BIOLOGICAL OVERVIEW

In Drosophila, Hedgehog (Hh) protein induces the transcription of target genes encoding secondary signals such as Decapentaplegic (Dpp) and Wingless (Wg) proteins by opposing a repressor system. The repressors include Costa (the more proper though less familiar name for Costal2 [or Cos2]), Protein kinase A, and the Hh receptor, Patched. A protein complex mediates signal transduction from Hh. The complex includes the products of at least three genes: fused (a protein-serine/threonine kinase), cubitus interruptus (a transcription factor), and the subject of this overview, costal2 (a kinesin-like protein). The complex binds with great affinity to microtubules in the absence of Hh, but binding is reversed by Hh. Cos2 is unlikely to possess a motor activity and consequently its role in Hh signal transduction probably does not involve energy dependent transport along microtubules (Sisson, 1997 and Robbins, 1997).

The function of a kinesin-like protein in Hedgehog signal transduction is unexpected, and the reason for its involvement is far from clear. Perhaps Cos2 serves to anchor Cubitus interruptus in the cytoplasm, preventing its transport into the nucleus where it functions as a transcription factor. Of particular interest is the observation that although Ci is found in a complex with Cos2, Cos2 activity reduces Ci staining in anterior compartment cells. Cos2 somatic clones in the anterior compartment of wing discs express high levels of cytoplasmic Ci staining and cause mirror-image duplications of the wing (Sisson, 1997). These results also conflict with the idea that Cos2 stabilises cytoplasmic Ci.

Whereas Cos2 activity is associated with decreased Ci levels, Cos2 levels are posttranscriptionally elevated in the anterior compartment. COS2 mRNA levels are uniform throughout discs. Therefore, the elevated level of Cos2 protein in anterior compartment cells must be due to differences between anterior and posterior cells in either the production or the stability of Cos2 protein. The uniform level of Cos2 throughout the anterior compartment of imaginal discs is inconsistent with Hh signal regulating its accumulation. Perhaps Ci stabilizes Cos2 in a macromolecular complex in anterior cells or Ci heightens translation of COS2 mRNA (Sisson, 1997).

The discovery of a multiprotein complex in the cytoplasm provides some of the explanation for regulation in the Hedgehog pathway, but the dynamic roles of Cos2 and Fused are not yet well understood and the fine details are still obscure. Stimulation of cells with Hh leads to an additional serine phosphorylation for both Fused and Cos2. The protein kinase(s) responsible for these phosphorylations have not been identified. The Hh-induced phosphorylation of Fused appears as long as 30 minutes after induction, suggesting that it represents a feedback device rather than an event in initial signal transduction. This leads in turn to the possibliity that Fused is not autophosphorylating, even though the phosphoryation can be abolished by mutations in the catalytic domain of Fused. Similarly, Fused is apparently not directly responsible for the phosphorylation of Cos2, which occurs even when inactivating mutations are present in the kinase domain of Fused (Robbins, 1997).

Maintaining the spotty understanding of regulation in the Hedgehog pathway, the targets of the Hh receptor (Patched) and the co-receptor (Smoothened) are not yet known. Because Ci lacks an obvious nuclear localization signal, its movement to the nucleus may be regulated by its ability to couple to a protein that carries it there. As the transcription factor Drosophila Creb binding protein (dCBP) has been found to be a transcriptional coactivator of Ci, perhaps dCBP plays a role in nuclear transport of Ci (Akimaru, 1997). Hedgehog signaling involves proteolysis of Cubitus interruptus (Aza-Blanc, 1997); this suggests that other components of the Hh pathway await discovery.

