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The Age of Crosstalk: Phosphorylation, Ubiquitination, and Beyond

2007; Elsevier BV; Volume: 28; Issue: 5 Linguagem: Inglês

10.1016/j.molcel.2007.11.019

1097-4164

Tony Hunter,

Glycosylation and Glycoproteins Research

Crosstalk between different types of posttranslational modification is an emerging theme in eukaryotic biology. Particularly prominent are the multiple connections between phosphorylation and ubiquitination, which act either positively or negatively in both directions to regulate these processes. Crosstalk between different types of posttranslational modification is an emerging theme in eukaryotic biology. Particularly prominent are the multiple connections between phosphorylation and ubiquitination, which act either positively or negatively in both directions to regulate these processes. Reversible posttranslational modification (PTM) is a versatile way to regulate protein activity. Historically, the best-studied PTM is phosphorylation, largely because of the relative ease of detecting protein phosphorylation in vivo and in vitro. However, many other types of reversible PTMs exist, with acetylation, methylation, O-GlcNacylation, and ubiquitination having become prominent more recently. Global mass spectrometric (MS) analysis is beginning to uncover the high density and variety of PTMs that exist, with many proteins being multiply modified by different covalently attached groups. For example, recent phosphoproteomic analysis has revealed that the majority of proteins in a mammalian cell are phosphorylated at one or more sites (e.g., 6000 phosphorylation sites identified in 2200 proteins in HeLa cells, with ∼15% changing in abundance within 15 min of EGF treatment (Olsen et al., 2006Olsen J.V. Blagoev B. Gnad F. Macek B. Kumar C. Mortensen P. Mann M. Global, in vivo, and site-specific phosphorylation dynamics in signaling networks.Cell. 2006; 127: 635-648Abstract Full Text Full Text PDF PubMed Scopus (2642) Google Scholar). Proteomic analysis of other types of PTM is less advanced but is also beginning to reveal extensive use of acetylation and methylation in nonhistone proteins and very widespread use of ubiquitination, both for protein degradation and other purposes. Indeed, the suggestion that the relative paucity of genes in vertebrate genomes may be compensated for by the extensive use of PTMs to generate multiple distinct protein states from a single gene product appears likely to be right. Single PTMs are capable of regulating protein function, either through creating new protein binding sites, by abrogating protein-protein interactions, or through allosteric effects. However, many proteins are multiply modified, and a significant increase in information content would be obtained if PTMs acted combinatorially. There are examples where this is known to be the case, but in evaluating this possibility, a general deficiency in the analysis of PTMs is the lack of direct information regarding whether different sites are simultaneously modified on an individual protein molecule. For this reason, it is clearly important to develop methods to determine which PTMs coexist on a single molecule so that one can define different protein modification states and assign functions to them. The use of electron transfer dissociation (ETD) MS, which can analyze large protein fragments for combinations of PTMs, may provide one solution to this problem. Indeed, a recent study using ETD-MS has revealed that the histone H3 N-terminal tail can be simultaneously methylated at K4 and acetylated at five different lysines (K9, K14, K18, K23, and K27) (Taverna et al., 2007Taverna S.D. Ueberheide B.M. Liu Y. Tackett A.J. Diaz R.L. Shabanowitz J. Chait B.T. Hunt D.F. Allis C.D. Long-distance combinatorial linkage between methylation and acetylation on histone H3 N termini.Proc. Natl. Acad. Sci. USA. 2007; 104: 2086-2091Crossref PubMed Scopus (137) Google Scholar). Histone tails provide one of the most remarkable examples of PTM density and variety, with Lys acetylation, mono-, di-, or trimethylation, biotinylation, ubiquitination, NEDDylation, SUMOylation; Arg methylation; Ser/Thr/Tyr phosphorylation; and Glu ADP ribosylation all occurring within 50–100 residues on the N-terminal and C-terminal