An intramolecular association between two domains of the protein kinase Fused is necessary for Hedgehog signaling - PubMed (original) (raw)
An intramolecular association between two domains of the protein kinase Fused is necessary for Hedgehog signaling
Manuel Ascano Jr et al. Mol Cell Biol. 2004 Dec.
Abstract
The protein kinase Fused (Fu) is an integral member of the Hedgehog (Hh) signaling pathway. Although genetic studies demonstrate that Fu is required for the regulation of the Hh pathway, the mechanistic role that it plays remains largely unknown. Given our difficulty in developing an in vitro kinase assay for Fu, we reasoned that the catalytic activity of Fu might be highly regulated. Several mechanisms are known to regulate protein kinases, including self-association in either an intra- or an intermolecular fashion. Here, we provide evidence that Hh regulates Fu through intramolecular association between its kinase domain (DeltaFu) and its carboxyl-terminal domain (Fu-tail). We show that DeltaFu and Fu-tail can interact in trans, with or without the kinesin-related protein Costal 2 (Cos2). However, since the majority of Fu is found associated with Cos2 in vivo, we hypothesized that Fu-tail, which binds Cos2 directly, would be able to tether DeltaFu to Cos2. We demonstrate that DeltaFu colocalizes with Cos2 in the presence of Fu-tail and that this colocalization occurs on a subset of membrane vesicles previously characterized to be important for Hh signal transduction. Additionally, expression of Fu-tail in fu mutant flies that normally express only the kinase domain rescues the fu wing phenotype. Therefore, reestablishing the association between these two domains of Fu in trans is sufficient to restore Hh signal transduction in vivo. In such a manner we validate our hypothesis, demonstrating that Fu self-associates and is functional in an Hh-dependent manner. Our results here enhance our understanding of one of the least characterized, yet critical, components of Hh signal transduction.
Figures
FIG. 1.
The protein kinase Fu associates with itself. (A) Purified recombinant Fu appears dimeric. Flag-tagged Fu was affinity purified and then fractionated on a Superose 12 gel filtration column. Samples obtained during the purification were resolved by SDS-PAGE and silver stained (left panel). The flow-through lane represents proteins which did not bind the anti-Flag resin. A single enriched protein was detected at a size consistent with it being Fu (arrow, elution lane). The eluted fractions from the Superose 12 column were resolved by SDS-PAGE and then immunoblotted using anti-Fu antibodies (right panel). Pure Fu elutes with a peak at fraction 55. Standard proteins were used to generate a calibration curve (see Materials and Methods); their elution peaks are marked (arrows). Based on this curve, the apparent molecular mass of Fu is 150 to 200 kDa, approximately twice the size predicted by its primary sequence. (B) Full-length Fu associates with two distinct Fu domains. Baculovirus expressing various Fu constructs or GST (as a negative control) were expressed in Sf21 cells, singly or in combination. The cellular lysates were resolved by SDS-PAGE and immunoblotted with the appropriate antibodies (left panel). The lysates were also immunoprecipitated with anti-Flag (α-Flag), anti-GST (α-GST), or mouse IgG1 isotype-matched antibodies. The immunoprecipitates were resolved by SDS-PAGE followed by immunoblotting with the appropriate antisera (right panel). GST did not significantly associate with any of the Fu proteins tested. (C) Fu-tail associates with ΔFu. Fu-tail and ΔFu were coexpressed, and the resulting lysates were resolved and immunoblotted (left panel) or immunoprecipitated (right panel) by using two different Fu antibodies. Fu-tail, specifically immunoprecipitated by anti-FuH (α-FuH) antibodies, is able to coimmunoprecipitate ΔFu. A similar result is observed when ΔFu is specifically immunoprecipitated with anti-FuKD (α-FuKD) antibodies. Control (Ctrl.) lanes represent normal rabbit serum, which was used as an antibody control. GST was also coexpressed with each of the Fu domains or full-length Fu, and no significant coimmunoprecipitation was observed. The bands which appear in the IgG and α-GST immunoprecipitation lanes represent mouse antibody cross-reactivity. Fu-tail consistently migrates faster than these antibody-cross-reacting proteins.
