Gene name - supernumerary limbs
Synonyms - slimb
Cytological map position - 93B10--11
Function - protein degradation \
Symbol - slmb
FlyBase ID: FBgn0283468
Genetic map position -
Classification - Beta-transducin family Trp-Asp repeats protein
Cellular location - cytoplasmic
|Recent literature||Hu, L., Wang, P., Zhao, R., Li, S., Wang, F.,
Li, C., Cao, L. and Wu, S. (2016). The
Drosophila F-box protein Slimb controls dSmurf protein turnover
to regulate the Hippo pathway. Biochem Biophys Res Commun [Epub
ahead of print]. PubMed ID: 27856247
SMAD ubiquitination regulatory factors 1 and 2 (Smurf1/2) are members of the HECT domain E3 ligase family which play crucial roles in the regulation of cell cycle progression, planar cell polarity, cancer metastasis and cell apoptosis. It has been previously shown that the Drosophila homolog dSmurf controls the stability of Warts kinase to regulate the Hippo pathway. This study found that the F-box protein Slimb controls dSmurf protein level to regulate the Hippo pathway. Slimb physically associates with dSmurf as revealed by co-immunoprecipitation assay in S2 cells. The C-terminal WD40 repeats of Slimb (188-510 amino acid) and the C-terminal HECT domain of dSmurf (723-1061 amino acid) are necessary for their binding. Interaction with Slimb leads to the ubiquitination and degradation of dSmurf, resulting in negative regulation of dSmurf-mediated Yki phosphorylation and activity in the Hippo pathway. These data reveal a new regulatory mechanism of the Hippo pathway which may provide implications for developing tumor treatment.
An important mechanism for regulating protein abundance in eukaryotes is the ubiquitin (Ub)-proteasome degradation pathway. The protein Supernumerary limbs (Slmb or more familiarly Slimb) is a subunit of a multi-protein complex that targets proteins for degradation by the ubiquitin-proteasome pathway. Slimb is an important regulator of several developmental pathways, in particular the Wingless, Hedgehog and Dorsal pathways. These developmental roles will be dealt with later, after the ubiquitin-proteosome pathway has been examined. To understand how Slimb functions, it helps to look at its yeast homolog, Cdc4c, because the involvement of this Slimb homolog in regulating the cell cycle in yeast is well understood. The pathway is rather complex, involving many proteins, five of which will be dealt with in some detail here. Briefly, the five protein targets of the pathway in yeast are: (1) cell cycle regulators such as Sic1p, a cyclin dependent kinase inhibitor (see Drosophila Dacapo; (2) the E2 ubiquitin conjugating enzyme Cdc34 that attaches ubiquitin to proteins, targeting them for destruction; (3) Cdc53, a scaffolding protein that binds Cdc34 and other components into a complex; (4) Skp1, an all purpose adaptor protein that holds together Cdc34 and various Slimb homologs, and (5) a dual domain protein, the Slimb homolog (Cdc4, for example), containing an F-box that attaches to Skp1, and a WD40 domain that attaches to the Cdc35 target (Sic1p, for example). A complex composed of Cdc53, Skp1, and an F-box protein (SCF complex) performs what is termed the E3 function, that is, activation of Cdc34 (Mathias, 1999 and references therein).
Ub is a member of a family of conserved polypeptides that are covalently attached to protein substrates. Multiple rounds of modification create a poly(Ub) chain on the substrate that targets the substrate for degradation by the proteasome. The transfer of free Ub onto a protein substrate is a multistep process. E1 activates free Ub at the expense of ATP. Ub is then transferred to an E2 (or ubiquitin protein-conjugating enzyme). Based on sequence comparison, yeast has 11 E2s: it is believed that each E2 is responsible for ubiquitinating distinct substrates. Although a free E2 enzyme may directly transfer Ub onto a substrate in a purified system, this reaction is promoted by additional proteins referred to as E3s or ubiquitin protein ligases. Some E3s act as intermediary Ub carriers in the transfer of Ub from E2 to substrate. Other E3s act as adapters, tethering E2 to E2's substrates. It turns out that a variety of structurally distinct E3 proteins each serve to regulate the interaction between E2 proteins and various distinct substrates (Mathias, 1999 and references therein).
In yeast, the ubiquitin-proteasome degradation pathway regulates two major cell cycle events: entry into S phase and entry into anaphase. Cell cycle progression is mediated by the activity of the cyclin-dependent kinase (CDK) Cdc28p (see Drosophila cdc2). The activation state and specificity of Cdc28p are determined by cyclins and CDK inhibitors. Association with cyclins Cln1 to Cln3 activates Cdc28p during G1, while the mitotic cyclins, Clb1 to Clb6 (see Drosophila CyclinA and CyclinB, are required for the S through M phases. Proteins regulating mitosis, including mitotic cyclins, are targeted for degradation by a cell cycle-regulated E3 complex, the anaphase-promoting complex. During G1, the CDK inhibitor Sic1p acts to inhibit CDK-Clb complex formation and prevent the initiation of S phase. Entry into S phase requires degradation of Sic1p by the ubiquitin-proteasome pathway. Multiple components of an E2-E3 complex are necessary to target Sic1p for degradation (Mathias, 1999 and references therein).