tails of H2A, H2B, H3, and H4 (Bhaumik et al., 2007Bhaumik S.R. Smith E. Shilatifard A. Covalent modifications of histones during development and disease pathogenesis.Nat. Struct. Mol. Biol. 2007; 14: 1008-1016Crossref PubMed Scopus (487) Google Scholar, Latham and Dent, 2007Latham J.A. Dent S.Y. Cross-regulation of histone modifications.Nat. Struct. Mol. Biol. 2007; 14: 1017-1024Crossref PubMed Scopus (316) Google Scholar). These “marks” are proposed to represent a code that is read by a series of transcriptional regulators and chromatin modifiers that affect local chromatin structure, leading to activation or repression of adjacent transcription units. Although there is little direct information regarding which PTMs occur simultaneously on a single histone molecule, functional analysis of histone tail PTMs has revealed many examples of crosstalk between different PTMs (see below). Nuclear coreceptors are another family of proteins in which PTM crosstalk is beginning to be uncovered (Lonard and O'Malley, 2007Lonard D.M. O'Malley B.W. Nuclear receptor coregulators: judges, juries, and executioners of cellular regulation.Mol. Cell. 2007; 27: 691-700Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar). Such crosstalk between PTMs is an emerging theme that I will consider in this perspective, using crosstalk between phosphorylation and ubiquitination as a primary example. There are several established principles through which PTMs mediate crosstalk. In this context, crosstalk can be either positive or negative in nature. Positive crosstalk is defined as a situation in which one PTM serves as a signal for the addition or removal of a second PTM or for recognition by a binding protein that carries out a second modification. Here, priming phosphorylation events (e.g., GSK3-mediated phosphorylations requiring a priming phosphate positioned at position +4, or SH2 domain-containing tyrosine kinase phosphorylation of pTyr-containing proteins), phosphorylation-dependent ubiquitination (e.g., many SCF E3 ligase substrates), and phosphorylation-dependent SUMOylation (e.g., HSF1 and MEF2C, wherein a ψKXEXXpSP motif is SUMOylated) are good examples. In the case of histone PTMs, both methylation and ubiquitination are able to trigger acetylation, and protein-protein interactions that involve two histone PTMs are known (see below). In the case of negative crosstalk, there can be direct competition for modification of a single residue in a protein, or indirect effects, wherein one modification masks the recognition site for a second PTM. For example, a single Lys can be modified by ubiquitination or SUMOylation (e.g., PCNA); by acetylation and ubiquitination (e.g., p53); or by acetylation, ubiquitination, and methylation (e.g., K302 in ERα). A recent example of direct competition is seen in the YopJ-mediated acetylation of Ser in the activation loops of MAP2Ks, which are normally phosphorylated by MAP3Ks, as a key signal transfer and amplification step in MAPK module activation. Stoichiometric acetylation of the two MAP2K activation loop Ser by YopJ, which is encoded by the virulence plasmid of Yersinia pestis, blocks phosphorylation by upstream MAP3Ks and is used by the bacterium to shut down MAPK signaling globally in the infected cell (Mukherjee et al., 2006Mukherjee S. Keitany G. Li Y. Wang Y. Ball H.L. Goldsmith E.J. Orth K. Yersinia YopJ acetylates and inhibits kinase activation by blocking phosphorylation.Science. 2006; 312: 1211-1214Crossref PubMed Scopus (435) Google Scholar). However, it is unclear whether analogous Ser acetylases are encoded by eukaryotic cells. Another example of a PTM competing with phosphorylation is attachment of O-linked N-acetylglucosamine residues, which are coupled to specific Ser/Thr in many types of proteins, with transcription factors being prominent (Hart et al., 2007Hart G.W. Housley M.P. Slawson C. Cycling of O-linked beta-N-acetylglucosamine on nucleocytoplasmic proteins.Nature. 