-Raf, B-Raf, and c-Raf-1 plays a complex and incompletely understood role in Raf activation and MAP kinase signaling, probably through a combination of stabilizing the active form of Raf, facilitating the interaction of Raf with downstream effectors, and maintaining inactive Raf in a reactivatable conformation [10–12]. Several 14-3-3-regulated ligands are physiological effectors of apoptosis, including phosphorylated forms of the kinase ASK1 [13], the Forkhead transcription factor FKHRL1 [14], and the Bcl-2 family member BAD [15]. Other 14-3-3 binding molecules include several transcriptional activators and coactivators [16], human telomerase [17], and histone deacetylases 4 and 5 [18]. Through what feat of structural gymnastics do 14-3-3 proteins accomplish their phosphospecific recognition while binding to such a diverse array of ligands? Some insight into this dilemma has emerged through studies of 14-3-3:phosphopeptide complexes. Each monomer of the dimeric 14-3-3 molecule contains nine ␣ helices, with the individual monomeric subunits related to each other by C2 symmetry [19, 20] (Figure 1). The three N-terminal helices of each monomer interact extensively with those of the opposing monomer through a combination of salt bridges, hydrogen bonds, and van der Waals interactions, forming a 35 ⫻ 35 ⫻ 20 A˚ wide central channel suitable for binding to peptide and protein ligands. The residues lining this central channel show extensive sequence conservation among all seven 14-3-3 isotypes found in mammalian cells [19]. Three basic residues, Lys49, Arg56, and Arg127, together with Tyr128, emerge from helices C
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Figure 1. 14-3-3 Proteins Bind Phosphoserine Peptides within a Large Central Channel Each monomeric subunit of the dimer, shaded blue and red, respectively, is composed of nine ␣ helices (␣A–␣I), with each monomer simultaneously binding a phosphopeptide. A single protein ligand, when phosphorylated on two sites, probably binds in a similar manner to both monomers of the 14-3-3 dimer. Alternatively, two proteins with single phosphorylation sites may bind to each monomer individually, allowing 14-3-3 to function as a molecular adaptor. The phosphoserine phosphate is coordinated by a basic pocket formed from the indicated residues emerging from two of the ␣ helices (bottom). A loosely similar arrangement of basic and hydroxyl-containing side chains coordinates the peptide phosphotyrosine phosphate in SH2 and some PTB domain complexes.
and E to form a positively charged pocket located toward one end of each edge of the central channel in what is otherwise a largely negatively charged molecule. Each of the residues in this basic pocket interacts directly with the peptide phosphate group, forming five ionic bonds and a hydrogen bond to three of the phosphate oxygens (Figure 1) [2, 3]. This strong network of interactions, like the fingers of a hand balancing a squash ball, forms the critical anchor that orients the remainder of the peptide, arguing for the importance of ligand phosphorylation in high-affinity 14-3-3 binding. The phosphopeptide main chain is stabilized in an extended conformation by a series of hydrogen bonds between the peptide backbone NH and CO groups with the side chains of residues in helices E, G, and I. However, a sharp alteration in peptide chain direction occurs at a position that is two residues C-terminal to the pSer. Most physiological 14-3-3 binding proteins as well as optimal 14-3-3 binding peptides contain a Pro residue at this position. The major effect of this kink is to redirect the chain direction away from 14-3-3 and back into the central channel. This type of four-fingered arrangement for “gripping”
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 highaffinity phospho-amino acid binding. WW Domains WW domains constitute one of the smallest modules found widely distributed in signaling proteins, often in association with HECT family ubiqitin ligase domains, proline isomerase domains, PTB domains, and PDZ domains. Comprised of ⵑ40 amino acids folded into three antiparallel  strands, WW domains bind to short proline-rich sequences containing PPXY, PPLP, or PPR motifs [25, 26] (Figure 2). Two proteins, the proline isomerase Pin1/Ess1 and the ubiquitin ligase Nedd4/Rsp5p, however, contain WW domains that can specifically recognize pSer-Pro and pThr-Pro motifs [27]. Intriguingly, 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 FKBP and cyclophilin families [28]. Since many Pin1 substrates are mitotic phosphoproteins, the conformational changes induced upon proline isomerization