Secreted Hedgehog (Hh) proteins control many aspects of growth and patterning in animal development. The mechanism by which the Hh signal is sent and transduced is still not well understood. A genetic screen is described that aimed at identifying positive regulators in the hh pathway. Multiple new alleles of hh and dispatched (disp) were recovered. In addition, a novel component in the hh pathway, name central missing (cmn), was identified. Central missing is an alternative name for Sightless, the transmembrane acyltransferase required to generate active Hedgehog protein (Lee, 2001b). Loss-of-function mutations in cmn cause patterning defects similar to those caused by hh or dispatched (disp) mutations. Moreover, cmn affects the expression of hh responsive genes but not the expression of hh itself. Like disp, cmn acts upstream of patched (ptc) and its activity is required only in the Hh secreting cells. However, unlike disp, which is required for the release of the cholesterol-modified form of Hh, cmn regulates the activity of Hh in a manner that is independent of cholesterol modification. cmn mutations bear molecular lesions in CG11495, which encodes a putative membrane bound acyltransferase related to Porcupine, a protein implicated in regulating the secretion of Wingless (Wg) signal (Amanai, 2001).
Several novel components in the hh pathway including PKA (also known as DCO) and supernumary limbs (slimb) have been identified in a genetic mosaic screen for mutations that affect patterning of adult structures. This screen utilized a heat inducible FLP/FRT system to randomly induce mutant clones and pattern abnormalities were examined in adult structures such as wings and legs. This screen efficiently identified inhibitory components in the hh pathway because clones of mutant cells ectopically activating the hh pathway induced pattern duplications if they were situated in the anterior compartment. In addition to PKA and slimb, new alleles of ptc and costal2 (cos2), two previously identified inhibitory components in the hh pathway, were identified. However, positive components in the hh pathway such as disp or tout-velu (ttv), which are involved in either sending or moving the Hh signal, were not isolated in this screen. A likely reason is that Hh signaling occurs only near the AP compartment boundary. Moreover, Hh as well as its major downstream effectors Dpp and Wg, acts cell non-autonomously. Thus, mutant clones that are defective in Hh signaling may cause significant phenotypes only when they are large enough and are situated near the AP compartment boundary, which are infrequent (Amanai, 2001).
To identify additional positive components that regulate either sending or receiving of the Hh signal, several factors suggested the usefulness of an exploration of the Drosophila compound eye. (1) Hh signaling activity is required for the initiation and progression of the morphogenic furrow. Conditional loss of Hh signaling in eyes prevents furrow progression, resulting in a small-eye phenotype. (2) It is possible to generate mosaic flies that have mutant eyes but wild-type bodies, using the eyFLP system. (3) Eyes are dispensable for viability and fertility; thus, mutant flies with eye defects can be recovered in the F1 generation. To test the potential of the eyFLP system for identifying positive components in the hh pathway, mosaic flies were generated with eyes mutant for smo or ttv using eyFLP. smo or ttv mutant eyes exhibit similar small-eye phenotypes to hh mutant eyes. A two-step screen was conducted. In the primary screen, eyFLP was used to generate mosaic flies and a screen was performed in the F1 generation for eye phenotypes similar to those caused by the smo or hh mutation. Once the mutants were bred true in the F2 generation, a secondary screen was conducted in which mosaic flies were generated carrying large mutant clones in the wing and these were screened for wing phenotypes similar to those caused by loss or reduction of Hh signaling activity. An extensive screen was conducted of randomly introduced mutations on the third chromosome and multiple new alleles of hh and disp were recovered. In addition, multiple alleles were isolated for a novel gene, which was named central missing (cmn) based on the wing phenotype. cmn mutant eyes, generated using eyFLP, exhibit similar small-eye phenotypes than those caused by mutations in other positive Hh signaling components (Amanai, 2001).
Nine cmn alleles from the mosaic screen and one additional allele were identified by complementation test with previously isolated lethal mutations mapped near the cmn locus. The cmnM82 allele was used for most of the analyses because cmnM82 homozygotes exhibit similar phenotypes as cmnM82 over deficiency, suggesting that cmnM82 is a genetically null allele. Wings carrying large clones of cmn mutant cells are often smaller and lack patterning elements in the central region such as vein 2, 3, 4 and 5. These phenotypes are similar to those caused by loss of hh in the posterior compartment. Despite the severe patterning defects along the AP axis, the wing margin appears normal, suggesting that cmn does not affect Wg signaling (Amanai, 2001).