2007; 446: 1017-1022Crossref PubMed Scopus (966) Google Scholar). O-GlcNacylation can in principle directly prevent phosphorylation at specific Ser/Thr (e.g., T58 in c-Myc) and can also influence phosphorylation of adjacent sites (e.g., O-GlcNacylation of S149 in p53 reduces phosphorylation of T155). However, both O-GlcNacylation and phosphorylation are usually substoichiometric, suggesting that only particular pools of any protein are crossregulated in this fashion and that indirect effects of O-GlcNacylation on phosphorylation could be more common (Forsythe et al., 2006Forsythe M.E. Love D.C. Lazarus B.D. Kim E.J. Prinz W.A. Ashwell G. Krause M.W. Hanover J.A. Caenorhabditis elegans ortholog of a diabetes susceptibility locus: oga-1 (O-GlcNAcase) knockout impacts O-GlcNAc cycling, metabolism, and dauer.Proc. Natl. Acad. Sci. USA. 2006; 103: 11952-11957Crossref PubMed Scopus (117) Google Scholar). Negative crosstalk of any sort between different PTMs can in principle be used as an OR logic gate in a signaling network. A major function of PTMs is to create binding sites for specific modular binding domains (e.g., SH2 domains binding to phosphorylated tyrosines), providing a mechanism for inducing protein-protein interactions and propagating signals (Seet et al., 2006Seet B.T. Dikic I. Zhou M.M. Pawson T. Reading protein modifications with interaction domains.Nat. Rev. Mol. Cell Biol. 2006; 7: 473-483Crossref PubMed Scopus (509) Google Scholar). In principle, different PTMs, of a single type or of distinct types, can cooperate to promote binding of proteins with PTM recognition domains; alternatively, one PTM can negatively regulate the interaction of a domain with another PTM (see below). Proteins commonly contain more than one protein interaction domain and in some cases more than one interaction domain that recognizes a PTM. Several proteins contain two phosphobinding domains, either of the same type (e.g., twin SH2 domains in the ZAP70/SYK tyrosine kinases, the SHP1/SHP2 PTPs, p85 regulatory subunits of the PI-3 kinases and PLCγ, and 14-3-3, which is a symmetric dimer) or of a different type (e.g., PTB and SH2 pTyr-binding domains in Shc, and FHA and BRCT repeat pSer/Thr binding domains in MDC1). Binding of both domains to a single phosphoprotein (or complex) can increase both affinity and specificity of interaction. Both SH2 domains in twin SH2 domain proteins can be occupied simultaneously by binding to pTyr residues on the same protein, and this principle is particularly important in the case of ITAM motifs present in the cytoplasmic domains of immunomodulatory receptors, where dually phosphorylated motifs recruit SYK/ZAP70 via bidentate binding of their tandem SH2 domains to pYxxI/Lx(6–12)pYxxI/L motifs. The same is true for 14-3-3, where two pSer/Thr residues in the same protein or same complex can be bound simultaneously, with one being essential for binding and serving as the gatekeeper and the second being required for specificity and full biological consequences (Yaffe, 2002Yaffe M.B. How do 14–3-3 proteins work? Gatekeeper phosphorylation and the molecular anvil hypothesis.FEBS Lett. 2002; 513: 53-57Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar). Whether proteins containing heterologous phosphobinding domains, such as MDC1, bind simultaneously to different phosphorylation sites in the same protein molecule or in two different protein molecules is largely unknown. Although PTMs are often thought of as acting independently, there are many examples in which two or more phosphorylation sites in a protein have a combinatorial effect on activity (e.g., inhibition of glycogen synthase activity by multisite phosphorylation, activation of MAP kinases by TXY motif diphosphorylation, and inhibition of Cdk activity by combined Thr14/Tyr15 phosphorylation). Recent global phosphoproteomic analyses have revealed many diphosphorylated peptides corresponding to different motifs (Villen et al., 2007Villen J. Beausoleil S.A. Gerber S.A. Gygi S.P. Large-scale phosphorylation analysis of mouse liver.Proc. Natl. Acad. Sci. USA. 2007; 104: 1488-1493Crossref PubMed Scopus (605) Google Scholar), and it seems likely that these double phosphorylations act combinatorially. In addition, proteins primed through phosphorylation by one protein kinase are often phosphorylated processively on the N-terminal side of the priming phosphate by GSK3 at a series of Ser/Thr spaced by three residues, with the cluster of phosphates regulating protein activity (e.g., glycogen synthase, β-catenin). If the two sites are phosphorylated by different protein kinases, then this can in