FIG. 2.
A head-to-tail monomer of Fu binds Cos2. (A) Two models that are consistent with the data presented in Fig. 1. Fu is either a head-to-tail dimer (left panel) or a monomer (right panel), where the kinase domain associates with an adjacent (dimer) or its own (monomer) carboxyl-terminal domain. (B) A Fu monomer binds to Cos2 at the expense of Fu dimer formation. Full-length Fu, six-His-tagged Fu-tail (6XHis Fu-tail) (which is monomeric), and increasing amounts of Cos2 (MOI of 2 and 4 of Cos2 baculovirus, respectively), were expressed in Sf21 cells in various combinations (left panel). The resulting lysates were immunoprecipitated specifically for Fu-tail using anti-His (α-His) or isotype-matched IgG antibodies (right panel). (C) Fu-tail can tether ΔFu to Cos2. After Flag-Cos2 (lanes C) or Flag-Cos2 with Fu-tail (lanes C/F) were immobilized on a Flag affinity resin, Sf21 lysate containing ΔFu was passed through each column and then specifically eluted with Flag peptide (see the text). The mock column (M) contained lysate-infected wt baculovirus and was treated as described above. ΔFu specifically associated with Cos2 from the C/F column compared to results with the C or M column.
FIG. 3.
ΔFu colocalizes with Cos2 in a Fu-tail-dependent manner. ΔFu was coexpressed with Fu-tail and/or eGFP-Cos2ΔN in S2 cells and prepared for immunofluorescence analysis. (A) ΔFu (red) singly expressed in S2 cells appears diffuse. (B and C) The ΔFu (B, red) staining pattern is unchanged upon coexpression with eGFP-Cos2ΔN (C, blue). (D) Merged image of panels B and C. (E to F) ΔFu (E, red) and Fu-tail (F, green) colocalize in punctate structures (arrows). (G) Merged image of panels E and F. (H to J) ΔFu (H, red), Fu-tail (I, green), and eGFP-Cos2ΔN (J, blue) all colocalize in punctate structures, consistent with vesicular localization (arrows). (K) Merged image of panels H to J.
FIG. 4.
Transgenic expression of Fu-tail rescues the wing vein phenotype of a fu class II mutant fly. (A) A wt wing showing the five distinct longitudinal veins (LV1 to LV5) of an adult Drosophila wing. (B) fuG3 is a strong class II fu allele that causes a fusion of LV3 and LV4. (C) Expression of Fu-tail, in a fuG3 mutant background, rescues the wing vein phenotype in 15% of the flies. (A′ to C′) A higher magnification of the wings in A to C. (A′) The double row of bristles (DR) does not normally extend past LV3 into the single-row (SR) domain between LV3 and LV4. (B′) fu mutant wings, like fuG3, exhibit a posterior extension of DR bristles into the SR domain. (C′) Expression of Fu-tail does not fully rescue the wing margin bristle phenotype, since some DR bristles can still be seen. The wings shown were taken from flies that were crossed at 25°C.
FIG. 5.
A model of Fu activation. In low to no Hh activation, Fu is held in a repressed inactive state. In the repressed state, the kinase and carboxyl-terminal domains of Fu are associated, preventing kinase activity and Cos2 activation. This state appears sufficient to facilitate processing of Ci to Ci75. Hh activation promotes dissociation of the kinase domain of Fu from its carboxyl-terminal domain, perhaps by phosphorylation, thereby activating the kinase domain of Fu. The HSC is then further activated by Fu phosphorylation of Cos2 and/or Su(fu), which leads to Ci maturation into its transcriptional activator forms, Ciact and Ci*.
References
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