Interactions with Costal2 and Suppressor of fused regulate nuclear translocation and activity of Cubitus interruptus

Costal2 (Cos2) and Suppressor of Fused [Su(fu)] inhibit Ci by tethering it in the cytoplasm, whereas Hh induces nuclear translocaltion of Ci through Fused (Fu). A 125 amino acid domain in the C-terminal part of Ci has been identiifed that mediates response to Cos2 inhibition. Cos2 binds Ci, prevents its nuclear import, and inhibits its activity via this domain. Su(fu) regulates Ci through two distinct mechanisms: (1) Su(fu) blocks Ci nuclear import through the N-terminal region of Ci, and (2) it inhibits the activity of Ci through a mechanism independent of Ci nuclear translocation. Cos2 is required for transducing high levels of Hh signaling activity, and it does so by alleviating the blockage of Ci activity imposed by Su(fu) (Wang, 2000).

Wild-type wing discs accumulate Ci in the nucleus in Hh receiving cells after treatment with Leptomycin B (LMB), a drug that blocks CRM1 dependent nuclear export. Ectopic hh expression in anterior (A) compartment cells away from the compartment boundary induces LMB-dependent nuclear translocation of Ci in these cells. The stimulation of LMB-dependent nuclear import by Hh appears to be much more efficient in the developing imaginal discs than in cultured cl-8 cells. One possible explanation is that cl-8 cells might not fully recapitulate all the Hh signaling properties. The ability of LMB to block nuclear export of Ci in cultured imaginal discs provides an opportunity to address the roles of Cos2 and other Hh signaling components in regulating Ci nuclear import. cos2 mutation results in constitutive nuclear translocation of Ci independent of Hh signaling. In contrast, fu mutation attenuates Ci nuclear translocation induced by Hh. Taken together, these experiments show that Cos2 and Hh have opposing influences on Ci nuclear import: Cos2 exerts a block on Ci nuclear translocation, whereas Hh stimulates Ci nuclear translocation through Fu (Wang, 2000).

Using deletion analysis coupled with in vivo coexpression assays, a 125 amino acid domain has been identified in the C-terminal part of Ci (aa 961-1065) that mediates transcriptional repression and cytoplasmic retention by Cos2. This domain has been named CORD for Cos2 responsive domain. Ci deletion mutants that lack CORD are insensitive to Cos2 repression and are no longer sequestered in the cytoplasm by Cos2 in these assays. Moreover, CORD is sufficient to mediate Cos2-dependent cytoplasmic retention when fused to a heterologous protein. In yeast two hybrid assay, CORD is found to be the only region of Ci that binds Cos2. Taken together, these data provide strong evidence that Cos2 inhibits Ci activity by tethering it in the cytoplasm via directly binding to CORD (Wang, 2000).

A Ci region from aa 703 to aa 850 can act to sequester heterologous proteins in the cytoplasm. However, this region does not mediate cytoplasmic retention by Cos2 because Ci deletion mutants that retain it fail to be sequestered by Cos2 and are resistant to Cos2 inhibition in the in vivo assay. Moreover, Ci fragments containing this region fail to bind Cos2 in yeast. Rather, this Ci domain appears to mediate Ci nuclear export as its effect on nuclear localization is abolished by LMB treatment (Wang, 2000).

The mechanism by which Su(fu) inhibits Hh signaling has remained controversial. Overexpression studies using mammalian cultured cells have shown that Su(fu) can sequester Gli1 in the cytoplasm. However, a different result was obtained from overexpression study using Drosophila cultured cells. For example, overexpressing Su(fu) in Drosophila cl-8 cells fails to block LMB-induced nuclear accumulation of Ci. In addition, several studies have revealed that Su(fu) can interact with Gli1 on DNA, raising the possibility that Su(fu) might affect Gli activity in the nucleus. In this study, genetic evidence is provided that Su(fu) regulates Ci/Gli by both blocking its nuclear import and affecting its activity after nuclear translocation. To overcome the problem of Ci instability in Su(fu) mutant cells, the effect of Su(fu) mutation was examined on nuclear translocation of overexpressed Ci that appears to saturate the mechanism responsible for degrading Ci in the absence of Su(fu). Overexpressed Ci is significantly retained in the cytoplasm in A compartment cells of wild-type wing discs but is largely accumulated in the nucleus in Su(fu) mutant wing discs. Removal of Su(fu) binding domain has a similar effect on Ci nuclear translocation to Su(fu) mutation, suggesting that Su(fu) sequesters Ci in the cytoplasm by directly binding to the N-terminal region of Ci. The ability of Su(fu) to sequester Ci in the cytoplasm appears to depend on Cos2, as Su(fu) does not prevent LMB-dependent Ci nuclear import in cos2 mutant cells (Wang, 2000).