The similarity of the cmn and hh phenotypes in both eyes and wings suggests that cmn may affect the hh pathway. To further test this possibility, whether cmn affects the expression of Hh-responsive genes including dpp and ptc, was examined. Since cmn mutants are pupal lethal, dpp and ptc expression were examined in late third instar wing discs homozygous for cmn. cmn mutant discs show reduced levels of dpp expression, as indicated by the expression of both Dpp protein and dpp-lacZ. As expected, the upregulation of ptc expression at the AP compartment boundary is nearly abolished. cmn does not regulate hh expression because hh-lacZ expression is not affected in cmn discs. These observations suggest that cmn acts in the hh pathway rather than upstream of hh to control its expression (Amanai, 2001).
To place cmn in the hh pathway, a genetic epistasis analysis was carried out. To determine whether cmn acts upstream or downstream of ptc, Hh responses were examined in cmn;ptc double mutant cells. To do this, ptc mutant clones were generated in cmn homozygous mutant discs. cmn singly mutant cells exhibit diminished levels of dpp-lacZ expression. In contrast, anteriorly situated cmn;ptc double mutant cells ectopically activate dpp-lacZ at wild-type levels. Thus, ptc mutation can bypass the requirement for cmn in activating the Hh signal transduction pathway, suggesting that cmn acts upstream of ptc (Amanai, 2001).
Cmn could act upstream of Ptc to regulate Hh movement or as a Hh coactivator in the Hh receiving cells. Alternatively, Cmn could regulate the production or secretion of Hh ligand in the Hh sending cells. To distinguish between these two possibilities, a mosaic analysis was carried out in which large clones of cmn mutant cells were generated in either the A- or P- compartment and their effects on ptc-lacZ expression were examined. Wing discs exhibit normal levels of ptc-lacZ expression even though anterior compartment cells near the AP compartment boundary are mutant for cmn. In contrast, wing discs lose ptc-lacZ expression if they contain large P-compartment cmn mutant clones that abut the AP compartment boundary. These results suggest that Cmn, like Disp, regulates the sending of Hh signal in the Hh producing cells (Amanai, 2001).
Whether cmn affects the secretion of Hh into the anterior compartment was investigated by examining Hh distribution in cmn mutant discs. To facilitate the detection of the Hh signal, Hh was overexpressed in P-compartment cells using the hh-gal4 driver line to activate a UAS-Hh transgene. Wild-type wing discs overexpressing Hh in P-compartment cells exhibit Hh staining in A-compartment cells near the AP compartment boundary. In these cells, Hh colocalizes with Ptc in intracellular vesicles. In contrast, cmn mutant discs that overexpress Hh in P-compartment cells exhibit little if any Hh signal in neighboring A-compartment cells. This observation suggests that secretion of Hh into the anterior compartment might be impeded in cmn mutant discs. cmn mutant discs exhibit lower levels of cell surface staining of Hh in the P-compartment. Moreover, cmn mutant P-compartment cells appear to accumulate more punctate intracellular staining of Hh than wild-type P-compartment cells. These observations suggest that cmn may affect Hh trafficking (Amanai, 2001).
It is possible that normal levels of active Hh are produced in P-compartment cells of cmn mutant discs but somehow Hh fails to be released into the anterior compartment. If this is true, one would expect that P-compartment cells should activate Hh responsive genes if provided with Ci. To test this possibility, an uncleavable form of Ci (CiU) was used that requires Hh for its activation. A wing-specific Gal4 driver (MS1096) was used to express UAS-CiU in wild-type, disp or cmn discs and these discs were examined for the expression of a Hh-responsive gene collier (col, a.k.a. knot). In wild-type discs, Hh induces col expression in a stripe of cells in the A-compartment abutting the AP compartment boundary. This col expression is reduced or abolished in disp and cmn mutant discs. Consistent with the previous finding that the activity of CiU depends on Hh, expressing CiU in wild-type wing discs ectopically activates col only in P-compartment cells. Misexpressing CiU in disp mutant discs activates col in P-compartment cells at levels comparable to those in wild-type discs. In contrast, P-compartment cells of cmn discs expressing CiU express diminished levels of col. Smo stabilization was also examined as a readout for Hh activity. Wild-type and disp mutant discs stabilize Smo in P-compartment cells at comparable levels. In contrast, cmn mutant discs stabilize Smo at levels much lower than wild-type or disp mutant discs. Taken together, these observations demonstrate that disp mutant discs produce normal levels of active Hh in P-compartment cells whereas cmn mutant P-compartment cells produce reduced levels of active Hh (Amanai, 2001).
