Development of multicellular organisms depends, at least in part, on mechanisms which specify, direct or maintain positional information to pattern cells, tissues, or organs. Various secreted signaling molecules, such as members of the transforming growth factor-beta (TGF-β), Wnt, fibroblast growth factors and hedgehog families have been associated with patterning activity of different cells and structures in Drosophila as well as in vertebrates. Perrimon, Cell: 80: 517-520 (1995).
Hedgehog (Hh) was first identified as a segment-polarity gene by a genetic screen in Drosophila melanogaster, Nusslein-Volhard et al., Roux. Arch. Dev. Biol. 193: 267-282 (1984), that plays a wide variety of developmental functions. Perrimon, supra.; Hammerschmidt et al., Trends Genet. 13: 14-21 (1997). Although only one Drosophila Hh gene has been identified, three mammalian Hh homologues have been isolated: Sonic Hh (Shh), Desert Hh (DHh) and Indian Hh (IHh). Reviewed by Hammerschmidt et al., Trends Genet. 13: 14-21 (1997). Shh is expressed at high level in the notochord and floor plate of developing vertebrate embryos where it plays a key role in neural tube patterning. Echelard et al., Cell 75: 1417-30 (1993), Ericson et al., Cell 81: 747-56 (1995), Hynes et al., Neuron 19: 15-26 (1997), Krauss et al., Cell 75, 1431-44 (1993), Marti et al., Nature 375: 322-25 (1995), Roelink et al, Cell 81: 445-55 (1995). Shh also plays a role in the development of limbs (Laufer et al., Cell 79, 993-1003 (1994)), somites (Fan and Tessier-Lavigne, Cell 79, 1175-86 (1994); Johnson et al., Cell 79: 1165-73 (1994)), gut (Roberts et al., Development 121: 3163-74 (1995), lungs (Bellusci et al., Develop. 124: 53-63 (1997) and skin (Oro et al., Science 276: 817-21 (1997), as well as the regulation of left-right asymmetry (reviewed by Ramsdell and Yost, Trends in Genetics 14: 459-65 (1998)). Likewise, IHh and DHh are involved in bone and germinal cell development, Vortkamp et al., Science 273: 613-22 (1996), Bitgood et al., Curr. Biol. 6: 298-304. Shh knockout mice further strengthened the notion that Shh is critical to many aspect of vertebrate development, Chiang et al., Nature 383: 407-13 (1996). These mice show defects in midline structures such as the notochord and the floor plate, absence of ventral cell types in neural tube, absence of distal limb structures, cyclopia, and absence of the spinal column and most of the ribs.
At the cell surface, the Hh signals is thought to be relayed by the 12 transmembrane domain protein Patched (Ptch) [Hooper and Scott, Cell 59: 751-65 (1989); Nakano et al., Nature 341: 508-13 (1989)] and the G-protein coupled like receptor Smoothened (Smo) [Alcedo et al., Cell 86: 221-232 (1996); van den Heuvel and Ingham, Nature 382: 547-551 (1996)]. Both genetic and biochemical evidence support a receptor model where Ptch and Smo are part of a multicomponent receptor complex, Chen and Struhl, Cell 87: 553-63 (1996); Marigo et al., Nature 384: 176-9 (1996); Stone et al., Nature 384: 129-34 (1996). Upon binding of Hh to Ptch, the normal inhibitory effect of Ptch on Smo is relieved, allowing Smo to transduce the Hh signal across the plasma membrane. Loss of function mutations in the Ptch gene have been identified in patients with the basal cell nevus syndrome (BCNS), a hereditary disease characterized by multiple basal cell carcinomas (BCCs). Disfunctional Ptch gene mutations have also been associated with a large percentage of sporadic basal cell carcinoma tumors, Chidambaram et al., Cancer Research 56: 4599-601 (1996); Gailani et al., Nature Genet. 14: 78-81 (1996); Hahn et al., Cell 85: 841-51 (1996); Johnson et al., Science 272: 1668-71 (1996); Unden et al., Cancer Res. 56: 4562-5 (1996); Wicking et al., Am. J. Hum. Genet. 60: 21-6 (1997). Loss of Ptch function is thought to cause an uncontrolled Smo signaling in basal cell carcinoma. Similarly, activating Smo mutations have been identified in sporatic BCC tumors (Xie et al., Nature 391: 90-2 (1998)), emphasizing the role of Smo as the signaling subunit in the receptor complex for Shh.