Evidence arguing that Su(fu) affects Ci transcriptional activity in the nucleus comes from analysis of cos2 mutant phenotypes. In wild-type wing discs, A compartment cells abutting the A/P compartment boundary transduce high levels of Hh signaling activity; these high levels convert Ci into a labile transcription activator by antagonizing the inhibitory role of Su(fu). As a consequence, these cells activate en and show low levels of Ci staining. cos2 mutant cells abutting the compartment boundary accumulate high levels of Ci and show low levels of Hh signaling activity as they fail to activate en. Thus, it appears that the majority of Ci in cos2 mutant cells remains in a latent stable form, likely in a complex with Su(fu). In support of this view, it has been shown that removal of Su(fu) from cos2 mutant cells restores high levels of Hh signaling activity and simultaneously decreases the concentration of Ci in these cells. Because Hh induction of Ci nuclear import is not affected by Su(fu) in cos2 mutant cells near the A/P compartment boundary, it is concluded that Su(fu) inhibits Ci activity at a step after it translocates into the nucleus. A possible mechanism by which Su(fu) inhibits Ci activity in the nucleus is to prevent it from forming an active transcriptional complex, since it has been shown that Su(fu) can interact with Gli on DNA (Wang, 2000).

Cos2 was identified as a negative component in the hh pathway by previous genetic studies. A novel, positive role for Cos2 in the hh pathway has now been uncovered. In addition to blocking Hh signal transduction in A compartment cells away from the compartment boundary, Cos2 is required for transducing high levels of Hh signaling activity by antagonizing Su(fu) in A compartment cells near the A/P compartment boundary. In addition, this requirement is a general property of Cos2 that applies to all A compartment cells. Thus, these results underscore an unusual relationship between Cos2 and Su(fu): in the absence of Hh signaling, Cos2 acts cooperatively with Su(fu) to block Ci nuclear import by forming a complex with Ci; in cells receiving high dose of Hh signal, Cos2 is required to alleviate the block on Ci transcriptional activity imposed by Su(fu) (Wang, 2000).

Based on the evidence presented here and elsewhere, a working model for how Cos2, Fu and Su(fu) regulate the nuclear translocation and activity of Ci is proposed. Su(fu) and Cos2 bind Ci via the N- and C-terminal domains, respectively, and the complex binds microtubules through Cos2 and retains Ci in the cytoplasm. In addition, Cos2 promotes the proteolysis of Ci to generate a truncated repressor form (Ci75), a process that also requires the activities of PKA, Slimb, and proteasome. Hh stimulates Ci nuclear translocation through Fu kinase and inhibits Ci processing possibly through dephosphorylating Ci. The transcriptional activity of full-length Ci is attenuated in the nucleus by Su(fu), which also stabilizes the latent form of Ci. High levels of Hh signaling activity convert Ci into a labile and active form, possibly by dissociating it from Su(fu), and this process requires the activities of Fu and Cos2 (Wang, 2000).

Several important issues regarding this model need to be addressed. For example, how Hh antagonizes Cos2 and Su(fu) to promote Ci nuclear translocation remains an important unsolved problem. It is likely that Hh stimulates Ci nuclear import by dissociating Ci complex from microtubules and, further, by releasing Ci from the complex. In support of this view, it has been shown that Hh can induce dissociation of Cos2 from microtubules. Moreover, it has also been implicated that dissociation of Ci tetrameric complex might proceed the nuclear translocation of Ci. However, no biochemical evidence has been obtained indicating that Hh induces dissociation of Ci from Cos2 and Su(fu) (Wang, 2000).

