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. 2004 Feb 20;279(8):7064-71.
doi: 10.1074/jbc.M311794200. Epub 2003 Nov 28.

The Kinesin-related protein Costal2 associates with membranes in a Hedgehog-sensitive, Smoothened-independent manner

Melanie A Stegman  1 , John A Goetz, Manuel Ascano Jr, Stacey K Ogden, Kent E Nybakken, David J Robbins
Affiliations

The Kinesin-related protein Costal2 associates with membranes in a Hedgehog-sensitive, Smoothened-independent manner

Melanie A Stegman et al. J Biol Chem. .

Abstract

In Drosophila, Hedgehog (Hh) signal transduction has been shown to require a multiprotein complex (Hedgehog signaling complex (HSC)), which includes the Kinesin-related protein Costal2 (Cos2), the serine/threonine protein kinase Fused (Fu), and the transcription factor Cubitus interruptus (Ci). We present evidence that a biologically relevant fraction of the HSC is found in association with cellular membranes. We demonstrate that Cos2 is capable of tethering an exogenous protein to vesicular membranes and that Cos2 association with membranes is Hh-sensitive. In addition, we demonstrate that Cos2 associates with membranes in cells that lack the transmembrane protein Smoothened (Smo) through a domain of Cos2 distinct from its recently characterized Smo binding domain. We suggest that an Hh-regulated membrane binding activity of Cos2 is part of the mechanism by which Cos2 contributes to Hh signaling. We propose a model in which there are two distinct HSCs with discrete subcellular localizations and activities: one is endosome-associated and facilitates production of a repressor form of Ci (HSC-R), and one is Smo-associated and promotes Ci activation (HSC-A). In response to Hh and through interaction with Cos2, Smo mediates both inhibition of the endosome-associated HSC-R and activation of HSC-A at the plasma membrane.