have been proposed to regulate mitotic entry and exit. For two of Pin1’s phosphorylated substrates, Cdc25C and Tau, proline isomerization facilitates their subsequent dephosphorylation by the Ser/Thr phosphatase PP2A [29], an event which requires the pSer-Pro bond to be in trans. Curiously, the Pin1 WW domain only binds to pSer-Pro peptides in the trans conformation [30], suggesting that one function of the WW domain may be to stabilize the trans-isomerase product in preparation for dephosphorylation. The Saccharomyces cerevisiae Pin1 homolog, Ess1, regulates transcription and mitosis through binding the hyperphosphorylated C-terminal domain of RNA polymerase II in a region that contains 26–52 repeats of the motif YSPTSPS [31]. The structure of Pin1 complexed to the serine-phosphorylated YpSPTpSPS peptide has provided a structural basis for understanding the specificity displayed by some WW domains toward phosphorylated ligands [30]. Overall, the number of specific contacts between the ligand’s phosphoserine phosphate and the WW domain’s side
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Figure 2. The Pin1 WW Domain Binds Phosphoserine-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 143-3 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 proteins (Figure 2). All of the phosphate contacts are made between the peptide’s second pSer and two residues in the 1–2 loop (Ser16 and Arg17) along with one in the 2 strand (Tyr23). The Arg guanidino group participates in a bidentate interaction with two of the phosphate oxygens along with a hydrogen bond from the Arg17 main chain NH, while the Ser residue forms a hydrogen bond with a third. Moreover, there is an additional water-mediated hydrogen bond between Tyr23 and one of the phosphate oxygens. Residues functionally equivalent to Ser16 and Arg17 are absent in the vast majority of WW domains, suggesting that the pSer/pThr binding function may be limited to a small subset of WW domains. FHA Domains Forkhead-associated (FHA) domains are found in a variety of transcriptional control proteins, DNA damage– activated protein kinases, cell cycle checkpoint proteins, phosphatases, kinesin motors, and regulators of small G proteins in organisms ranging from mycobacteria and yeast to higher plants and humans. In humans, many FHA domain–containing proteins appear to be involved in processes protecting against cancer, since mutations in, or inactivation of, several FHA proteins, including Chk2, p95/Nbs1, and Chfr, have been postulated to play a role in tumor formation. Originally identified as 55–75 amino acid modules by sequence profiling [32], a variety of biophysical studies have now demonstrated that FHA domains are considerably larger, encompassing 130–140 amino acids. Several independent lines of evidence led to the recognition that FHA domains were phosphothreonine binding modules, including work showing that FHA domains played critical roles in the interaction between the protein phosphatase KAPP and phosphorylated receptor-like kinases in Arabidopsis [33, 34] and between the cell cycle checkpoint kinase Rad53p and the phosphorylated DNA damage control protein Rad9p in S. cerevesiae [35, 36]. Nevertheless, the work of Tsai and coworkers [37] has shown that Rad53 FHA2 is able to bind peptides containing phosphotyrosine [37, 38]. Although the biological significance of pTyr-dependent interactions in budding yeast remains to be demonstrated, these data indicate that FHA domains may constitute a rather promiscuous class of phospho-dependent modules that may indeed partic-
ipate in phosphotyrosine signaling processes in higher eukaryotes. The in vivo binding partners for most FHA domain proteins are unknown. Data regarding the specificity of different FHA domains for phosphothreonine-based sequence motifs, however, emerged from peptide library experiments [39]. FHA domains were found to recognize short sequence motifs bracketing the central phosphothreonine residue, with selection extending from the pT⫺4 to the pT⫹3 position. In many cases, the selection was strongest in the pT⫹3 position, allowing a tentative grouping of FHA domains into discrete classes on the basis of pT⫹3 specificity. All FHA domains reported to date are pThr-specific, as pSer substitution eliminates peptide binding [39]. NMR analysis of the C-terminal FHA domain (FHA2) from S. cerevisiae Rad53 revealed the overall fold and identified a likely interacting surface for phosphopeptide binding [37]. The high-resolution crystal structure of FHA1 from Rad53p in complex with a phosphothreonine-containing peptide ligand (SLEVpTEADATFAKK) derived