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52 Wattenberg, B. and Lithgow, T. (2001) Targeting of C-terminal (tail)-anchored proteins: understanding how cytoplasmic activities are anchored to intracellular membranes. Traffic 2, 66–71 53 Walewski, J.L. et al. (2001) Evidence for a new hepatitis C virus antigen encoded in an overlapping reading frame. RNA 7, 710–721 54 Xu, Z. et al. (2001) Synthesis of a novel hepatitis C virus protein by ribosomal frameshift. EMBO J. 20, 3840–3848 55 Varaklioti, A. et al. (2002) Alternate translation occurs within the core coding region of the hepatitis C viral genome. J. Biol. Chem. 277, 17713–17721

56 Carrère-Kremer, S. et al. (2002) Subcellular localization and topology of the p7 polypeptide of hepatitis C virus. J. Virol. 76, 3720–3730 57 Elbers, K. et al. (1996) Processing in the pestivirus E2-NS2 region: identification of proteins p7 and E2p7. J. Virol. 70, 4131–4135 58 Harada, T. et al. (2000) E2-p7 region of the bovine viral diarrhea virus polyprotein: processing and functional studies. J. Virol. 74, 9498–9506 59 Santolini, E. et al. (1995) The NS2 protein of hepatitis C virus is a transmembrane polypeptide. J. Virol. 69, 7461–7471 60 Yamaga, A.K. and Ou, J.H. (2002) Membrane topology of the hepatitis C virus NS2 protein. J. Biol. Chem. 277, 33228–33234

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61 Mizushima, H. et al. (1994) Analysis of N-terminal processing of hepatitis C virus nonstructural protein 2. J. Virol. 68, 2731–2734 62 Pietschmann, T. et al. (2001) Characterization of cell lines carrying self-replicating hepatitis C virus RNAs. J. Virol. 75, 1252–1264 63 Mottola, G. et al. (2002) Hepatitis C virus nonstructural proteins are localized in a modified endoplasmic reticulum of cells expressing viral subgenomic replicons. Virology 293, 31–43 64 Pfeifer, U. et al. (1980) Experimental non-A, non-B hepatitis: four types of cytoplasmic alteration in hepatocytes of infected chimpanzees. Virchows Arch. B Cell Pathol. Incl. Mol. Pathol. 33, 233–243

Similarities between the Hedgehog and Wnt signaling pathways Daniel Kalderon Hedgehog and Wnt proteins are signaling molecules that direct many aspects of metazoan development through signal transduction pathways that are just beginning to be understood. Recently, the common use of glycogen synthase kinase 3 and casein kinase 1 has been added to a growing list of straightforward similarities between Hedgehog and Wnt signaling pathways. These kinases silence both pathways by labeling a key transcription factor (Cubitus interruptus) β-catenin) for proteolysis, and it is possible that reversal of or co-activator (β these phosphorylation events is, in each case, central to pathway activation. This review compares the two pathways to explore whether our more extensive knowledge of Wnt pathways can be of predictive value for investigating Hedgehog signaling.

Daniel Kalderon Dept of Biological Sciences, Columbia University, New York NY 10027, USA. e-mail: [email protected]

The Wnts are a large family of secreted proteins that regulate a huge variety of developmental processes in vertebrates and invertebrates by inducing transcriptional or morphological changes in responding cells [1]. Hedgehog (Hh) proteins make up a smaller family and are notably absent from some invertebrates (Caenorhabditis elegans), but they also have numerous transcriptionally mediated developmental roles [2]. Furthermore, inappropriate activation of Wnt and Hh signaling pathways has been linked definitively to the initiation of specific human cancers [3]. Some progress has been made in understanding how Wnt and Hh signals are transduced into a response, but this still falls short of describing a complete chain of events, let alone explaining how different levels of ligand elicit distinct responses in equivalent cells, or how and why the signaling pathways evolved to their present forms. Over the past several years, various discoveries have suggested that there are fundamental similarities between Wnt and Hh signaling pathways [1,2]. Each is activated through a membrane protein http://tcb.trends.com