Fu kinase appears to be required for Hh to stimulate Ci nuclear translocation, because Ci is retained significantly in the cytoplasm in fu mutant cells that receive Hh signal. The substrate for Fu kinase still remains a mystery. One attractive candidate is Su(fu), which binds Fu and whose function is antagonized by Fu. Since Su(fu) is a PEST domain protein, phosphorylation of Su(fu) might cause its degradation and subsequent disassembly of Ci complex. Another good candidate for a Fu substrate is Cos2, which also interacts with Fu. It has been shown that Hh induces phosphorylation of Cos2; however, the kinase responsible for Hh-dependent phosphorylation of Cos2 has not been identified. It remains to be determined if Fu contributes to Cos2 phosphorylation. Although the biological significance of Cos2 phosphorylation has not been shown yet, it is conceivable that such phosphorylation could cause dissociation of Cos2 from microtubules or from Ci, leading to Ci nuclear translocation. In support of this view, it has been found that the effect of fu mutation on Ci nuclear translocation can be suppressed by removal of Cos2, arguing that Fu promotes Ci nuclear import by antagonizing Cos2 (Wang, 2000).

Finally, how Cos2 positively regulates Hh signaling activity remains to be determined. The finding that Cos2 is required for Hh to antagonize Su(fu) could be explained by the observation that Cos2 forms a complex with Fu and Su(fu). One scenario is that Cos2 might simply play a structural role in which it antagonizes Su(fu) by recruiting Fu. Alternatively, Cos2 might play a more active role in which it recruits other positive components in close proximity to Fu while also regulating Fused activity in response to Hh signaling. Structure and functional analysis of Cos2 and identifying other Cos2 interacting proteins may help to resolve this important issue (Wang, 2000).

Regulation of mammalian Gli proteins by Costal 2 and PKA in Drosophila reveals Hedgehog pathway conservation

Hedgehog (Hh) signaling activates full-length Ci/Gli family transcription factors and prevents Ci/Gli proteolytic processing to repressor forms. In the absence of Hh, Ci/Gli processing is initiated by direct Pka phosphorylation. Despite those fundamental similarities between Drosophila and mammalian Hh pathways, the differential reliance on cilia and some key signal transduction components had suggested a major divergence in the mechanisms that regulate Ci/Gli protein activities, including the role of the kinesin-family protein Costal 2 (Cos2), which directs Ci processing in Drosophila. This study shows that Cos2 binds to three regions of Gli1, just as for Ci, and that Cos2 functions to silence mammalian Gli1 in Drosophila in a Hh-regulated manner. Cos2 and the mammalian kinesin Kif7 can also direct Gli3 and Ci processing in fly, underscoring a fundamental conserved role for Cos2 family proteins in Hh signaling. Direct PKA phosphorylation regulates the activity, rather than the proteolysis of Gli in Drosophilia, and evidence is provided for an analogous action of PKA on Ci (Marks, 2011).

There are clearly many conserved features of Hh signaling between Drosophila and mammals, and some explicit differences, most obviously with regard to the employment of cilia. However, incomplete understanding can lead to premature conclusions as to exactly what is conserved. When studies of Gli activity were initiated in Drosophila, the absence of key fly Smo activation and Cos2-binding domains from mammalian Smo, coupled to studies showing no effect of Kif7 and Kif27 RNAi on the Hh pathway in tissue culture cells, had led to the suggestion that a Cos2-like molecule was not important in regulating Gli activity in mammals. This study has shown that Cos2 is essential for Gli3 processing into a repressor form in Drosophila, that Cos2 prevents constitutive Gli1 activity while permitting Gli1 activation by Hh in Drosophila and that Cos2 binds to three distinct regions of Gli1, as observed for Ci. These results strongly indicated that a Cos2-like molecule must regulate the activity of Gli proteins in mammals. Indeed, genetic elimination of Kif7 in mouse was subsequently found to reduce processing of Gli3 and was also inferred to result in partial activation of Gli2 in the absence of a Hh ligand. The fundamental demonstration of conservation of Cos2 function focuses attention on more precise questions about how Cos2-family proteins regulate Ci/Gli protein function. Some of these questions have been addressed by comparing the regulation of Ci and Gli proteins in Drosophila (Marks, 2011).