principle provide a logical AND gate in a downstream response. Histone marks are commonly studied in isolation through the use of state-specific antibodies, but modification of one site can in principle promote or prevent modification or recognition of an adjacent site so that they are coupled. For example, acetylation and methylation of H3K9 are mutually exclusive, providing an example of direct competition. Indirect effects of one PTM on another are also found. For instance, phosphorylation of H3S10 prevents HP1 chromodomain recognition of H3K9m3e; conversely, a chromodomain bound to H3K9me3 would preclude phosphorylation of S10. The same would hold for acetylation sites recognized by bromodomains that are adjacent to phosphorylation sites. This realization led to the idea of binary switches in which phosphorylation modulates access to or modification of adjacent acetylation or methylation sites and vice versa (Fischle et al., 2003Fischle W. Wang Y. Allis C.D. Binary switches and modification cassettes in histone biology and beyond.Nature. 2003; 425: 475-479Crossref PubMed Scopus (526) Google Scholar). In the last few years, many additional examples of crosstalk between histone tail PTMs occurring both in cis and trans have emerged (Latham and Dent, 2007Latham J.A. Dent S.Y. Cross-regulation of histone modifications.Nat. Struct. Mol. Biol. 2007; 14: 1017-1024Crossref PubMed Scopus (316) Google Scholar). Phosphorylation can prevent or promote Lys acetylation or methylation and vice versa, methylation and ubiquitination can stimulate Lys acetylation, and SUMOylation antagonizes histone acetylation. The further elucidation of histone PTM crosstalk calls for more extensive information regarding which modifications coexist on a single histone molecule and on histone tails in the same nucleosome. The extent to which the principles underlying histone PTM crosstalk are utilized by nonhistone proteins remains to be determined. More interesting from the perspective of PTM crosstalk are proteins that have two interaction domains that bind two different types of PTMs. Good examples here are proteins that bind specific marks on histone tails, where many proteins that have various combinations of chromo, PHD, Tudor, MBD, bromo, PWWP, WD40, FHA, and BRCT domains are known, all of which bind to specific histone PTMs in a context-dependent manner. For instance, the BPTF subunit of the NURF chromatin-remodeling complex has a PHD domain that binds trimethyl-Lys and an adjacent bromodomain that binds acetyl-Lys. A recent structure of the BTPF PHD domain bound to an H3K4me3 peptide demonstrates how the adjacent bromodomain could in principle bind simultaneously to H3K4me3 and H4K16ac in the same nucleosome (Li et al., 2006Li H. Ilin S. Wang W. Duncan E.M. Wysocka J. Allis C.D. Patel D.J. Molecular basis for site-specific read-out of histone H3K4me3 by the BPTF PHD finger of NURF.Nature. 2006; 442: 91-95Crossref PubMed Scopus (170) Google Scholar, Ruthenburg et al., 2007Ruthenburg A.J. Li H. Patel D.J. Allis C.D. Multivalent engagement of chromatin modifications by linked binding modules.Nat. Rev. Mol. Cell Biol. 2007; 8: 983-994Crossref PubMed Scopus (778) Google Scholar). Several chromatin-binding factors have this type of tandem PHD/bromodomain organization, and these could, in general, be utilized to recognize a doubly modified histone tail or nucleosome. This type of dual recognition system could function as another type of AND logic gate by allowing stable binding of an effector protein only when both histone sites are modified on the same molecule. Alternatively, in the case of a nucleosome, one protein could bridge between different PTMs on two histone tails (Ruthenburg et al., 2007Ruthenburg A.J. Li H. Patel D.J. Allis C.D. Multivalent engagement of chromatin modifications by linked binding modules.Nat. Rev. Mol. Cell Biol. 2007; 8: 983-994Crossref PubMed Scopus (778) Google Scholar). This raises another challenge, namely how one determines the modification status of histones in a single nucleosome. In principle, the two PTM-binding domains do not have to be part of the same protein but could belong to two different subunits within a protein complex, of which there are many among the chromatin modifiers. How general simultaneous binding of two different