[Frizzled (Fz) or Smoothened (Smo)] that is related to G-protein-coupled receptors (GPCRs). In both pathways, signaling prevents phosphorylationdependent proteolysis of a key effector [Cubitus interruptus (Ci) or β-catenin], which, in turn, converts a DNA-binding protein from a repressor to an activator of transcription. In addition, in each case, pathway silencing in the absence of ligand requires the same specific component (Slimb–β-TRCP–FWD-1) of an SCF ubiquitin ligase complex and, as most recently discovered, the two protein kinases, glycogen synthase kinase 3 (GSK3) and casein kinase 1 (CK1) [4–8]. Despite these similarities, the receptor ligands themselves are unrelated, there are a host of other components that are apparently pathway specific, and it is not clear if the related Fz and Smo molecules actually function in the same way. This review examines current evidence for similarities and differences between the two pathways, starting with the transmembrane molecules, Fz and Smo. Current evidence suggests a single basic Smo-dependent Hh signaling pathway, but several different Fz pathways with different regulators and effectors have been recognized [1,9,10]. The three major pathways (Fig. 1) are commonly referred to as canonical (Wnt–β-catenin), Wnt–Ca2+, and planar cell polarity (PCP). There are differences among Fz molecules that either permit or bias participation in specific pathways. For example, vertebrate Wnts and Fzs both fall into two largely non-overlapping sets that are able to stimulate either the Ca2+ or the β-catenin pathway efficiently [9]. Similarly, Drosophila Fz – but not DFz2 – functions in the PCP pathway [11]. However, some single Fz isotypes can

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Fig. 1. Three modes of Frizzled (Fz) signaling. It is not known exactly which G proteins are used in Fz–Ca2+ signaling (here drawn as Goα, Goβ and Goγ) and authentication of the proposed mechanism requires further genetic tests. Consequent proposed effectors include diacylglycerol (DAG), inositol tris phosphate (IP3), protein kinase C (PKC) and Ca2+-calmodulin-dependent protein kinase II (CaMKII). Planar cell polarity signaling might involve a Wnt, but cadherins (Fat or Flamingo) might suffice to localize Frizzled (Fz) and Disheveled (Dsh) activities within cells. Downstream effectors could include Dsh and formin family members such as Daam1, RhoA, Rho kinase (RhoK) and Jun N-terminal kinase (JNK). Wnt–β-catenin signaling probably involves recruitment of axin to LRP5/6, but whether Fz acts through a G protein or recruits Dsh are equivocal issues. Signaling releases β-catenin from a destruction complex that includes axin, adenomatous polyposis coli protein (APC), glycogen synthase kinase 3 (GSK3) and casein kinase 1 (CK1). Dickkopf antagonizes pathway activity by binding to Kremen and LRP5/6, stimulating endocytosis of LRP5/6. Both Fz and Smo (see Fig. 2) have a large extracellular cysteine-rich domain (CRD)

mediate more than one response flawlessly, as demonstrated by the sufficiency of either Fz or DFz2 to regulate the β-catenin pathway in Drosophila [11]. The key question when comparing Fz with Smo is: what are the molecular contacts of the entire family of http://tcb.trends.com

The most obvious regulators of Fz activity are Wnts. For Wnt–β-catenin signaling to occur, Wnt must also bind a co-receptor (LRP5 or LRP6 in vertebrates; Arrow in Drosophila) [12,13]. As the presence of Wnt enables binding of Fz to LRP5/6 [14], apposition of the two receptors might be key to initiating signal transduction. By contrast, there is no evidence that Hh or any other ligand binds to Smo. Instead, Smo activity appears to be regulated indirectly by the twelve-transmembrane domain protein, Patched (Ptc) [2] (Fig. 2). Patched inhibits Smo activity, but this repression is relieved by binding of Hh to Ptc. As for possible co-receptors, megalin, a protein in the same low-density lipoprotein (LDL) receptor family as LRP5/6, was recently found to bind Sonic hedgehog (Shh). Furthermore, megalin mutant mice present holoprosencephaly phenotypes resembling those of Shh mutant mice [12,15]. Whether megalin associates with Smo or is required specifically for transduction of a Hh signal has not yet been explored. An essential receptor role for megalin as a Smo partner would be surprising given that Smo activity is not influenced by Hh in the absence of Ptc. Megalin might affect Shh signaling in other ways, perhaps by promoting movement of Shh molecules between cells. Is there any evidence that Fzs, in a similar way to Smo, can be regulated by mechanisms other than direct binding to ligand? There are two situations where this appears to happen. First, during PCP signaling in Drosophila, Wingless, the most widely used Drosophila Wnt, does not directly activate Fz and there is no positive evidence implicating other Wnts [16]. One suggestion is that Fz is directly regulated by a transmembrane cadherin-like molecule, known as Fat [17]. In these PCP settings, Fz and other pathway components show marked asymmetries in plasma membrane concentration within individual cells or between adjacent cells [10,16]. This localization involves an Fz-dependent feedback mechanism [18] and might therefore be accomplished by spatial modulation of Fz activity. However, it is also possible that Fz is constitutively active and that initiation and amplification of the response to polarity cues involves polarized Fz localization without any alteration in Fz specific activity. Second, in vertebrates, Fz–β-catenin signaling can be inhibited by a non-Wnt secreted factor, Dickkopf (Dkk). Dickkopf binds to LRP6 and a transmembrane co-receptor, Kremen, leading to accelerated endocytosis of LRP6 [19] (Fig. 1). This presumably leaves insufficient LRP6 surface protein to act as a co-receptor for Wnts. These mechanisms, involving regulated subcellular localization and, perhaps, constitutively active GPCR-like molecules, bear at least some resemblance to mechanisms currently postulated for regulation of Smo activity by Ptc (Fig. 2).