Although physiological regulation of Gli1 protein activity by Shh was demonstrated by knocking Gli1 coding sequence into the mouse Gli2 locus, those mice have not been extensively characterized further to test which specific factors regulate Gli1 activity in mouse. In mice with wild-type Gli genes, the dependence of Gli1 transcription on Hh pathway activity coupled with the ubiquitous presence of Gli2 as a major sensor of pathway activity has prevented an assessment of post-transcriptional regulation of Gli1. For example, genetic loss of mouse suppressor of fused (Sufu) leads to substantial activation of Shh-target genes and transcriptional induction of Gli1, leading only to the conclusion that Sufu normally limits Gli2 activity. Similarly, the effects of mutations affecting cilia or Kif7 have been interpreted only with respect to Gli3 and Gli2 activities (Marks, 2011).

This study found that silencing Gli1 in the absence of Hh signaling requires PKA, PKA sites S544 and S560, Cos2 and the Cos2-binding region equivalent to CORD in Ci, but that T374 and the DSGVEM motif, which has been previously implicated in Gli1 regulation, are not required for Gli1 silencing or activation by Hh. Although the known dependence of Ci phosphorylation on Cos2 suggests that Cos2 might be required to promote Gli1 phosphorylation by PKA, the greater activity of Gli1 lacking the CORD region compared with Gli1 lacking PKA sites in anterior wing disc cells suggests that Cos2 may do more than simply promote Gli1 phosphorylation (Marks, 2011).

How is Gli1 silenced by PKA phosphorylation in Drosophila? Ci silencing involves equivalent PKA sites (in position and immediate sequence context) and creates a Slimb-binding site essential for Ci-155 processing. However, additional PKA and PKA-primed phosphorylation sites important for efficient Ci and Gli3 proteolysis are absent from Gli1. Thus, it would be difficult to predict a priori whether PKA and Hh alter Gli1 activity by regulating its proteolysis (Marks, 2011).

Direct assessment of Gli1 protein levels (using a C-terminal HA tag) in wing disc cells indicated that neither Hh stimulation nor PKA inhibition increased Gli1 levels. However, Ci-155 levels can be a deceptive indicator of PKA-dependent proteolysis because Ci-155 can also be degraded by an activation-dependent mechanism that is independent of PKA. Hence, Ci-155 levels are elevated at intermediate levels of Hh signaling, but in cells exposed to the highest levels of Hh, where PKA-dependent processing is strongly inhibited, Ci-155 levels are very similar to those in anterior cells with no Hh exposure. The observation that Gli1 levels may actually be slightly reduced in posterior cells relative to anterior cells, and in anterior clones deficient for PKA, suggests that activation may also de-stabilize Gli1 and could disguise any putative, opposite contribution from PKA-dependent proteolysis. Consequently, evidence of PKA-dependent proteolysis was sought by creating a Ci-Gli1 fusion protein for which PKA-dependent proteasome digestion would be expected to be arrested and produce Ci-75 repressor. The activity of the Ci-Gli1 fusion protein was regulated by PKA, Cos2 and Hh, as for Gli1 and Ci, but no repressor was formed. Hence, no evidence was found of PKA-dependent proteolysis of Gli1. Weak induction of ptc-lacZ expression in anterior cells was found to require much higher levels of Gli1 protein than evident for equivalent (or greater) ptc-lacZ induction by Gli1 lacking PKA sites 544 and 560, or by wild-type Gli1 in response to Hh. Thus, both PKA inhibition and Hh clearly increase the specific activity of Gli1 and do not appear to stabilize Gli1 protein (Marks, 2011). If PKA principally regulates the specific activity of Gli1 in Drosophila, it might be expected that the same mechanism can regulate Ci-155 activation. Direct evidence for such a role was not observed previously when comparing the activity of low levels of proteolysis-resistant Ci (Ci-S849A) in posterior smo and smo pka mutant clones, even though it is clear that endogenous Ci-155 is activated more strongly by loss of PKA than by loss of Slimb. Likewise, loss of Cos2 activates Ci-155 more strongly than loss of Slimb but did not detectably increase the activity of Ci S849A in posterior smo mutant clones. Su(fu) also has the potential to limit Ci-155 activity. By eliminating Su(fu), this study revealed a repressive influence of both PKA and Cos2 on Ci-S849A in posterior smo mutant clones. It is concluded that the potentially redundant effects of PKA, Cos2 and Su(fu) limiting Ci-155 activity can each be exposed in the absence of Ci-155 processing but are probably most readily seen at high Ci-155 levels. The experiments with Ci do not reveal the mechanism by which PKA and Cos2 can limit Ci-155 activity. An equivalent regulatory influence was much clearer for Gli1; it involves direct Cos2 binding and phosphorylation of defined PKA sites, providing an important starting point for further investigation (Marks, 2011).