interaction domains in one protein to distinct PTMs will prove to be needs to be ascertained and requires further bioinformatic analysis of the encoded proteome to identify proteins with these properties. One prominent intersection between PTMs is between phosphorylation and ubiquitination (for an earlier review, see Gao and Karin, 2005Gao M. Karin M. Regulating the regulators: control of protein ubiquitination and ubiquitin-like modifications by extracellular stimuli.Mol. Cell. 2005; 19: 581-593Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). There are many parallels between phosphorylation and ubiquitination. Both modifications are employed in a wide variety of cellular processes; they are catalyzed by large numbers of transferases (>500 protein kinases and >600 E3 ubiquitin [Ub] ligases, respectively) and are reversed by large numbers of hydrolases (>140 protein phosphatases and >100 deubiquitinating enzymes [DUBs], respectively). In consequence, very large numbers of different proteins in the cell are phosphorylated and ubiquitinated; recent estimates based on phosphoproteomic analysis indicate that the majority of proteins in a cell can be phosphorylated at one or more sites under the right circumstances, and the number of proteins known to be subject to ubiquitination (including both polyubiquitinated proteins destined for degradation and monoubiquitinated proteins) is steadily increasing. Both systems utilize ATP, albeit in different ways; phosphorylation transfers the γ-phosphate of ATP onto the target protein generating ADP, whereas ATP is used in the first step of Ub activation by the E1 enzyme generating AMP and PPi and an E1∼Ub adduct, which transfers its Ub onto an E2-conjugating enzyme. However, there are also differences. In general, phosphorylation is a monoadduct (i.e., a phosphate monoester), although in rare cases pyrophosphate may be generated by transfer of phosphate from an inositol pyrophosphate onto an existing phosphoserine in a protein (Bhandari et al., 2007Bhandari R. Saiardi A. Ahmadibeni Y. Snowman A.M. Resnick A.C. Kristiansen T.Z. Molina H. Pandey A. Werner Jr., J.K. Juluri K.R. et al.Protein pyrophosphorylation by inositol pyrophosphates is a posttranslational event.Proc. Natl. Acad. Sci. USA. 2007; 104: 15305-15310Crossref PubMed Scopus (151) Google Scholar). In contrast, ubiquitination is more commonly a polyadduct, although monoUb is increasingly recognized as an important modification. Added diversity in the ubiquitin system comes from the possibility of generating branched Ub chains, in which branches occur at any one of the seven Lys in Ub. In this way, Ub presents multiple chemical surfaces allowing branched Ub chains to be interpreted in different ways; K48-branched chains are recognized by the proteasome leading to degradation of the conjugated protein, and K63-branched chains are used for multiple purposes, including for activation of protein kinases (see below). Ub is almost exclusively added to a single amino acid in target proteins, namely Lys, through the formation of an isopeptide bond between the Ub C terminus and the ɛ-NH2 group of Lys (more rarely, Ub is attached to a free α-NH2 group of a protein or a Cys). In contrast, phosphate can be linked to nine of the 20 amino acids (Ser/Thr, Tyr, His, Lys, Arg, Asp/Glu, and Cys). In general, protein kinases exhibit quite strong selectivity for the primary sequence around the residues that they phosphorylate, although secondary interactions with docking sites elsewhere in target proteins usually play a cardinal role as well. By contrast, there appears to be little primary sequence selectivity for most E3 ubiquitin ligases surrounding the target Lys; in the cases in which specific Lys are highly preferred, this might be dictated by accessibility and the topology of the E3 ligase/target complex. Both protein-linked phosphate and ubiquitin are recognized by modular domains, which mediate inducible protein-protein interaction. Phosphobinding domains of several types are known, with some specific for pSer/pThr (e.g., FHA, BRCT, and 14-3-3) and others for pTyr (e.g., SH2 and PTB) (Seet et al., 2006Seet B.T. Dikic I. Zhou M.M. Pawson T. Reading protein modifications with interaction domains.Nat. Rev. Mol. Cell Biol. 2006; 7: 473-483Crossref