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‘sterol-sensing’ domain [22]. Additionally, it has been shown directly that Ptc limits accumulation of Smo protein, especially at the plasma membrane [23], and that Hh binding to Ptc can divert Smo into endosomes that escape a lysosomal fate [24]. What remains to be proved is that the observed changes in Smo localization and concentration underlie the regulation of Smo signaling activity; however, inhibition of Shh signaling by antibodies to endosomal membrane components and by mutations in the Rab23 GTPase – a potential regulator of vesicle fusions – support this contention [24,25]. Thus, although certain general features (e.g. involvement of additional receptors and regulation of localization within the cell) are common to regulation of both Smo and Fz activities, no obviously conserved mechanism has yet been revealed. Fz and Smo signaling: G proteins and protein recruitment?

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Fig. 2. Hypothetical scheme for Hedgehog (Hh) signaling. Patched (Ptc) indirectly clears Smoothened (Smo) from the plasma membrane. In the presence of Hedgehog (Hh), Ptc–Hh complexes are endocytosed and directed to lysosomes, but Smo becomes hyperphosphorylated (‘PPP’) and is sorted into vesicles that lack Ptc; Smoothened (Smo) might be active either in vesicles or if returned to the plasma membrane. It is not clear whether G proteins mediate Smo signaling or whether binding of Hh to megalin affects signal transduction or, for example, Hh transport between cells.

How Ptc regulates Smo activity remains a matter of conjecture. It is still conceivable that Ptc inhibits Smo through direct contact, and that binding of Ptc to Hh alters this association, as originally postulated when binding interactions between Ptc and Smo were first detected [20]. However, several lines of evidence suggest that Ptc can inhibit Smo activity catalytically [2,21]. Non-exclusive mechanisms that have been proposed include regulation of Smo phosphorylation, Smo stability, Smo subcellular localization, or the concentration of an unidentified small-molecule modulator of Smo activity that can be pumped across membranes by Ptc. The latter suggestion rests largely on sequence similarities among Ptc, Niemann-Pick type C1 disease protein (NPC1) and prokaryotic multi-drug permeases [22]. The subcellular localization hypothesis is also based partly on the functions of other proteins that, like Ptc, include a http://tcb.trends.com

How do Fz and Smo molecules alter cytoplasmic events? Although crucial features of the Fz–Ca2+ pathway have not yet been authenticated by requisite genetic loss-of-function tests, a variety of observations implicate direct activation of a G protein that is consistent with the GPCR-related structure of Fz [9]. Either the native Rat Fz2 (RFz2) or a chimera of RFz2 with extracellular regions of the β2-adrenergic receptor can mediate agonist-induced release of intracellular Ca2+. In F9 teratocarcinoma cells, this could be inhibited either by pertussis toxin (which inactivates Goα, Gtα and Giα protein families), or by depletion of specific G protein subunits (including Gβ3, Goα and Gtα, but not Gqα) [26], and was accompanied by activation of protein kinase C and calcium-calmodulin-dependent protein kinase II, suggesting Gβγ-mediated activation of phospholipase C [9] (Fig. 1). Is G protein activation evident or instrumental in all Fz pathways? Surprisingly, it is still hard to deliver a definitive answer. On the one hand, a Rat Fz1 (RFz1) chimera with β2-adrenergic receptor mediates ligand-dependent activation of a β-catenin pathway reporter gene in F9 cells. This can be blocked by pertussis toxin or by depletion of G-protein subunits (Gqα or Goα) and can be partially phenocopied by activated Gqα and Goα [27]. On the other hand, there is little published evidence that Fz–β-catenin or Fz–PCP signaling can be disrupted or phenocopied in other settings by manipulation of G protein activity. It would be interesting to use the RFz1–β2-adrenergic receptor chimeras (or, if possible, DFz chimeras) to identify a variety of point mutations that do not elicit G-protein-dependent responses in F9 cells. These variants of intact Fz molecules could then be tested in physiological assays for β-catenin and PCP pathway signaling. If G protein activation does not mediate signal transduction, what else might respond directly to Fz in the β-catenin and PCP pathways? Many other activated receptors nucleate the assembly of a protein complex that juxtaposes, and thereby stimulates,

