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).

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).

date revised: 5 February 2001  

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