Cells lacking the gene encoding the E2 enzyme Cdc34p remain in G1, develop multiple elongated buds, and fail to separate duplicated spindle pole bodies. This phenotype is consistent with failure to activate the Clb-CDK complexes because of the inability to degrade Sic1p. Mutations in two other genes, CDC4 and CDC53, cause phenotypes indistinguishable from mutations in CDC34. Cdc53p is a member of the so-called 'cullin' family of proteins, characterized by the presence of WD-40 repeats and a second motif known as the F-box. The entry into S phase requires CDC34 to act in concert with CDC4 and CDC53. Indeed, CDC34, CDC4, and CDC53 gene products form a complex in vivo and that complex formation is necessary for S phase entry. A fourth gene, SKP1 encodes yet another member of this complex, and recombinant Cdc34p, together with Cdc4p, Cdc53p, and Skp1p (produced in insect cells) ubiquitinate Sic1p in vitro. Thus, the Cdc4p-Cdc53p-Skp1p complex is an E3 for Cdc34p (Mathias, 1999 and references therein).
In addition to Cdc4p, two other F-box-containing proteins in yeast, Met30p and Grr1p, have been proposed to interact with Skp1p and Cdc53p to form complexes referred as SCFs. Like Cdc4p, both Met30p (50) and Grr1p (9) contain repetitive domains, WD-40 repeats, and leucine-rich repeats, respectively (20), which potentially interact with distinct Cdc34p substrates. Thus, a family of potential E3 complexes has been identified that is distinguished by a component that has been proposed to recognize distinct substrates. It has been suggested that each SCF complex contains only one F-box protein. Thus, Cdc4p, Met30p, and Grr1p interact with a common set of components: Skp1p and Cdc53p (Mathias, 1999 and references therein).
Members of the Hedgehog (Hh) and Wnt/Wingless (Wg) families of secreted proteins control many aspects of growth and patterning during animal development. Hh signal transduction leads to increased stability of the transcription factor Cubitus interruptus (Ci), whereas Wg signal transduction causes increased stability of Armadillo (Arm/beta-catenin), a possible co-factor for the transcriptional regulator Lef1/TCF, known as Pangolin. slimb, negatively regulates both of these signal transduction pathways. Loss of slimb function results in a cell-autonomous accumulation of high levels of both Ci and Arm, and the ectopic expression of both Hh- and Wg- responsive genes. Clones of slimb1 cells in the leg or wing disc ectopically express dpp or wg when they arise in the anterior (but not the posterior) compartments of these discs. Anterior clones reorganize normal limb pattern, creating supernumerary 'double-anterior' limbs. Slimb, like PKA, is a negative regulator that normally prevents activity of the Hh signal transduction pathway in the absence of ligand. slimb mutant cells that arise in the presumptive wing blade ectopically express Scute and differentiate ectopic sensory bristles instead of epidermal hairs on the surface of the wing blade. Both phenotypes are strictly autonomous to the mutant cells, as is the case when the Wg signal transduction pathway is constitutively activated, but not when Wg is ectopically expressed. It is proposed that Slimb normally targets Ci and Arm for processing or degradation by the ubiquitin/proteasome pathway, and that Hh and Wg regulate gene expression, at least in part, by inducing changes in Ci and Arm, which protect both Ci and Arm from Slimb-mediated proteolysis (Jiang, 1998).
In Drosophila, Hh transduces its signal via Cubitus interruptus (Ci), a transcription factor present in two forms: a full-length activator and a carboxy-terminally truncated repressor that is derived from the full-length form by proteolytic processing. The proteolytic processing of Ci is promoted by the activities of Protein kinase A (PKA) and Slimb, whereas it is inhibited by Hh. PKA inhibits the activity of the full-length Ci in addition to its role in regulating Ci proteolysis. Whereas Ci processing is blocked in both PKA and slimb mutant cells, the accumulated full-length Ci becomes activated only in PKA but not in slimb mutant cells. Moreover, PKA inhibits an uncleavable activator form of Ci. These observations suggest that PKA regulates the activity of the full-length Ci independent of its proteolytic processing. Evidence exists that PKA regulates both the proteolytic processing and transcriptional activity of Ci by directly phosphorylating Ci. It is proposed that phosphorylation of Ci by PKA has two separable roles: (1) it blocks the transcription activity of the full-length activator form of Ci, and (2) it targets Ci for Slimb-mediated proteolytic processing to generate the truncated form that functions as a repressor (G. Wang, 1999).