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Fig. 3. Scaffolds for tethering and phosphorylation. (a) Axin brings casein kinase 1α (CK1α) and glycogen synthase kinase 3 (GSK3) to the vicinity of adenomatous polyposis coli protein (APC) and β-catenin, promoting phosphorylation of APC and β-catenin. Adenomatous polyposis coli protein can favor nuclear exclusion of β-catenin through nuclear export sequences (NES) and microtubule binding. Axin and APC bind through an RGS domain and SAMP repeats, respectively. Disheveled (Dsh) can bind the DIX domain of axin and binds GSK3 binding protein (GBP) through its PDZ domain. This promotes binding of CBP to GSK3, and displacement of GSK3 from axin. It is not known whether binding of Dsh to either GBP or axin is modulated by Wnt signaling. Axin and APC also bind different subunits of phosphatase 2A. (b) Mutual interactions between Ci-155, Cos2 and Fu have been observed but it is not known whether protein kinase A (PKA), GSK3, CK1 or phosphatases are recruited into this complex.

interaction between downstream signaling components. Such a hypothesis for Fz must include mention of Disheveled (Dsh). Disheveled is required in both β-catenin and PCP pathways, has credible connections to other pathway components, and, according to simple genetic tests, is upstream of those components [28]. Wnt–β-catenin signaling is generally accompanied by recruitment of Dsh to the plasma membrane [28,29], and during Drosophila PCP signaling there are marked asymmetries between membrane-associated Dsh and Fz that are likely to be instrumental to the transduction of directional information [10,16,18]. Disheveled consists largely of three distinct homology domains known as DIX, PDZ and DEP (Fig. 3a). The DEP domain is required – and can suffice – for Wnt-induced recruitment to the plasma membrane and might therefore serve as a sensor of Fz activity [30,31]. However, there is no evidence that http://tcb.trends.com

Dsh can bind directly to Fz. Fz–β-catenin signaling is less sensitive than Fz–PCP signaling to alterations of the Dsh DEP domain, and β-catenin pathway activity does not always correlate well with robust membrane localization of Dsh, suggesting that Dsh might be activated in fundamentally different ways in PCP and β-catenin signaling [30,31]. Indeed, demonstrable molecular interactions between Fz and LRP5/6, LRP5/6 and axin [14], and axin and Dsh [32–34], might be sufficient both to bring Dsh to the plasma membrane, and to initiate a change in β-catenin pathway activity (Fig. 3a). Perhaps analogous interactions involving different Fz assistants, such as the cadherin family proteins, Fat or Flamingo, recruit Dsh in PCP signaling. However, it is more appealing to believe that Fz activity itself can recruit Dsh by a mechanism that is common to both PCP and β-catenin pathways. Whether Wnt binding to Fz can recruit Dsh or otherwise modify the phosphorylation status or binding properties of Dsh without participation of LRP5/6 would be an important test of this conjecture. Could Smo signaling fit into any of the above paradigms? There is evidence that, in heterologous cells, Smo can couple to a pertussis-toxin-sensitive G protein (Gi) [35] but there are no substantial indications that this is how Smo activates signaling in its normal setting. Whether there is any spatial apposition of Smo and its effectors within the cell has not been thoroughly investigated. There are no known Smo-binding proteins that could nucleate a series of protein interactions, and neither Dsh nor axin has been implicated in Smo signaling. It is conceivable that the C-terminus of Smo, which is considerably larger than in Fzs, plays a role analogous to that of Dsh. The protein kinases CK1ε and PAR-1 have been shown to both bind to and phosphorylate Dsh (inter alia), and to promote and be at least partially required for Wnt–β-catenin signaling (as determined by limited genetic tests) [36,37]. This suggests a positive role for phosphorylation in the early steps of Wnt–β-catenin signaling that is perhaps focused on Dsh. Specific mechanisms could include CK1ε-stimulated association of Dsh with GSK3 binding protein (GBP; see later) as observed in Xenopus extracts [38], or binding of β-arrestin to phosphorylated Dsh [39], perhaps initiating further protein interactions. Smo also becomes hyperphosphorylated during Hh signaling, co-incident with increased surface accumulation [23]. Smo contains many serines and threonines in its large cytoplasmic tail, including a cluster of protein kinase A (PKA)-primed GSK3 and CK1 sites, as in Ci-155 (see later). Phosphorylation of such sites might regulate recycling through endosomes (and hence Smo activity), perhaps involving β-arrestin, as observed for several GPCRs [40]. Alternatively, the Smo C-terminal domain might have a direct effector function that is promoted by phosphorylation, as hypothesized for Dsh in Wnt signaling. No candidate phospho-dependent Smo partners have been