Hh is produced as a full-length precursor, which undergoes an auto-processing event to generate a cholesterol-modified N-terminal fragment that functions as a ligand. To determine if cmn affects Hh processing, a test was made to determine whether a pre-cleaved form of Hh (HhN) could rescue cmn mutant phenotypes. The actin>CD2>Gal4 driver line was used to express UAS-HhN uniformly in wild-type, disp or cmn mutant discs and ptc upregulation was examined as a readout for the Hh signaling activity. Indiscriminately expressing HhN in either wild-type or disp mutant discs causes ectopic ptc upregulation in the entire A-compartment. In contrast, uniformly expressing HhN in cmn mutant discs fails to induce upregulation of ptc. These results suggest that Cmn does not regulate the cleavage of the Hh precursor into the mature form of Hh. Since HhN is no longer modified by cholesterol, this result also suggests that cmn is required for the activity of Hh, independent of cholesterol modification (Amanai, 2001).
These experiments suggest that cmn acts upstream of ptc and its function is required in the Hh sending cells but not in cells that receive the Hh signal. Thus, cmn represents a second gene after disp that regulates sending of the Hh signal. In cmn mutant discs, no Hh signal is detected in anterior compartment cells near the AP compartment boundary. However, unlike the case of disp, no accumulation of Hh staining is observed in P-compartment cmn mutant cells. In contrast, cmn mutant P-compartment cells consistently exhibited lower levels of cell surface staining of Hh with concomitant increase in the number and size of intracellular Hh aggregates as compared with wild-type cells. This observation implies that cmn might affect cellular trafficking of Hh. Consistent with lower levels of cell surface Hh staining, it was found that cmn mutant P-compartment cells produce lower levels of active Hh as compared with wild-type or disp mutant cells (Amanai, 2001).
It has been shown that mammalian Sonic hedgehog (Shh) acquires a palmitoyl modification on an N-terminal cysteine in cell culture. N-terminal fatty acid modification of Shh appears to enhance its activity in certain developmental settings. In addition, mutation of a conserved cysteine residue in Drosophila Hh (C84S-Hh) impairs its function in vivo, implying that Hh may also be modified by palmitoylation at its N-terminal region. Thus, one possible role for Cmn is to regulate Hh palmitoylation, which may control Hh activity or intracellular trafficking. Indeed, C84S-Hh acts in a dominant negative fashion, implying that it is defective in signaling. Thus, the lack of detectable Hh signal in A-compartment cells of cmn mutant discs could be explained if palmitoyl-free Hh fails to bind and internalize Ptc efficiently (Amanai, 2001 and references therein).
It is interesting to note that Cmn is related to Porcupine, which is required for the secretion of Wg signal. Like Cmn, Porcupine also belongs to the membrane bound acyltransferase family, suggesting that acylation of secreted proteins may be a more general mechanism than previously thought for regulating the activity or secretion of signaling molecules involved in animal development (Amanai, 2001).
One of the most dominant influences in the patterning of multicellular embryos is exerted by the Hedgehog family of secreted signaling proteins. A segment polarity gene has been identified in Drosophila, skinny hedgehog (ski, a. k. a. sightless); its product is required in Hh-expressing cells for production of appropriate signaling activity in embryos and in the imaginal precursors of adult tissues. The ski gene encodes an apparent acyltransferase, and genetic and biochemical evidence is provided that Hh proteins from ski mutant cells retain carboxyl-terminal cholesterol modification but lack amino-terminal palmitate modification. These results suggest that ski encodes an enzyme that acts within the secretory pathway to catalyze amino-terminal palmitoylation of Hh, and further demonstrate that this lipid modification is required for the embryonic and larval patterning activities of the Hh signal (Chamoun, 2001).