A recent study by Methot (1999) demonstrates the existence of distinct activator and repressor forms of Ci. These two forms play separable roles in Drosophila limb development by regulating different sets of Hh target genes. In the developing wing, the expression of patched and engrailed appears to be exclusively controlled by the activator form of Ci whereas decapentaplegic expression is governed by both the activator and repressor forms. Interestingly, preventing Ci proteolysis with an uncleavable form of Ci is not sufficient to convert Ci into a constitutive activator, suggesting that the full-length activator form of Ci encounters additional regulatory block(s) that need to be alleviated by Hh signaling (Methot, 1999). Evidence is provided that PKA activity exerts such a block. Initial evidence that PKA regulates the activator form of Ci comes from a close examination of PKA and slimb mutant phenotypes. In slimb mutant cells, Ci processing is nearly abolished, and, as a consequence, full-length Ci accumulates. However, the expression of ptc-lacZ and en is not induced, suggesting that the full-length form of Ci that accumulates in slimb mutant cells is transcriptionally silent. In contrast, PKA mutant cells express ptc-lacZ and en even though they accumulate full-length Ci at levels comparable to slimb mutant cells. This suggests that the full-length Ci that accumulates in PKA mutant cells is transcriptionally active. Furthermore, slimb mutant cells with reduced PKA activity ectopically express ptc-lacZ, arguing that the lack of Ci activity in slimb mutant cells is due to an inhibitory role for PKA rather than a positive requirement for Slimb in the Hh signaling pathway. Further evidence that PKA regulates the activator form of Ci independent of its processing come from the gain-of-function studies. The ectopic expression of ptc-lacZ induced by the uncleavable activator form of Ci (CiU) is suppressed by overexpression of a constitutively active form of PKA (mC*) (G. Wang, 1999).
In support of the view that Ci is a direct target for PKA in regulating Hh signaling, it was found that a modified form of Ci with three PKA phosphorylation consensus sites mutated is not processed but exhibits constitutive activity when expressed in the developing wings. Although these observations suggest that PKA antagonizes Hh signaling by directly phosphorylating Ci and targeting it for proteolysis, they do not to address whether phosphorylation of Ci by PKA also regulates the activity of the full-length activator form of Ci. The low levels of constitutive activity exhibited by the PKA phosphorylation-deficient form of Ci could be secondary to the lack of Ci processing, which results in a dramatic increase in the levels of the full-length activator form of Ci, since it has been shown that overexpression of a full-length wild type form of Ci can activate ptc expression in wing discs. To define the role of PKA phosphorylation in regulating the activity of the full-length Ci, advantage was taken of the uncleavable activator form of Ci (CiU). Mutating multiple PKA phosphorylation sites in CiU dramatically alters its transcriptional activity and renders it constitutively active. This observation suggests that the activity of CiU is normally blocked by PKA phosphorylation, even though its processing is impaired. This result provides compelling evidence that PKA phosphorylation of Ci inhibits the activator form of Ci, independent of its role in promoting Ci processing. Taken together, these results suggest the following working model for the inhibitory function of PKA in the Hh pathway. It is proposed that PKA phosphorylation of Ci in its carboxy-terminal region has two separable roles: (1) it blocks the activity of the full-length activator form of Ci, and (2) it targets the full-length Ci for Slimb-mediated proteolysis to generate the truncated repressor form of Ci. Such a dual regulation ensures that only the repressor form of Ci is active in the absence of any Hh signaling. This model accounts for the difference between PKA and slimb mutant phenotypes. In slimb mutant cells, Ci is not processed to the repressor form but accumulates in an inactive phosphorylated form, and, as a consequence, dpp is derepressed at low levels but ptc and en are not activated. In PKA mutant cells, however, Ci accumulates in an unphosphorylated or hypophosphorylated active form, and, as a consequence, ptc and en are activated (G. Wang, 1999).
How phosphorylation of Ci regulates its activity and proteolytic processing remains to be explored. It has been proposed that Su(fu) attenuates Hh signaling activity by blocking a maturation step that converts Ci into a short-lived nuclear transcriptional activator. Analyses of slimb Su(fu) double mutant and slimb Su(fu) PKA triple mutant phenotypes suggest that inhibition of Ci activity by PKA is independent of Su(fu). When Ci processing is blocked, removing Su(fu) only partially stimulates the activity of the full-length Ci whereas simultaneously removing Su(fu) function and reducing PKA activity leads to virtually full activation of Ci. These observations suggest that PKA and Su(fu) act in parallel through independent mechanisms to regulate the activity of the full-length Ci. In slimb Su(fu) double mutant cells, the majority of unprocessed full-length Ci appears to be transformed into a labile nuclear form, and yet the activity of this nuclear form of Ci seems to be inhibited by PKA. This implies that PKA might inhibit Ci at a step after it enters the nucleus. For example, phosphorylation of Ci by PKA might prevent the formation of an active Ci transcription complex or might attenuate its ability to bind DNA. Another possible mechanism by which PKA exerts its influence on Ci is to regulate its nuclear trafficking. It has been shown recently that Hh signaling increases the nuclear import of full-length Ci. As PKA and Hh act antagonistically, it is possible that PKA phosphorylation of Ci might tether the full-length Ci in the cytoplasm in the absence of Hh signaling (G. Wang, 1999).