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Fig. 4. Competition for β-catenin. β-Catenin bound to E-cadherin is quite stable and sequestered from other interactions. Glycogen synthase kinase 3 (GSK3)-phosphorylated adenomatous polyposis coli protein (APC) enhances affinity of β-catenin for axin, drawing β-catenin from potential association with TCF. β-Catenin bound to axin is phosphorylated, ubiquitinated and degraded, regenerating a high affinity β-catenin destruction complex. The phosphorylation-driven cycles of complex formation and proteolysis are interrupted by Wnt signaling. Theoretically, this could be achieved by inhibiting or opposing any of the phosphorylation events shown here, or by impairing any of the interactions between APC, β-catenin, axin, GSK3 and casein kinase 1 (CK1).

documented, but the presence of similar PKA–GSK3–CK1 motifs on Ci-155 and Smo suggests the possibility of a common binding partner to bridge these molecules. Clearly, there is insufficient information to compare immediate Smo and Fz effector functions. At this point, it might be reasonable to speculate that Smo and Fz–β-catenin pathways each use both G protein coupling to enzymatic effectors, and nucleation of protein associations to activate downstream signaling. This could be most economically achieved if the G proteins themselves nucleate protein assemblies. Cytoplasmic events: regulated proteolysis and sequestration Fz–β-catenin signaling

Downstream events in Smo and Fz–β-catenin pathways appear initially to be reasonably analogous. In the absence of Wnt signaling, β-catenin is found in stable cadherin-containing adhesive junction complexes. Elsewhere, β-catenin has a rapid turnover in a process http://tcb.trends.com

that requires the activity of axin (or the related axil–conductin protein), adenomatous polyposis coli protein (APC), GSK3, CK1, the SCF-complex component β-TRCP, and the 26S proteasome. In this process, binding interactions between axin, APC, CK1 and GSK3 facilitate phosphorylation of β-catenin at multiple sites, culminating in two GSK3 sites that constitute a phospho-dependent binding site for β-TRCP [41] (Fig. 4). β-TRCP promotes polyubiquitination and complete proteolysis of β-catenin. During Wnt signaling, cadherin-free β-catenin increases in abundance, is substantially unphosphorylated at sites crucial for degradation [42], partitions between the nucleus and cytoplasm [43], and can be found associated with the TCF–LEF family of DNA-binding proteins [44]. This results in altered transcription, either as a result of displacing co-repressors from TCF–LEF, or through β-catenin acting as a co-activator for TCF–LEF [44]. What biochemical changes underlie the altered fate of cytoplasmic β-catenin initiated by Wnt signaling? It is often stated that Wnt signaling inhibits GSK3 activity. Indeed, modest reductions in GSK3 activity (approx. twofold) in extracts of mammalian and Drosophila tissue culture cells were detected following exposure of cells to Wnts [45,46]. Similarly small changes in GSK3 activity are considered sufficient for a specific cell-fate decision during Dictyostelium development [47] and for the response of mammalian cells to insulin [48]. Wnt signaling does not, however, appear to regulate GSK3 activity by altering tyrosine phosphorylation of GSK3 (as used by the cAMP receptors CAR3 and CAR4 in Dictyostelium) or by