However, the exact mechanism by which Ptch controls Smo activity still has yet to be clarified and the signaling mechanisms by which the Hh signal is transmitted from the receptor to downstream targets is unclear. Genetic epistatic analysis in Drosophila has identified several segment-polarity genes which appear to function as components of the Hh signal transduction pathway, Ingham, Curr. Opin. Genent. Dev. 5: 492-98 (1995); Perrimon, supra.
Signaling by hedgehog has been shown to be transduced in vertebrates through the Gli family of zinc finger transcription factors, Hynes et al., Neuron 19: 15-26 (1997); Lee et al., Development 124: 2537-52 (1997); Sasaki et al., Development 124: 1313-22 (1997); Ruiz, i Altaba, Development 125: 2203-12 (1998), and in Drosophila by the Gli homologue Cubitus interruptus (Ci) (Orenic et al., Genes Dev. 4: 1053-67 (1990); Alexandre et al., Genes Dev. 10: 2003-13 (1996); Dominquez et al., Science 272: 1621-25 (1996). Consistent with a pivotal role for Ci in transducing the Hh signal, several genes have been identified genetically in Drosophila and shown to modulate Ci activity (reviewed by Goodrich and Scott, Neuron 21: 1243-57 (1998); Ingham, Embo. J. 17: 3505-11 (1998). These include the putative serine threonine kinase fused (Fu), Preat et al., Genetics 135: 1047-62 (1993), a novel protein designated Suppressor of fused (Su(fu)) [Pham et al., Genetics 140: 587-98 (1995); Preat, Genetics 132: 725-36 (1992)] protein kinase A (PKA), Li et al., Cell 80: 553-562 (1995); Pan and Rubin, Cell 80: 543-52 (1995)], the kinesin-like molecule, Costal-2 (Cos-2) [Robbins et al., Cell 90: 225-34 (1997); Sisson et al., Cell 90: 235-45 (1997)], and the F-box/WD40 repeat protein slimb [Jiang and Struhl, Nature 391: 493-496 (1998)]. Additional elements implicated in Hh signaling include the transcription factor CBP [Akimaru et al., Nature 386: 735-738 (1997)], and the Shh response element COUP-TFII [Krishnan et al., Science 278: 1947-1950 (1997)].
Mutations in Cos-2 are embryonicly lethal and display a phenotype similar to Hh over expression, including duplications of the central component of each segment and expansion domain of Hh responsive genes. In contrast, mutant embryos for Ci of fused show a phenotype similar to Hh loss of function, while mutations in negative regulators of the Hh pathway, such as ptch or PKA, induce ectopic expression of Hh-target genes (reviewed by Ingham, Embo. J. 17: 3505-11 (1998)). For example, fused and Ci mutants exhibited deletion of the posterior part of each segment and replacement of a mirror-like image duplication of the anterior part or each segment and replacement of a mirror-like duplication of the anterior part, Busson et al., Roux. Arch. Dev. Biol. 197: 221-230 (1988). Molecular characterizations of Ci suggested that it is a transcription factor which directly activates Hh responsive genes such as Wingless and Dpp, Alexandre et al., (1996) supra, Dominguez et al., (1996) supra. Likewise, molecular analysis of fused reveals that it is structurally related to serine threonine kinases and that both intact N-terminal kinase domain and a C-terminal regulatory region are required for its proper function, Preat et al., Nature 347: 87-9 (1990); Robbins et al., (1997), supra; Therond et al., Proc. Natl. Acad. Sci. USA 93: 4224-8 (1996). However, whereas fused null mutations and N-terminal kinase domain mutations can be fully suppressed by Suppressor of fused mutations, C-terminus mutations of fused display a strong Cos-2 phenotype in a Suppressor of fused background. This suggests that the fused kinase domain can act as a constitutive activator of Shh signaling when Suppressor of Fused is not present.