Kif7 is known to promote the proteolysis of Gli2 and Gli3 in mouse. This study has shown that Kif7 can also promote Ci-155 and Gli3 processing in Drosophila. Cos2 promotes Gli3 processing and limits Gli1 activity in Drosophila. In all of these cases, PKA and conserved PKA sites have analogous effects to Cos2, consistent with the common hypothesis that Cos2 and Kif7 can promote phosphorylation of Ci-155 and Gli proteins at specific PKA and PKA-primed sites (Marks, 2011).

This study has found that Cos2 effects on Gli1 required the CORD-equivalent region of Gli1. Regulation of Ci-155 by Cos2 does not require the CORD region of Ci. In fact, Ci-155 can be processed and silenced in the absence of both CDN and CORD domains, provided a third Cos2 interaction domain within its zinc fingers is present. Gli1 shares all three Cos2 interaction domains. Moreover, Kif7 can promote processing of Ci lacking CDN and CORD regions, implying that a single Ci/Gli interface with a Cos2/Kif7 protein can silence Ci-155 but not Gli1. It is possible that a single Cos2/Ci-155 interface suffices because it is stabilized by additional common binding partners, Fu and Su(fu), and that these proteins do not contribute efficiently to Cos2/Gli1 binding (Marks, 2011).

Most importantly, Hh inhibits the Cos2-dependent processing of Gli3 and Cos2-dependent silencing of Gli1 in Drosophila. The mechanisms by which Hh opposes Cos2 actions on Ci-155 are not yet fully resolved. One potential mechanism, the dissociation of PKA, CK1 and GSK3 from Cos2, would be expected to influence both Ci and Gli protein activities. However, there is probably also a role for Hh-dependent dissociation of Cos2 from Ci, implying that the interactions between Cos2 (or Kif7) and Gli proteins may be regulated by an analogous mechanism (Marks, 2011).

GENE STRUCTURE

PROTEIN STRUCTURE

Amino Acids - 1201

Structural Domains

The N-terminal (residues 1-450) and C-terminal (residues 1050-1201) regions are predicted to form globular structures consisting of alternating alpha helices and beta sheets. The central region (residues 643-990) contains 36 heptad repeats that are predicted to mediate the formation of a stable homodimer through a parallel coiled coil. Cos2 is similar to members of the kinesin protein family. Over a span of 254 N-terminal residues (residues 136-389) Cos2 is 25% to 30% identical to the motor domains of different members of the kinesin family. Several motor domain motifs implicated in nucleotide or microtubule binding are highly conserved within the kinesin family and are generally conserved in Cos2. At least two motifs have been tentatively implicated in microtubule binding: the strictly conserved DLL motif and the L12 motif, both located in the N-terminal region (Sisson, 1997).