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stimulating phosphorylation of GSK3 at the PKB site affected by insulin signaling [46,48]. Furthermore, insulin signaling does not stabilize β-catenin. Thus, both the mechanism and the significance of a generalized reduction in GSK3 activity are open to question. The idea that Wnt signaling regulates either access of β-catenin to GSK3, or the activity of GSK3 specifically within a sequestered complex, is more appealing because it would preserve pathway specificity. This concept could now be equally well extended to CK1α. Casein kinase 1α phosphorylation of residue 45 in β-catenin was recently shown to be required in vivo to prime GSK3 phosphorylation of β-catenin, although it is not yet clear which of these phosphorylation events is directly regulated by Wnt signaling [4,6,8]. In the absence of Wnts, several interactions contribute to β-catenin phosphorylation (Fig. 4). First, axin binds CK1α, GSK3 and β-catenin [6,49]. Before the priming role of CK1α was appreciated, it was shown, using purified components, that axin significantly facilitated phosphorylation of (unprimed) β-catenin by GSK3 [49]. Whether axin similarly assists CK1α, or whether CK1α-primed β-catenin phosphorylation by GSK3 is significantly enhanced by axin need to be tested with pure components, but positive results seem probable. Second, the affinity of axin for β-catenin is markedly increased by APC [50]. Furthermore, axin binds APC directly and enhances phosphorylation of APC by GSK3, which, in turn, increases direct binding of APC to β-catenin [51]. Thus, axin-bound GSK3 acts within a positive-feedback loop both to assemble a complex that brings β-catenin together with CK1 and GSK3, and to phosphorylate β-catenin directly (Fig. 4). Why is this system so complicated? Perhaps positive feedback ensures efficient capture and phosphorylation of β-catenin, thus keeping the pathway silent in the absence of stimulation. It also provides many opportunities for a signal-induced event to disrupt efficient phosphorylation and, incidentally, makes it harder to establish which Wnt-induced change is primary. One proposed mechanism for Wnt signaling involves a GSK3 binding protein (GBP–FRAT) that can displace GSK3 from axin [52,53]. To do so efficiently, GBP must bind to Dsh (PDZ domain) and Dsh must bind (through its DIX domain) to axin [32,34,50,52] (Fig. 3a). As tagged axin and Dsh co-localize extensively in the absence of a Wnt signal [30,32,34], it is not obvious which, if any, of these binding events is induced by Fz–LRP5/6 activation. Although GBP appears crucial to Fz–β-catenin signaling in some settings, no GBP homolog has been found in Drosophila. Whether or not Dsh associates with an analogous molecule in Drosophila, it seems probable that Dsh can, in some way, disrupt associations among APC, axin, GSK3, CK1α and β-catenin. Smo signaling

Phosphorylation-dependent proteolysis also plays a key role in Hh signaling. In the absence of Hh, http://tcb.trends.com

full-length Cubitus interruptus (Ci-155) is readily detected, but slowly undergoes partial proteolysis to form a transcriptional repressor (Ci-75) that retains the DNA-binding domain but not the C-terminal transactivation region of Ci-155 [2]. This partial proteolysis requires the activity of PKA, GSK3, Slimb and the 26S proteasome, and the presence of three clustered PKA sites on Ci-155 as well as adjacent GSK3 and CK1 sites that can be primed by PKA phosphorylation [5,7]. In the presence of Hh, Ci-155 is no longer converted to Ci-75. Partial proteolysis of Ci-155 requires multi-site phosphorylation. It is therefore probable that, in the same way as β-catenin, a scaffolding protein brings protein kinases to the vicinity of Ci-155. Costal-2 (Cos2) is a good candidate for this because, like axin, it is a large protein, it is known to bind to Ci-155 and it is required for proteolysis of Ci-155 to Ci-75 [54–57] (Fig. 3b). Could there also be a protein – analogous to APC – that could facilitate assembly of a Ci-155 complex that favors phosphorylation? Fused (Fu) is a protein kinase that can bind independently to Cos2 and Ci-155 through its regulatory domain, which is required for normal Ci-155 proteolysis [57–59]. It is not known if associations between Cos2, Fu and Ci-155 are dependent on phosphorylation by GSK3 or any other protein kinases, but both Fu and Cos2 are phospho-proteins, and their degree of phosphorylation changes rapidly in response to Hh signaling [60]. Does Hh signaling affect Ci-155-complex assembly or the phosphorylation status of Ci-155? Hh signaling can reduce Ci-155 phosphorylation [61] but the specific sites have not been identified, and whether altered phosphorylation is instrumental in inhibition of proteolysis by Hh has not yet been established. The majority of Ci-155 still appears to be in complexes with Fu and Cos2 during Hh signaling. However, these complexes no longer retain microtubule binding activity [57]. Furthermore, only the Ci-155 complexes that do not bind microtubules are found to also contain Suppressor of Fused [Su(fu)] [58], suggesting that Hh signaling might also stimulate changes in the composition of the Ci-155–Fu–Cos2 complex. Mechanisms of proteolysis