In a genetic screen for components of the Drosophila Hh signaling pathway mutations in the skinny hedgehog (ski) gene were found to produce phenotypes typical of those resulting from loss of hh or wingless function. Mutant individuals lacking zygotic function of ski survive until early pupal stages. However, lethality during the embryonic period and a strong segment polarity phenotype result from additional loss of the maternal component of ski function. To determine whether ski is required specifically for either the Hh or Wg pathway, larval tissues, in which the two pathways function independently of each other in distinct subpopulations of cells, were examined. Mutant imaginal discs show wild-type levels of Wg target gene expression but are abnormally small. The expression of the Hh target genes decapentaplegic (dpp) and patched (ptc), respectively, is strongly reduced or absent in these discs. Thus, it is concluded that ski acts in the Hh signaling pathway and that ski mutant discs are undersized because dpp is expressed at abnormally low levels (Chamoun, 2001).
To determine whether ski is required for the production of active Hh signal or for transduction of this signal, Hh target gene expression was examined in genetic mosaics. Clones lacking ski function in the anterior compartment of wing imaginal discs show no effect on dpp or ptc expression. In contrast, discs with large clones of posterior compartment cells lacking ski display reduced expression of Hh target genes in adjacent anterior cells. Since this requirement for ski function is similar to that of hh itself, these results are interpreted as evidence that Ski is essential for effective production of the Hh signal. Ski appears not to be required for expression of the hh gene, since wild-type levels of hh transcription are observed in mutant discs. Neither does Ski appear to be required for secretion of the Hh protein, since abnormal Hh protein accumulation in ski mutant cells is not observed. One remaining possibility is that the ski gene product controls a maturation event critical for activity of the Hh signal (Chamoun, 2001).
Members of the Hedgehog (Hh) family encode secreted molecules that act as potent organizers during vertebrate and invertebrate development. Post-translational modification regulates both the range and efficacy of Hh protein. One such modification is the acylation of the N-terminal cysteine of Hh. In a screen for zygotic lethal mutations associated with maternal effects, rasp, a novel Drosophila segment polarity gene identical to sightless, has been identified. Analysis of the rasp/sightless mutant phenotype, in both the embryo and wing imaginal disc demonstrates that rasp does not disrupt Wnt/Wingless signaling but is specifically required for Hh signaling. The requirement of rasp is restricted only to those cells that produce Hh; hh transcription, protein levels and distribution are not affected by the loss of rasp. Molecular analysis reveals that rasp encodes a multipass transmembrane protein that has homology to a family of membrane bound O-acyl transferases. These results suggest that Rasp-dependent acylation is necessary to generate a fully active Hh protein (Micchelli, 2002).
In a large genetic screen for EMS-induced, zygotic lethal mutations with maternal effects, two allelic mutations on the third chromosome were identified: 7F21 and 9B15. Normally, the ventral cuticle of a wild-type embryo displays a segmentally repeating pattern of denticle belts and naked cuticle. However, 7F21/7F21 and 9B15/9B15 embryos derived from germline clone (GLC) females display a lawn of denticles and have little or no naked cuticle -- the segment polarity phenotype. Based on the resemblance these mutant embryos have with a coarse file, this mutation was named rasp. 7F21/7F21 and 9B15/9B15 embryos that lack both the maternal and zygotic gene products are referred to here as'rasp mutant embryos'. By contrast, rasp/+ embryos, derived from germ-line clone (GLC) mothers that received a wild-type paternal chromosome often display a cuticle that is indistinguishable from wild type. Finally, rasp/rasp mutant animals derived from heterozygous females survive embryogenesis but die later during larval or pupal stages (Micchelli, 2002).
To characterize the rasp segment polarity phenotype further, the Wg and En protein distribution was examined in rasp mutant embryos. In the ventral embryonic epidermis, Wg signaling is required for maintenance of en transcription during stage 10. En promotes the expression of hh, which is subsequently required to maintain wg transcription. In rasp mutant embryos, both the levels of Wg and En protein fail to be maintained and fade prematurely. The observed segment polarity phenotype and the failure to maintain Wg and En protein in rasp mutant embryos is consistent with a role for Rasp in Wg signaling, Hh signaling, or both (Micchelli, 2002).