EVOLUTIONARY HOMOLOGS

The Hedgehog (Hh) signaling pathway regulates development in animals ranging from flies to humans. Although its framework is conserved, differences in pathway components have been reported. A kinesin-like protein, Costal2 (Cos2), plays a central role in the Hh pathway in flies. Knockdown of a zebrafish homolog of Cos2, Kif7, results in ectopic Hh signaling, suggesting that Kif7 acts primarily as a negative regulator of Hh signal transduction. However, in vitro analysis of the function of mammalian Kif7 and the closely related Kif27 has led to the conclusion that neither protein has a role in Hh signaling. Using Kif7 knockout mice, this study demonstrates that mouse Kif7, like its zebrafish and Drosophila homologs, plays a role in transducing the Hh signal. Kif7 accumulates at the distal tip of the primary cilia in a Hh-dependent manner. A requirement was also demonstrated for Kif7 in the efficient localization of Gli3 to cilia in response to Hh and for the processing of Gli3 to its repressor form. These results suggest a role for Kif7 in coordinating Hh signal transduction at the tip of cilia and preventing Gli3 cleavage into a repressor form in the presence of Hh (Endoh-Yamagami, 2009).

Positive and negative regulation of gli activity by kif7 in the zebrafish embryo

Loss of function mutations of Kif7, the vertebrate orthologue of the Drosophila Hh pathway component Costal2, cause defects in the limbs and neural tubes of mice, attributable to ectopic expression of Hh target genes. While this implies a functional conservation of Cos2 and Kif7 between flies and vertebrates, the association of Kif7 with the primary cilium, an organelle absent from most Drosophila cells, suggests their mechanisms of action may have diverged. Using mutant alleles induced by Zinc Finger Nuclease-mediated targeted mutagenesis, this study shows that in zebrafish, Kif7 acts principally to suppress the activity of the Gli1 transcription factor. Notably, endogenous Kif7 protein accumulates not only in the primary cilium, as previously observed in mammalian cells, but also in cytoplasmic puncta that disperse in response to Hh pathway activation. Moreover, Drosophila Costal2 can substitute for Kif7, suggesting a conserved mode of action of the two proteins. Kif7 interacts with both Gli1 and Gli2a, suggest that it functions to sequester Gli proteins in the cytoplasm, in a manner analogous to the regulation of Ci by Cos2 in Drosophila. Zebrafish Kif7 potentiates Gli2a activity by promoting its dissociation from the Suppressor of Fused (Sufu) protein and mediates a Smo dependent modification of the full length form of Gli2a. Surprisingly, the function of Kif7 in the zebrafish embryo appears restricted principally to mesodermal derivatives, its inactivation having little effect on neural tube patterning, even when Sufu protein levels are depleted. Remarkably, zebrafish lacking all Kif7 function are viable, in contrast to the peri-natal lethality of mouse kif7 mutants but similar to some Acrocallosal or Joubert syndrome patients who are homozygous for loss of function KIF7 alleles (Maurya, 2013).

Mammalian Sonic hedgehog (Shh) signaling is essential for embryonic development and stem cell maintenance and has critical roles in tumorigenesis. Although core components of the Shh pathway are conserved in evolution, important aspects of mammalian Shh signaling are not shared with the Drosophila pathway. Perhaps the most dramatic difference between the Drosophila and mammalian pathways is that Shh signaling in the mouse requires a microtubule-based organelle, the primary cilium. Proteins that are required for the response to Shh are enriched in the cilium, but it is not clear why the cilium provides an appropriate venue for signal transduction. This study demonstrates that Kif7, a mammalian homologue of Drosophila Costal2 (Cos2), is a cilia-associated protein that regulates signaling from the membrane protein Smoothened (Smo) to Gli transcription factors. By using a Kif7 mutant allele identified in a reporter-based genetic screen, this study shows that, similar to Drosophila and zebrafish Cos2, mouse Kif7 acts downstream of Smo and upstream of Gli2 and has both negative and positive roles in Shh signal transduction. Mouse Kif7 activity depends on the presence of cilia and Kif7-eGFP localizes to base of the primary cilium in the absence of Shh. Activation of the Shh pathway promotes trafficking of Kif7-eGFP from the base to the tip of the cilium, and localization to the tip of the cilium is disrupted in a motor domain mutant. It is concluded that Kif7 is a core regulator of Shh signaling that may also act as a ciliary motor (Liem, 2009).

date revised: 2 September 2022  

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