PubMed Scopus (509) Google Scholar). There are at least 16 types of ubiquitin-binding domains (UBDs) (e.g., UBA, UIM, CUE, UBZ, etc.), and some of these are specific for particular branch types in polyUb chains (Kirkin and Dikic, 2007Kirkin V. Dikic I. Role of ubiquitin- and Ubl-binding proteins in cell signaling.Curr. Opin. Cell Biol. 2007; 19: 199-205Crossref PubMed Scopus (162) Google Scholar). A major difference in recognition of phosphorylated and monoubiquitinated proteins is that pSer/Thr and pTyr recognition by phosphobinding domains generally depends on the sequence of amino acids immediately around the phosphorylated residue, whereas recognition of monoUb by UBDs does not appear to be influenced by the primary sequence embedding the ubiquitinated Lys. This raises the question of how specificity is determined. One likely possibility is that proteins harboring a UBD have weak but direct interaction with proteins that are ubiquitinated. Such a bipartite interaction would provide increased affinity and specificity. Crosstalk between phosphorylation and ubiquitination occurs at several levels (Figure 1). Phosphorylation can promote or inhibit ubiquitination, which in turn can lead to proteasomal degradation (polyUb, e.g., cyclin E) or processing (polyUb, e.g., Ci or NF-κB2/p100) or regulate intracellular trafficking of membrane proteins (monoUb, e.g., Ste2 and EGFR). Phosphorylation can regulate ubiquitination of a protein in three main ways. First, phosphorylation positively or negatively regulates the activity of the E3 ligase responsible for Ub transfer. Second, phosphorylation promotes recognition by an E3 ligase by creating a phosphodegron. Third, phosphorylation can influence ubiquitination by regulating substrate/ligase interaction at the level of subcellular compartmentalization. Both Ser/Thr and Tyr phosphorylation of E3 ligases can regulate substrate ubiquitination either positively or negatively. There are two main types of E3 ligases—RING finger (single or multisubunit) and U box adaptor E3 ligases and HECT domain catalytic ligases. In the case of HECT domain ligases, phosphorylation can affect target protein binding (e.g., Nedd4-2 phosphorylation in the WW domain region by Sgk1 induces 14-3-3 binding and thereby blocks binding of the ENaC sodium channel substrate to the WW domains [Ichimura et al., 2005Ichimura T. Yamamura H. Sasamoto K. Tominaga Y. Taoka M. Kakiuchi K. Shinkawa T. Takahashi N. Shimada S. Isobe T. 14-3-3 proteins modulate the expression of epithelial Na+ channels by phosphorylation-dependent interaction with Nedd4-2 ubiquitin ligase.J. Biol. Chem. 2005; 280: 13187-13194Crossref PubMed Scopus (157) Google Scholar]) or E3 ligase phosphorylation can cause allosteric activation/inhibition (e.g., Itch activation through JNK Ser/Thr phosphorylation [Gallagher et al., 2006Gallagher E. Gao M. Liu Y.C. Karin M. Activation of the E3 ubiquitin ligase Itch through a phosphorylation-induced conformational change.Proc. Natl. Acad. Sci. USA. 2006; 103: 1717-1722Crossref PubMed Scopus (213) Google Scholar] or inhibition through Fyn-mediated Tyr phosphorylation in the WW domain region [Yang et al., 2006Yang C. Zhou W. Jeon M.S. Demydenko D. Harada Y. Zhou H. Liu Y.C. Negative regulation of the E3 ubiquitin ligase itch via Fyn-mediated tyrosine phosphorylation.Mol. Cell. 2006; 21: 135-141Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar]). RING finger ligases, which lack catalytic activity and act as adaptors by recruiting both an E2∼Ub conjugate and a target protein, are also tightly regulated through phosphorylation. Phosphorylation can in principle promote or inhibit binding of substrate proteins or E2∼Ub to RING finger ligases. For instance, the activity of Mdm2 toward p53 is increased by Akt/PKB Ser phosphorylation through reduced autoubiquitination, increased USP7/HAUSP deubiquitination, and also nuclear accumulation; conversely, Mdm2 activity is inhibited by c-Abl phosphorylation of Y394. COP1 autoubiquitination is stimulated by ATM phosphorylation of S387 through an allosteric mechanism; c-Cbl activity is stimulated by Tyr phosphorylation, possibly via an allosteric mechanism. Multisubunit RING finger E3 ligases, such as SCF and APC/C, can also be positively and negatively regulated by phosphorylation. For