The requirement of rasp for Hh target gene activation was examined. In the wing disc patched (ptc) and dpp are expressed in a narrow anterior stripe along the AP boundary. Of these, ptc expression requires higher levels of Hh signaling. In wing discs homozygous for rasp, the level of ptc expression is reduced, as shown by both the ptc-lacZ and ptc Gal4, UAS-GFP reporters. A striking feature of the rasp wing disc phenotype is their markedly reduced size. This suggests that dpp expression might also be reduced in this mutant background, since reception of dpp is normally required for cell proliferation within the wing disc. In situ hybridization of RNA probes to wing discs homozygous for rasp has revealed a reduction in dpp expression levels. Thus, rasp activity is necessary for the expression of two direct transcriptional targets of Hh (Micchelli, 2002).
Sequence analysis of the predicted protein reveals that Rasp is quite hydrophobic and contains at least eleven membrane-spanning regions. An invariant histidine residue was identified that may mark the position of the active site. Members of a conserved family of membrane bound MBOAT proteins have two characteristics in common: (1) these proteins typically contain between eight and ten membrane-spanning regions; (2) they share a region of sequence similarity in common that includes an invariant histidine residue within a long hydrophobic region. Based on these criteria, Rasp appears to be a bona fide MBOAT protein (Micchelli, 2002).
In conclusion this study describes the requirement of rasp in patterning the Drosophila embryo and wing imaginal disc. These results indicate that rasp is required for Hh signaling and that post-translational lipid modification is crucial for long and short range, Hh-dependent patterning. These conclusions are based on the following observations:
(1) The expression of direct transcriptional targets of Hh signaling are reduced in the absence of rasp.
(2) Using clonal analysis, the requirement of rasp was tested in Hh-sending and -receiving cells. Loss of rasp in posterior, Hh-sending cells, has a non-autonomous effect on Hh target gene expression, while no requirement for rasp in receiving cells was detected.
(3) hh transcription, protein levels and distribution do not depend on rasp function.
(4) rasp is required for the production of an active Hh protein.
(5) rasp encodes a protein with homology to acyltransferases (Micchelli, 2002).
What, then, is the role of Rasp in modifying the Hh protein? One possibility is that Rasp is required directly for the cholesterol modification of Hh, and that rasp mutant cells produce Hh, but that it lacks a cholesterol moity. Elegant studies in Drosophila have examined the role of cholesterol modification for Hh activity. These experiments demonstrate that Hh proteins, which are not cholesterol modified, can still activate Hh target genes, even in the absence of endogenous Hh. This, however, is not consistent with what is observed, since lack of rasp activity leads to a reduction of target gene expression. Therefore, the data do not appear to be consistent with a role for rasp in cholesterol modification (Micchelli, 2002).
Members of the MBOAT superfamily that have been well-characterized biochemically encode enzymes that transfer fatty acids onto hydroxyl groups of membrane-tethered targets. Palmitoylation, the attachment of saturated 16-carbon fatty acyl chain to a protein by a thioester bond, is the only other lipid modification of Hh that has been described. A second possibility, then, is that Rasp is directly required for Hh palmitoylation. Studies of mutant variants in which the N-terminal Cys of Drosophila Hh was mutated, fail to be palmitoylated, and lead to phenotypes very similar to those reported here for rasp. While this study does not directly address this possibility, these observations suggest that Rasp may be required directly for Hh palmitoylation. Interestingly, biochemical analysis suggests that the palmitoyl moiety of Hh may not be attached to the thiol group of the N-terminal Cys, but rather to the alpha-amino group. Thus, if Rasp does palmitoylate Hh, this would constitute a difference in the enzymatic specificity between Rasp and other members of the MBOAT superfamily. Finally, it is possible that Rasp is required for additional lipid modifications that are required for Hh activity, but have yet to be characterized (Micchelli, 2002).
Among acylated proteins, there are a growing number that regulate developmental signaling events. This study has highlighted the importance of lipid modification for Hh signal transduction. Such lipid modifications appear to be important for protein-membrane interactions and for targeting proteins to membrane microdomains. Palmitoylation, in particular, has been the subject of increasing interest. This may be due to the fact that, unlike other lipid modifications, palmitoylation can be reversible and thus, constitutes a regulatable step in signal transduction. Finally, like rasp, porcupine (porc) also encodes a protein with homology to members of the MBOAT superfamily; porc is required for sending the Wg/Wnt signal. Thus, some acylating enzymes appear to be highly specific, and may prove to be of general importance in regulating the function of secreted proteins during development (Micchelli, 2002).