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Figures

Fig. 1
Fig. 1. The HSC associates with membranes
A, following hypotonic lysis and fractionation of Drosophila embryos, total cellular membranes were sequentially extracted by homogenization with a Dounce in lysis buffer containing the indicated concentrations of NaCl or finally 1% Nonidet P-40 (NP40). The cytosol and sequential extractions were normalized to protein, separated by SDS-PAGE, and immunoblotted for the indicated proteins. B, total embryo membranes were washed in lysis buffer containing 0.15 m NaCl and then extracted with lysis buffer containing 0.5 m NaCl (producing the S4 fraction). This salt extraction was immunoprecipitated with antibodies to Fu or Cos2 or with rabbit IgG or mouse IgG1 as controls (Ctrl.). The immunoprecipitates were separated by SDS-PAGE and immunoblotted for the indicated proteins. C, the cytosolic fraction (top panel) and S4 extraction of embryo cellular membranes (middle and bottom panels) were fractionated by size on a Superose 6 column. The size exclusion column was equilibrated in lysis buffer containing 0.15 m NaCl (top panel), 0.5 m NaCl (middle panel), or 1 m NaCl (bottom panel). Every other fraction was immunoblotted for Fu. Fractions where standard size markers elute are indicated. Peaks A and B of Fu elution are referred to in the text.
Fig. 2
Fig. 2. Cos2 associates with vesicles and plasma membrane
A, total membranes prepared from S2 cells were washed in 0.15 m NaCl, then resuspended in 1.4 m sucrose, and overlaid with 1.22 m sucrose and then with 0.1 m sucrose. Following equilibrium density centrifugation (112,000 × g for 18 h), fractions were taken from the top of the tube. Each fraction was separated by SDS-PAGE and immunoblotted. Approximately 30% of Kinesin associates with vesicular membranes and is used here as a vesicular membrane marker (fractions 6–10) (61, 63). Fasciclin is a glycosylphosphatidylinositol-anchored protein and is used here as a plasma membrane marker (fractions 2–4) (66). The apparent accumulation of Fu in the plasma membrane to a greater degree than Cos2 is not consistently observed. B, S2 cells were transfected with HA-Cos2, then fixed, and stained with anti-HA-Alexa 488 and with 4,6-diamidino-2-phenylindole (not shown) to visualize DNA followed by confocal microscopy.
Fig. 3
Fig. 3. A central region of Cos2 contains a membrane binding domain
A, S2 cells were transfected with expression vectors containing HA-tagged Cos2 (residues 1–1201), Cos2ΔC, Cos2ΔN, or Cos2M. Two days post-transfection, cells were lysed in hypotonic lysis buffer. Total lysate (Tot), postnuclear cytosol (Cyto), and total membrane (Memb) fractions were normalized to volume, separated by SDS-PAGE, and immunoblotted with α-HA antibody (top panel). An immunoblot of endogenous Cos2 is also shown (bottom panel) to demonstrate that endogenous Cos2 is not displaced by overexpressed Cos2. B, S2 cells transfected with eGFP-Cos2ΔN were lysed, and total membranes were separated by equilibrium density centrifugation. eGFP-Cos2ΔN fractionates in a manner similar to endogenous Cos2. C, electron microscopy of the vesicular membrane fraction (fraction 6) compared with the pellet (fraction 10) of the sucrose gradient in B.
Fig. 4
Fig. 4. The Cos2 membrane binding domain can tether an exogenous protein to vesicles
A, S2 cells expressing eGFP-Cos2ΔN or eGFP were fixed, stained with 4,6-diamidino-2-phenylindole (DAPI) (blue) and observed by fluorescence microscopy. The distribution of eGFP-Cos2ΔN is cytoplasmic and punctate similar to full-length Cos2 (see Fig. 2B). However, eGFP is predominantly nuclear with a diffuse cytoplasmic pattern. B, S2 cells were transfected with eGFP-Cos2ΔN, fixed, and stained with α-Rab11 (red) and observed by confocal microscopy. Rab11 is a marker for early/recycling endosomes (73). Plain arrows indicate eGFP-Cos2ΔN signal alone; filled arrows indicate yellow puncta where co-localization of Rab11 and Cos2 signals occurs.
Fig. 5
Fig. 5. The HSC association with membranes is Hh-sensitive and independent of Smo
A, S2 cells were transfected with Hh or a control vector. Two days post-transfection, cells were lysed in hypotonic lysis buffer and fractionated as described under “Experimental Procedures.” 0.5× total lysate, 1× postnuclear cytosol (Cyto), and 1× total membrane (Memb) samples were normalized to volume, separated by SDS-PAGE, and immunoblotted. Kinesin and Cadherin serve as positive controls for soluble and membrane-associated proteins, respectively. Kinesin is generally about 70% soluble upon hypotonic lysis, while the remainder associates with vesicles (61, 63). Cos2 and Fu are more cytoplasmic in Hh-treated cells. B, Cl8 cells were transfected with a ptc-Luciferase reporter construct and treated with or without dsRNA homologous to Smo in the presence or absence of Hh. Relative luciferase activity was measured post-lysis and demonstrates reduced Hh-activated transcription in cells treated with Smo dsRNA. White bars indicate −Hh; black bars indicate +Hh. C, S2 cells were transfected with Hh or a control vector with or without the addition of Smo dsRNA. Two days post-transfection, cells were hypotonically lysed and fractionated into postnuclear cytosol and total cellular membranes. Left panel, total cellular membranes separated by SDS-PAGE and immunoblotted for Fu, Cos2, and Kinesin. Fu and Cos2 continue to fractionate with membranes despite Smo dsRNA treatment. Right panel, postnuclear cytosols are immunoblotted for Fu and Tubulin. This panel demonstrates that the Hh-induced phosphorylation of Fu does not occur in cells treated with Smo dsRNA, verifying depletion of Smo in these cells. D, S2 cells were transfected with eGFP-Cos2-(501–800), which expresses a fusion protein that contains the membrane binding domain of Cos2 but not the Smo association domain. Confocal microscopy reveals that eGFP-Cos2-(501–800) distribution is similar to full-length Cos2; eGFP-Cos2-(501–800) is both cytoplasmic and punctate. Thus Cos2 does not require a Smo association domain to tether eGFP to vesicles. Rel., relative.
Fig. 6
Fig. 6. Model
See text for details.
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