instance, association of the Cdh1 substrate selectivity subunit with the APC/C core complex is prevented by Cdk-mediated phosphorylation of Cdh1, thus decreasing APC/C activity toward a subset of targets. Phosphorylation can also regulate E2 activity (e.g., in yeast, phosphorylation by the Bur1/Bur2 Cdk complex activates Rad6, which functions with the Bre1 RING finger E3 ligase in histone monoubiquitination [Wood et al., 2005Wood A. Schneider J. Dover J. Johnston M. Shilatifard A. The Bur1/Bur2 complex is required for histone H2B monoubiquitination by Rad6/Bre1 and histone methylation by COMPASS.Mol. Cell. 2005; 20: 589-599Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar]). In addition, reports of regulation of DUB activity by phosphorylation are beginning to appear (e.g., USP44 phosphorylation by an unknown kinase in mitotic extracts enhances its DUB activity to prevent premature activation of APC/C; CYLD and USP8 are also reportedly regulated by phosphorylation). Inevitably, there will be other ways in which phosphorylation can regulate ubiquitination. For instance, Ub itself may be phosphorylated. A major insight into inducible Ub-mediated protein degradation came with the realization that phosphorylation itself can create a recognition signal for binding of an E3 ligase. Short motifs that mediate phosphorylation-dependent recognition by an E3 ligase have become known as phosphodegrons (in retrospect, the majority of PEST instability sequences [Rogers et al., 1986Rogers S. Wells R. Rechsteiner M. Amino acid sequences common to rapidly degraded proteins: the PEST hypothesis.Science. 1986; 234: 364-368Crossref PubMed Scopus (1900) Google Scholar], containing Pro, Glu, Ser, and Thr bounded by basic residues, are in reality phosphodegrons). This concept was derived from studies of multisubunit SCF (Skp1/cullin/F box protein) RING finger E3 ligases, in which the F box subunit associates with the phosphorylated forms of their substrates (e.g., Skp1/Cdc53/Cdc4/Rbx1 with multiply phosphorylated forms of the Sic1 Cdk inhibitor via the Cdc4 F box protein and Skp1/Cdc53/Grr1/Rbx1 with phosphorylated Cln1 and Cln2 via the Grr1 F box protein). Similarly, analysis of the c-Cbl single-subunit RING finger E3 ligase showed that autophosphorylation of the EGF and PDGF receptor tyrosine kinases (RTKs) creates binding sites for the variant SH2 domain in c-Cbl, allowing c-Cbl recruitment to activated RTKs to promote receptor ubiquitination through association of E2∼Ub conjugates with the RING finger domain that lies just downstream of the SH2 domain in c-Cbl. The majority of Ser/Thr phosphorylation-dependent ubiquitination targets are recognized by members of the SCF family of E3 ligases, which contain a Skp1/cullin core associated with the Rbx1 RING finger protein, and an F box protein, which acts as a substrate specificity determinant (Cardozo and Pagano, 2004Cardozo T. Pagano M. The SCF ubiquitin ligase: insights into a molecular machine.Nat. Rev. Mol. Cell Biol. 2004; 5: 739-751Crossref PubMed Scopus (825) Google Scholar). Two subfamilies of F box proteins are known to recognize phosphodegrons—WD40 repeat and leucine-rich repeat (LRR) F box proteins. WD40 repeat F box proteins, such as Cdc4/Fbw7 and β-TrCP1/2, bind phosphodegrons in a sequence-specific manner via the WD40 β propeller structure, with Cdc4/Fbw7 recognizing LLpTPXXD/pT/S motifs and β-TrCP1/2 binding a diphosphorylated motif with the consensus DpSGxxpS (Table 1). In most but not all cases, the two phosphates in SCF-β-TrCP phosphodegrons are contributed by different protein kinases, and in principle this provides an AND logic gate for degradation of a target protein, which requires that both protein kinases are active at the same time before the protein can be ubiquitinated. LRR F box proteins, such as Skp2, also recognize specific phosphodegron sequences. Elegant structures of complexes of these F box proteins in complex with a target phosphopeptide and additional subunits of the SCF complex establish how phosphorylation-dependent recognition is achieved with such precision (e.g., Wu et al., 2003Wu G. Xu G. Schulman B.A. Jeffrey P.D. Harper J.W. Pavletich N.P. Structure of a beta-TrCP1-Skp1-beta-catenin complex: destruction motif binding and lysine specificity of the SCF(beta-TrCP1)