from Rad9p revealed the detailed architecture of this class of signaling module and the unusual features of phospho-dependent binding [39]. Both structures show the FHA domain to consist of an 11-stranded  sandwich that has an identical  strand topology to the MH2 domains of Smad tumor suppressor molecules (Figure 3). In both structures, the FHA homology region encompasses only six of the  strands that largely form one side of the  sandwich. The N- and C-terminal  strands lie adjacent to one another on the nonconserved face of the domain. Indeed, the proximity of the N and C termini is consistent with the modular nature of the FHA domain as an independently folded binding module. The Rad53 FHA1:phosphopeptide complex reveals that ligand binding occurs at one end of the molecule, where it interacts with residues from the loops between 3/4, 4/5, and 6/7 (Figure 3). The conserved residues within the FHA region either interact directly with the peptide or form the structural framework upon which the peptide binding loops are presented. Of the six most highly conserved residues in the FHA family, three make interactions with the peptide and, of these, Arg70 and Ser85 bind directly to the pThr residue itself. Most of the phospho-independent contacts are formed between
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Figure 3. Structure of the Rad53 FHA1:Rad9 Phosphopeptide Complex The fold topology of the FHA domain, an 11stranded  sandwich, and the phosphopeptide binding loops are indicated in the left panel. Two critical Arg residues (green) interact with the phosphothreonine phosphate and the pT⫹3 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 peptide. In this way, the peptide chain is held in an extended conformation similar to that observed in SH2, 14-3-3, and WW domain complexes. The phosphothreonine side chain makes a total of five hydrogen bonds with main and side chain atoms from Thr106 and Asn86 and two of the highly conserved residues, Arg70 and Ser85. Arg70 forms the side of a basic pocket and makes a charged hydrogen bond to the OG1 atom of the phosphothreonine The importance of this interaction is demonstrated by mutagenesis of Arg70 to alanine, which considerably lowers the binding affinity [39]. These observations are largely consistent with the recent NMR structure of Rad53 FHA2 in complex with a phosphotyrosine peptide [38], although in this case, the phosphate moiety appears to be somewhat displaced from the observed position in the pThr complex, presumably due to differences in the size of the phosphorylated side chain. At present, the bulk of available biological and biochemical data support the notions that in most cases, FHA domains function by specifically recognizing phosphorylated threonine residues in target proteins, and substitution of pSer for pThr in model peptides abrogates or severely compromises binding. It appears that the pThr rotamer observed in the Rad53 FHA1 is determined by steric clashes and by hydrogen bonding between the main chain N and a phosphate O. The threonine ␥-methyl group sits in a cavity formed by side chain and main chains atoms from Ser85, the side chains of Thr 106 and Asn 107, and backbone atoms encompassing Ser82–Ile84, forming extensive van der Waals interactions that would not be possible with a pSer substitution (Figure 3). As mentioned, many FHA domains show the strongest amino acid discrimination in the pT⫹3 position in oriented peptide screening experiments. In the case of Rad53 FHA1, these methods select strongly for aspartic acid in the ⫹3 position [39, 40]. Again, the X-ray structure nicely explains this observation, since the carboxylate
oxygens of Asp⫹3 form salt bridging interactions with N⑀ and N atoms of Arg83 from the 4/5 loop. Intriguingly, the type of residue selected at this position varies considerably in other FHA domains [39], but the basis for selection at the ⫹3 position in these cases awaits further structural and biochemical analyses of FHA domain complexes. WD40 and LRR Domains of F-Box Proteins Two other domains may function, in some circumstances, as pSer/Thr binding modules: the WD40 domains and leucine-rich regions present within F-box proteins. F-box proteins recognize substrates of SCF (Skp1Cdc53/Cullin-F-box) ubiquitin ligases, targeting them for phosphorylation-dependent ubiquitination and proteasomal degradation [41, 42]. In addition to the F-boxes from which F-box proteins derive their names, most also contain a WD40 or LRR (leucine-rich repeat) domain that may be responsible for phosphosubstrate recognition. However, unlike 14-3-3, WW, and FHA domains, structural