Su(fu), Cos2, and the Ser/Thr kinase Fused form a multiprotein complex with Ci and the complex associates with microtubules in a manner regulated by Hh. It has been proposed that the assembly of the microtubule-associated Ci complex is critical for inhibiting Ci activity, possibly by tethering Ci in the cytoplasm. The relationship between PKA phosphorylation and the formation of Ci complex is not known. It is not clear whether they are two independent processes or whether one step might regulate the other. The nearly identical phenotypes caused by loss of PKA or Cos2 function in limb development suggest that these two regulatory events might be intimately related. For example, Cos2 might target Ci for efficient phosphorylation by PKA, allowing PKA to exert its negative regulation on Ci. Alternatively, phosphorylation of Ci by PKA might regulate the complex formation, allowing Cos2 to exert its influence on Ci. Further genetic and biochemical studies are required to resolve this important issue (G. Wang, 1999).
It has been proposed that phosphorylation of Ci by PKA allows Slimb to bind Ci and target it for ubiquitin/proteasome-mediated proteolysis. The epistatic relationship between PKA and Slimb defined by this study is consistent with this hypothesis. Moreover, it has been shown recently that proteasome is involved in Ci proteolytic processing. However, no evidence has been obtained to indicate that Ci is ubiquitinated. It is possible that the polyubiquitin chains added to Ci might be unstable and thus might escape detection. Alternatively, the proteolytic processing of Ci might not be directly mediated by ubiquitination, and Slimb might regulate Ci processing indirectly. For example, the so-called SCF (Skp1, Cdc53, and F-box) ubiquitin ligase complex (SCFSlimb) might promote the ubiquitination and degradation of an inhibitor of a protease that cleaves Ci (G. Wang, 1999).
Another important question that remains largely unanswered is how Hh antagonizes PKA. The structural similarity between Smo and G protein-coupled seven-transmembrane receptors suggests that Hh signaling might antagonize PKA by down-regulating its cAMP dependent kinase activity. However, the observations that a constitutively active cAMP independent form of PKA (mC*) can rescue PKA mutant phenotypes without perturbing normal Hh signaling both in embryos and in imaginal discs strongly argue against this possibility. The finding that high but not low levels of mC* are able to override Hh signaling is more consistent with a model in which Hh and PKA act competitively and antagonistically on Ci. For example, Hh may activate a phosphatase that removes the phosphates added to Ci by PKA. In support of this view, pharmacological evidence suggests that Hh stimulates target gene expression via a PP2A-like phosphatase in tissue culture cells. However, there is no genetic evidence for the involvement of a phosphatase in the Hh pathway (G. Wang, 1999).
In vertebrates, Hh signaling is mediated by three members of the Gli family of transcription factors: Gli1, Gli2, and Gli3. Like Ci, all three Gli proteins contain multiple PKA phosphorylation consensus sites at conserved positions, so they are likely to be direct targets for PKA regulation in the vertebrate Hh signaling pathway. Among the three Gli proteins, Gli3 is both structurally and functionally related to Ci. Gli3 has been implicated to have both activator and repressor function depending on the developmental contexts. Moreover, PKA appears to promote Gli3 processing to generate a putative repressor form. Thus, the mechanism by which PKA targets Ci for Slimb-mediated processing may well be conserved from invertebrates to vertebrates. Gli1 and Gli2 appear to function mainly as positive regulators in the vertebrate Hh signaling pathway. Unlike Gli3 and Ci, Gli1 and Gli2 do not undergo PKA-dependent processing, however, their activities are likely to be regulated by PKA. For example, it has been shown that overexpression of a constitutive active form of PKA represses the transcriptional activity of Gli1 in mammalian culture cells. Thus, whereas the mechanism by which PKA regulates Ci processing may only apply to Gli3, the processing-independent inhibitory mechanism defined by this study may well apply to all three Gli proteins and is likely to be a more general mechanism by which PKA negatively regulates Hh signaling (G. Wang, 1999 and references therein).
The slimb gene was identified in two laboratories (Jiang, 1998 and Theodosiou, 1998). The transcript encodes a Cdc4-related protein containing F-box and WD-40 motifs.
date revised: 14 February 2000
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