Lipid modifications such as palmitoylation or myristoylation target intracellular proteins to cell membranes. Secreted ligands of the Hedgehog and Wnt families are also palmitoylated; this modification, which requires the related transmembrane acyltransferases Rasp (Sightless) and Porcupine, can enhance their secretion, transport, or activity. rasp is also essential for the developmental functions of Spitz, a ligand for the Drosophila epidermal growth factor receptor (EGFR). In cultured cells, Rasp promotes palmitate addition to the N-terminal cysteine residue of Spitz, and this cysteine is required for Spitz activity in vivo. Palmitoylation reduces Spitz secretion and enhances its plasma membrane association, but does not alter its ability to activate the EGFR in vitro. In vivo, overexpressed unpalmitoylated Spitz has an increased range of action but reduced activity. These data suggest a role for palmitoylation in restricting Spitz diffusion, allowing its local concentration to reach the threshold required for biological function (Miura, 2006).
This study shows that the acyltransferase Rasp promotes palmitoylation of the Spi in addition to its previously reported substrate Hh. rasp mutants show phenotypes similar to spi mutants, and rasp is required for the activity of ectopic sSpi produced either by cleavage of endogenous Spi or by expression of a truncated protein. Rasp is also necessary for the hydrophobic character of Spi expressed in S2 cells. Palmitoylation of Spi by Rasp can be reproduced in COS cells, which do not contain any endogenous Spi palmitoyltransferase activity; either these cells do not express a Rasp homolog, or the homolog is too divergent to recognize Drosophila Spi. Mutation of the predicted active site histidine of Rasp blocks palmitate incorporation into Spi, suggesting that Spi may be a direct target of Rasp. However, the possibility cannot be excluded that other proteins present within COS cells contribute to the acyltransferase activity (Miura, 2006).
The basis for substrate recognition by Rasp is not obvious. There is little sequence homology between Hh and Spi following the palmitoylated cysteine, although both proteins have several basic amino acids in the vicinity; basic amino acids follow the palmitoylation site of some classes of intracellular proteins. Myc-tagged Skn, the mouse homolog of Rasp, has been reported to localize to the endoplasmic reticulum (ER) in CHO cells; in S2 cells, colocalization has been seen of HA-Rasp with markers of the Golgi apparatus. If Hh and Spi are palmitoylated in the same cellular compartment, they later follow different paths; Hh is released from the cell through the activity of the membrane protein Dispatched, whereas Spi requires Star for export from the ER and is then activated by Rho-mediated cleavage. Because sSpi can be palmitoylated, cleavage by Rho is not a prerequisite for palmitoylation. However, the effect of palmitoylation on secretion is more dramatic for full-length Spi than for sSpi, suggesting that palmitoylation may be more efficient when Spi undergoes its normal processing (Miura, 2006).
rasp is also required for processes mediated by EGFR ligands other than Spi. The observation that lack of rasp in the germline causes ventralization of the follicle cells suggests that Grk might be palmitoylated. Consistent with this possibility, it was observed that the extracellular domain of Grk also fractionates into the Triton X-114 layer when expressed in S2 cells, although to a lesser extent than Spi. The rasp phenotype is relatively mild compared to loss of grk, suggesting that Grk has a less stringent requirement for palmitoylation than Spi (Miura, 2006).
Wing vein development, which is affected in rasp mutants, requires both Rho and Vein, but not Spi. Since Vein is not synthesized as a transmembrane precursor, the requirement for Rho may suggest the involvement of Krn, a ligand closely related to Spi. Grk and Krn have cysteine residues immediately following the signal peptide, making them likely substrates for Rasp, but Vein does not, consistent with the observation that rasp is not required for the expression of the Vein target gene mirr. It is unclear whether vertebrate EGFR ligands undergo a similar palmitoylation, since none of the known ligands has an N-terminal cysteine residue; TGF-α is palmitoylated on two cysteines in the cytoplasmic domain of the transmembrane precursor, but this is likely to involve a different mechanism. It will be interesting to determine whether EGFR signaling is affected in mice mutant (Chen, 2004) for the rasp homolog Skn (Miura, 2006).