proof for direct binding of pSer/Thr peptides to purified WD40 or LRR domains is lacking. LRR domains form multiple repeats of single ␣ helix/ strand units that align to form a large surface area for interaction with substrates [43]. How such a structure might recognize short pSer/Thr peptides is unclear. In contrast, WD40 domains adopt a  propeller structure, which has precedence for binding linear peptide motifs within grooves formed by adjacent propeller blades [44]. Whether or not contributions from other components within the ubiquitination complex play important roles in phosphospecific binding is currently unknown. Concluding Remarks The identification of several families of pSer/Thr binding modules within the last four years presents new paradigms for how cell signaling events can be regulated by Ser/Thr phosphorylation. The remarkable diversity of protein folds displayed by these domains, together with differences in how the ionized phosphate group is rec-
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ognized, suggests that their phospho binding functions evolved independently through a process of convergent evolution. Challenges for the future include the identification of additional pSer/Thr binding modules, the development of a deeper molecular understanding into their phosphospecificity along with the design of specific molecular inhibitors, and the elucidation of their physiological role within signaling pathways relevant to human health and disease. Acknowledgments Andrew E. H. Elia contributed to the section on WD40/LRR modules. We thank Drs. Mark Verdecia and Joseph Noel for providing the Pin1 WW domain:phosphopeptide coordinates used in Figure 2 and Dr. Ming-Daw Tsai for discussion on FHA domains. We apologize to the many investigators whose work was not cited due to space limitations. References 1. Fu, H., Subramanian, R.R., and Masters, S.C. (2000). 14-3-3 proteins: structure, function, and regulation. Annu. Rev. Pharmacol. Toxicol. 40, 617–647. 2. Yaffe, M.B., et al., and Cantley, L.C. (1997). The structural basis for 14-3-3:phosphopeptide binding specificity. Cell 91, 961–971. 3. Rittinger, K., et al., and Yaffe, M.B. (1999). Structural analysis of 14-3-3 phosphopeptide complexes identifies a dual role for the nuclear export signal of 14-3-3 in ligand binding. Mol. Cell 4, 153–166. 4. Dalal, S.N., Schweitzer, C.M., Gan, J., and DeCaprio, J.A. (1999). Cytoplasmic localization of human cdc25C during interphase requires an intact 14-3-3 binding site. Mol. Cell. Biol. 19, 4465– 4479. 5. Peng, C.Y., Graves, P.R., Thoma, R.S., Wu, Z., Shaw, A.S., and Piwnica-Worms, H. (1997). Mitotic and G2 checkpoint control: regulation of 14-3-3 protein binding by phosphorylation of Cdc25C on serine-216. Science 277, 1501–1505. 6. Zeng, Y., and Piwnica-Worms, H. (1999). DNA damage and replication checkpoints in fission yeast require nuclear exclusion of the Cdc25 phosphatase via 14-3-3 binding. Mol. Cell. Biol. 19, 7410–7419. 7. Yang, J., Winkler, K., Yoshida, M., and Kornbluth, S. (1999). Maintenance of G2 arrest in the Xenopus oocyte: a role for 143-3-mediated inhibition of Cdc25 nuclear import. EMBO J. 18, 2174–2183. 8. Kumagai, A., and Dunphy, W.G. (1999). Binding of 14-3-3 proteins and nuclear export control the intracellular localization of the mitotic inducer Cdc25. Genes Dev. 13, 1067–1072. 9. Lopez-Girona, A., Furnari, B., Mondesert, O., and Russell, P. (1999). Nuclear localization of Cdc25 is regulated by DNA damage and a 14-3-3 protein. Nature 397, 172–175. 10. Roy, S., et al., and Hancock, J.F. (1998). 14-3-3 facilitates Rasdependent Raf-1 activation in vitro and in vivo. Mol. Cell. Biol. 18, 3947–3955. 11. Tzivion, G., Luo, Z., and Avruch, J. (1998). A dimeric 14-3-3 protein is an essential cofactor for Raf kinase activity. Nature 394, 88–92. 12. Van Der Hoeven, P.C., Van Der Wal, J.C., Ruurs, P., Van Dijk, M.C., and Van Blitterswijk, J. (2000). 14-3-3 isotypes facilitate coupling of protein kinase C-zeta to Raf-1: negative regulation by 14-3-3 phosphorylation. Biochem. J. 345 Pt 2, 297–306. 13. Zhang, L., Chen, J., and Fu, H. (1999). Suppression of apoptosis signal-regulating kinase 1-induced cell death by 14-3-3 proteins. Proc. Natl. Acad. Sci. USA 96, 8511–8515. 14. Brunet, A., et al., and Greenberg, M.E. (1999). Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell 96, 857–868. 15. Zha, J., Harada, H., Yang, E., Jockel, J., and Korsmeyer, S.J. (1996). Serine phosphorylation of death agonist BAD in response to survival factor results in binding to 14-3-3 not BCLX(L). Cell 87, 619–628.
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