Su(fu) was originally isolated as a gene, which when activated, was able to suppress the embryonic and adult phenotypes of fused mutants, and when duplicated, enhanced the fused mutant phenotype, suggesting that fused and Su(fu) have antagonistic roles. [Preat, Genetics 132: 725-36 (1992); Preat et al., Genetics 135: 1047-62 (1993)]. Su(fu) mutant flies have a wing phenotype similar to but not as strong as patched or PKA mutants (Ohlmeyer and Kalderon, Nature 396: 749-53 (1998). The combination of patched or PKA mutations in a Su(fu) mutant background enhances the mutant phenotype of patched and PKA, suggesting a cooperative effect of these genes in modulating hedgehog signaling. Ohlmeyer and Kalderon, supra. Fused, Su(fu), Cos-2 and Ci have been shown to form a microtubule-associated multiprotein complex and hedgehog signaling leads to dissociation of this complex from microtubules. Robbins et al., Cell 90: 225-34 (1997); Sisson et al., Cell 90: 235-45 (1997); Monnier et al., Curr. Biol. 8: 583-86 (1998).
Both fused and Cos-2 become phosphorylated in response to Hh treatment, Robbins et al., supra; Therond et al., Genetics 142: 1181-98 (1996), but the kinase(s) responsible for this activity(ies) remain(s) to be characterized. To date, the only known vertebrate homologues for these components are members of the Gli protein family (e.g., Gli-1, Gli-2 and Gli-3). These are zinc finger putative transcription factors that are structurally related to Ci. Among these, Gli-1 was shown to be a candidate mediator of the Shh signal [Hynes et al., Neuron 15: 35-44 (1995), Lee et al., Development 124: 2537-52 (1997); Alexandre et al., Genes Dev. 10: 2003-13 (1996)] suggesting that the mechanism of gene activation in response to Hh may be conserved between Drosophila and vertebrates.
In the absence of hedgehog, full length Ci (Ci-155) is proteolytically processed into an N-terminal repressor fragment (Ci-75). Aza-Blanc et al., Cell 89: 1043-53 (1997). Recent studies demonstrate that complex formation is necessary to target Ci for proteolysis. Methot and Basler, Cell 96: 819-31 (1999). The cleavage of Ci potentially requires PKA phosphorylation of Ci and ubiquitination by Slimb, which targets Ci to the proteosome. Chen et al., Proc. Natl. Acad. Sci. USA 95: 2349-54 (1998); Jiang and Struhl, Nature 391: 493-96 (1998). In response to Hh, Ci cleavage is blocked and Ci-155 is activated into a labile but still uncharacterized form. Ohlmeyer and Kalderon, supra; Methot and Basler, Cell 96: 819-31 (1999).
To determine whether other signaling components in the Hh cascade are evolutionarily conserved and to examine the function of fused in the Hh signaling cascade on the biochemical level, Applicants have isolated and characterized human used cDNA, a kinase homologous the Drosophila Fu (dFu). Tissue distribution on the mouse indicates that fused is expressed in Shh and other hedgehog responsive tissues, and also displays the same subcellular localization as human Gli1 (hGli1) and hSu(fu), the human homologue of Drosophila Su(fu) (dSu(fu)).
Biochemical studies demonstrate that fused is a functional kinase and that it forms a complex with hSu(fu) and hGli1. Functional studies provide evidence that fused is an activator of Gli and that a dominant negative form of fused is capable of blocking Shh signaling in Xenopus embryos. Applicant also herein show that Shh signaling leads to the reversible dissociation of human Su(fu) from human Gli-1 (hGli-1) in mammalian cells. Applicants also demonstrate herein that the catalytic subunit of protein kinase A (PKAc) is present in a complex in association with hSu(fu). PKAc phosphorylates both hSu(fu) and Gli, and thereby promotes the binding of hSu(fu) to Gli, while ectopic hFu or Shh stimulation trigger the dissociation of hSu(fu) from Gli. These biochemical observations correlate with data obtained in a functional readout where fused abrogates hSu(fu)-mediated repression of Gli in a Gli reporter assay. Together this data demonstrated generally that fused is directly involved in Hh signaling and specifically that fused antagonizes PKAc activity, thereby triggering the dissociation of hSu(fu) from hGli-1. Regulation of the hSu(fu)-hGli-1 interaction is central to the control of hGli-1 activity and is promoted by PKAc and inhibited by Shh and hFu.
Applicants have identified a cDNA encoding a human fused (hfused) polypeptide and thus have provided for the first time a vertebrate fused molecule.