How similar are the mechanisms that effect proteolysis of phosphorylated Ci-155 and β-catenin? For β-catenin, the evidence for direct Slimb–β-TRCP binding to the phospho-epitope DSpGXXSp, consequent polyubiquitination, and proteolysis, is compelling. Additional significant interactions among β-catenin complexes and SCF complex components are suggested by the increased affinity of β-TRCP for β-catenin in the presence of axin [62], and the phenotype of Roc1a mutants in Drosophila [63]. Roc1 family proteins contain RING finger motifs and act within SCF complexes to stimulate substrate ubiquitinylation [64]. Roc1a-deficient Drosophila cells accumulate high levels of Ci-155 but levels of β-catenin are not

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significantly altered [63]. This implies that distinct Slimb-containing SCF complexes primarily regulate Ci-155 and β-catenin, and suggests a role for non F-box components in substrate recognition. For Ci-155, there are more fundamental uncertainties surrounding the connection between phosphorylation and proteolysis. First, phospho-epitopes in Ci-155 created by PKA, GSK3 and CK1 do not match those known to be efficiently recognized by Slimb, and there is no published evidence of Slimb binding directly to Ci-155. Second, Ci-155 has not to-date been found to be detectably ubiquitinated, even in cells where proteolysis was prevented by 26S proteasome inhibitors [61]. Perhaps another component of the Ci-155 complex is ubiquitinated to direct Ci-155 to the proteasome, or the 26S proteasome http://tcb.trends.com

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directly or indirectly activates a Ci-155 protease, rather than acting directly on Ci-155. However, if the proteasome does act directly on Ci-155, then why does partial proteolysis ensue? The NFκB precursor, p105, presents the only well-documented precedent for ubiquitin- and proteasome-mediated partial proteolysis [65]. Proteolysis of full-length p105 depends on two short stretches of polypeptide, one of which can recruit a β-TRCP-containing SCF complex when phosphorylated (despite the absence of a consensus β-TRCP binding site). In addition, a glycine-rich region of p105 is essential to stop the apparent step-wise degradation towards the amino-terminus, yielding a stable p50 product. Although the topology is similar, no related sequence is evident in Ci-155. Holding Ci-155 and β-catenin at bay

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Fig. 5. Two-step model for activation of Cubitus interruptus (Ci)-155 by Hedgehog (Hh). Ci-155 partners promote cytoplasmic anchorage and phosphorylation, leading to proteolysis. Hedgehog might induce interdependent changes in the Ci-155 complex and Ci-155 phosphorylation that prevent proteolysis, reduce association with microtubules, and promote Suppressor of Fused [Su(fu)] association. A second Hh-initiated process requiring Fused (Fu) kinase activity promotes nuclear access of Ci-155 and also, perhaps, association with co-activators, such as CBP. Whether this is accompanied by dissociation of some or all Ci-155 partners is not clear.

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Is there more to Hh and Wnt signaling than simply regulating the proteolysis of Ci-155 and β-catenin, respectively? For Hh signaling, the answer is clearly ‘yes’. In several artificial situations, Ci-155 is not proteolyzed to Ci-75 even in the absence of Hh, but ectopic induction of Hh target genes is nevertheless limited [2]. In most of these cases, Ci-155 activity can be enhanced by Su(fu) inactivation and the subcellular localization of Ci-155 correlates well – but not perfectly – with activity. In physiological settings, Ci-155 is always largely cytoplasmic, but Hh stimulates a barely detectable accumulation of Ci-155 in the nucleus [61]. This effect is significantly enhanced when CRM-1-mediated nuclear export is inhibited by leptomycin B (LMB). Ci-155 has a recognizable nuclear export sequence (NES), and the analogous sequence in the related vertebrate Gli-1 protein has been shown to function as an NES [66]. Strong nuclear accumulation of Ci-155 in LMB-treated wing-discs is still dependent on Hh in cells lacking either PKA or Su(fu), but occurs without Hh in cells lacking Cos2 or both PKA and Su(fu) together, and is abrogated even in the presence of Hh when cells lack normal Fu activity [54,59,67,68]. Thus, Ci-155 binding partners not only promote proteolysis but also retain Ci-155 in the cytoplasm and restrict its activity. Hh signaling opposes all of these actions. A two-step mechanism has often been proposed for the Hh response because Ci-155 can be activated to different degrees; however, this is always accompanied (and therefore might be preceded) by inhibition of proteolysis. In my speculative characterization (Fig. 5), the first step both prevents Ci-155 proteolysis by altering Ci-155 phosphorylation, and weakens the interactions between microtubules, Cos2, Fu and Ci-155 that normally restrict Ci-155 very effectively to the cytoplasm. I classify this as a single step because I am speculating that these protein interactions and Ci-155 phosphorylation are interdependent, in a similar way to axin, APC, β-catenin, GSK3 and CK1. The altered Ci-155 complex might incorporate Su(fu)