Both the acyltransferase Rasp and cysteine 29 are essential for the activity in vivo of endogenous or overexpressed full-length Spi, and both significantly enhance the activity of overexpressed truncated Spi. By contrast, in vitro studies with sSpiCS clearly argue that loss of palmitoylation has no effect on EGFR binding or activation, or on Argos binding. Thus, it is likely that palmitoylation defines biologically critical spatial or temporal aspects of Spi distribution, rather than affecting its inherent binding properties. Indeed, mutating cysteine 29 in either full-length or truncated Spi allows greater recovery of secreted Spi from cell culture media. In addition, wild-type tagged sSpi shows strong membrane localization both in S2 cells and in imaginal discs, while unpalmitoylated sSpi is not membrane associated in S2 cells and can reach and act on distant cells in vivo. It is therefore suggested that palmitoylation is required to maintain a high local concentration of Spi, perhaps by directly tethering Spi to the plasma membrane or allowing it to form a complex with other factors that restrict its diffusion (Miura, 2006).
Palmitoylation might have additional effects on Spi signaling; its strong effect on secretion of mSpi could be partially due to an inhibitory effect on Spi cleavage, although this would be unlikely to lead to increased Spi activity. It is also possible that palmitoylation contributes to endocytosis and recycling of Spi, a mechanism that has been reported to enhance Wg signaling. Palmitoylation is unlikely to affect ER retention of sSpi, as this occurs in both COS cells and S2 cells. In addition, no effect of palmitoylation has been observed on the intracellular distribution of tagged sSpi in S2 or COS cells (Miura, 2006).
Spi acts as a short-range signal in vivo, in part due to its induction of the secreted feedback inhibitor Argos. Palmitoylation of Spi does not affect its binding to Argos, as expected because this binding is mediated by the EGF domain of Spi. In addition, rasp is required for Spi function even in the complete absence of argos. It is therefore suggested that a high concentration of Spi is necessary simply to reach the level of EGFR activation required for biological function, irrespective of the presence of Argos. The results suggest that palmitoylation is the mechanism used to achieve this local accumulation of Spi (Miura, 2006).
Although Hh, Wg, and Spi all carry palmitate modifications essential for their function, palmitoylation appears to have different effects on each molecule. Wg, though not Wnt3a, requires palmitoylation for its secretion. Shh requires palmitoylation for incorporation into a lipoprotein complex that enhances its transport; Wg is also found in a similar complex. In addition to its effects on transport, palmitoylation enhances Hh activity in assays that do not require transport. It is noted that sSpiCS does not show the dominant-negative effects described for HhC84S, suggesting that palmitoylation does not affect Spi activity in the same way (Miura, 2006).
Palmitoylation of intracellular proteins frequently promotes membrane association, though it usually does so in conjunction with a second lipid modification. This raises the possibility that palmitoylated Spi is associated with the plasma membrane, rather than binding to lipoprotein particles like those that transport Hh and Wg. If so, it will be interesting to learn whether membrane-tethered sSpi can directly bind the EGFR. Full-length transmembrane Spi, in which the EGF domain is adjacent to the membrane, is inactive in the absence of Rhomboid, but membrane association of sSpi through its N-terminal palmitate group would place the EGF domain at a distance from the membrane. If membrane-bound Spi cannot activate the EGFR, Spi may be released from the membrane by depalmitoylation. Cycles of palmitoylation and depalmitoylation have been shown to regulate the intracellular localization of Ras. However, the N-terminal palmitate modification is likely to form a stable amide linkage as in Hh, rather than a labile thioester bond. Alternatively, release of Spi could be accomplished by proteolytic processing. Interestingly, it was found that the sequence of wild-type sSpi released into the media from S2 cells begins at methionine 45, whereas sSpiCS begins with the serine at position 29, immediately after the signal peptide (Miura, 2006).
The observation that palmitoylation of Spi is essential in vivo extends the importance of this modification of extracellular secreted proteins to a third class of ligands. However, its function appears to vary between different molecules and across species. Further study of membrane-bound palmitoyltransferases and their substrates is likely to yield new insights into the regulation of ligand secretion, transport, and activity (Miura, 2006).
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date revised: 25 February 2008
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