530

Acknowledgements I thank the NIH for support provided for my research.

Review

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and would still be largely retained in the cytoplasm through associations dependent on Cos2 and Su(fu). In a second step, which requires Fu kinase activity within the complex and is therefore probably driven by phosphorylation events, Su(fu) or other components might dissociate or change their properties, thereby permitting faster nuclear uptake of Ci-155 and also, perhaps, facilitating interaction of Ci-155 with co-activators such as CBP. It is thought that low levels of Hh efficiently promote the first step, blocking Ci-155 proteolysis and modestly activating Ci-155, whereas high levels of Hh are required for efficient execution of the second step. Whether the specific activity of cadherin-free β-catenin is similarly subject to regulation by Wnt signaling is not so clear. Certainly, β-catenin cannot bind to TCF if the Armadillo-repeat superhelix is already occupied by APC in a stable axin–APC–β-catenin complex [50,69]. Subcellular sequestration might also contribute to restricting access of β-catenin to TCF. Adenomatous polyposis coli protein can associate both directly and indirectly with microtubules [70] and has several CRM-1-dependent nuclear export sequences [71,72]. However, it is not known whether Wnt signaling sometimes blocks β-catenin proteolysis without liberating β-catenin from its partners, perhaps by influencing phosphorylation of β-catenin but not APC. There is evidence that suggests that: (1) the affinity of TCF for β-catenin can be modified by phosphorylation [38] and acetylation [44]; (2) β-catenin can be sequestered from TCF by additional partners [69]; and (3) β-catenin–TCF associates with additional molecules, Pygopus and Legless, which are required for Wnt signaling [73]. However, it is not known whether Wnt signaling regulates these processes. Especially intriguing is an indication that Su(fu) participates in Wnt–β-catenin signaling. Suppressor of Fused has been found to associate with β-catenin in cell extracts (although not necessarily directly), and overexpression of Su(fu) limits TCF–LEF induced

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transcription and nuclear accumulation of β-catenin in APC mutant cell lines [74]. It has not been established whether loss of Su(fu) has the opposite effect or whether Wnt-induced changes can effectively counteract the effects of Su(fu). These tests are crucial in the evaluation of whether Su(fu) has a similar function in Wnt and Hh pathways. Nevertheless, the physical association of Su(fu) with β-catenin suggests the possibility that cells contain some complexes that include both Ci-155 and β-catenin, along with their familiar associates. Concluding remarks

Hedgehog and Wnt–β-catenin pathways share the principle of keeping potent transcriptional activators in check in the absence of receptor ligand. As this is achieved through stoichiometric binding partners, the degradation of inactive β-catenin and Ci-155 is also essential. Ci-75 is an important product of proteolysis, maintaining pathway silence by repressing transcriptional targets. The same result is achieved in the Wnt–β-catenin pathway by association of TCF with co-repressors. Phosphorylation by kinases within a complex contributes to the cooperative association of β-catenin with its restraining partners, and it is probable that this feature will also be found in Hh signaling. Signaling disrupts β-catenin and Ci-155 complexes sufficiently to prevent proteolysis but can also elicit further changes that increase the specific activity of spared Ci-155, and perhaps β-catenin also. How Fz, LRP5/6 and Smo relay a signal is not at all clear. It is hard to dismiss some involvement of G proteins, but the body of evidence is slim and does not reveal the complete pathway. Similarly, changes in protein phosphorylation and subcellular localization of Smo and the Fz effector, Dsh, might be crucial, but there is no definitive evidence for causality or necessity. Finally, there are several indications that the cytoskeleton and vesicle trafficking play an active role in Fz and Smo signaling and in their activation.

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