Yasuhiko Y et al. (2001), Calmodulin binds to inv protein: implication fo...

XB-ART-7996

Dev Growth Differ 2001 Dec 01;436:671-81. doi: 10.1046/j.1440-169x.2001.00604.x.

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Calmodulin binds to inv protein: implication for the regulation of inv function.

Yasuhiko Y , Imai F , Ookubo K , Takakuwa Y , Shiokawa K , Yokoyama T .

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Establishment of the left-right asymmetry of internal organs is essential for the normal development of vertebrates. The inv mutant in mice shows a constant reversal of left-right asymmetry and although the inv gene has been cloned, its biochemical and cell biological functions have not been defined. Here, we show that calmodulin binds to mouse inv protein at two sites (IQ1 and IQ2). The binding of calmodulin to the IQ2 site occurs in the absence of Ca(2+) and is not observed in the presence of Ca(2+). Injection of mouse inv mRNA into the right blastomere of Xenopus embryos at the two-cell stage randomized the left-right asymmetry of the embryo and altered the patterns of Xnr-1 and Pitx2 expression. Importantly, inv mRNA that lacked the region encoding the IQ2 site was unable to randomize left-right asymmetry in Xenopus embryos, implying that the IQ2 site is essential for inv to randomize left-right asymmetry in Xenopus. These results suggest that calmodulin binding may regulate inv function. Based on our findings, we propose a model for the regulation of inv function by calcium-calmodulin and discuss its implications.

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Species referenced: Xenopus
Genes referenced: ank1 avd lgals4.2 nodal1 pitx2

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	Fig. 1. Yeast two-hybrid assay. (a) Mouse inv regions fused to the GAL4-binding domain. Horizontal bars above the schematic representations of mouse inv indicate regions fused to the GAL4- binding domain. Ank, ankyrin repeat domain; NLS, nuclear localization signals; IQ, IQ motif. (b) -Galactosidase activity in yeast cells cotransformed with GAL4-binding domain fusion plasmids (pAS-MP, pAS-E286 or pVA3-1) and GAL4 activation domain fusion plasmids (pCaM8 (calmodulin) or pTD1-1). Blue staining indicates a positive interaction.
	Fig. 2. Gel overlay assays for calmodulin–inv protein binding. (a) Schematic representations of glutathione S-transferase (GST)–inv fusion plasmids. Ank, ankyrin repeat domain; NLS, nuclear localization signals; IQ, IQ motif. Coomassie staining (b,d,f) and gel overlay assays (c,e,g) for the binding of calmodulin to GST fusion proteins. (+), (–),bacterial cultures with and without isopropyl-—D-thiogalactoside (IPTG) induction, respectively. Fusion proteins are indicated above the gels. Arrowheads point to proteins expressed. Gel overlay assays were performed without Ca2+ (c), or with 10mM (e) or 5mM (g) EGTA.
	Fig. 3. Calcium dependency of the binding of calmodulin–inv (B285) protein. (a) Gel overlay assay in the presence and absence of Ca2+. B285 fusion proteins were electrophoresed in sodium dodecyl sulfate (SDS)–polyacrylamide gels and transferred onto polyvinylidene fluoride (PVDF) membranes. Coomassie staining (panel 1). Calmodulin binding was assessed in the presence of 500 μM Ca2+ (panel 2), 100 μM Ca2+ (panel 3), in the absence of Ca2+ (5mM EGTA; panel 4) and in the presence of 5mM EGTA and 5.5mM Ca2+ (panel 5). Arrowheads point to the protein expressed. The asterisk indicates a positive signal. (+), (–), bacterial cultures with and without isopropyl-— D-thiogalactoside (IPTG) induction, respectively. (b) Calmodulin– inv protein complex precipitation by magnetic beads in the presence and absence of Ca2+. Lane 1, B285 protein digested with Precission protease (Amersham Pharmacia Biotech, Piscataway, NJ, USA); lane 2, presence of 100 μM Ca2+ with calmodulin; lane 3, absence of Ca2+ (presence of 5mM EGTA) with calmodulin. Lanes 2, 3, the biotinylated calmodulin–inv (B285) fusion protein complex was collected with avidin-conjugated magnetic beads. Complexes were electrophoresed in SDS-polyacrylamide gel and transferred onto PVDF membranes. Anti-inv antibody was used to detect B285 protein.
	Fig. 4. Xenopus embryos exhibiting situs solitus and abnormal situs. Embryos are shown in the ventral view. (a) An embryo with normal cardiac looping and gut coiling. (b) An embryo with inverted heart and gut coiling (situs inversus). (c) An example of disturbed gut coiling that cannot be classified as either clockwise or counterclockwise.
	Fig. 5. (a) Reversal of visceral asymmetry by injection of inv mRNA or -globin mRNA into Xenopus embryos at the two-cell stage. Horizontal bars indicate the percentage of embryos with reversed heart orientation. The embryos with inverted heart orientation are further divided into those with inverted gut coiling (), disturbed gut coiling ( ), and normal gut coiling (). (b) Relationship between reversal of cardiac orientation and dosage of inv mRNA. Error bars represent SEM values from two to six independent experiments. (), injected into a right blastomere; (), injected into a left blasomere.
	Fig. 6. Whole-mount in situ hybridization of Xenopus embryos probed with Xnr-1 (a–d) or Pitx2 (e–g). All embryos are viewed from the ventral side. Arrows point to positive staining areas. (a) Normal left-sided expression of Xnr-1 in an uninjected embryo. (b–d) Alteration of Xnr-1 expression in embryos whose right blastomere was injected with mouse inv mRNA. Xnr-1 is expressed on the right side (b), bilaterally (c) or absent (d). (e) Normal left-sided expression of XPitx2 in an uninjected embryo. Alteration of XPitx2 expression in embryos whose right blastomere was injected with mouse inv mRNA into the right blastomere. XPitx2 is expressed on the right side (f) or bilaterally (g).
	Fig. 7. (a) Cardiac reversal in embryos injected with full-length inv, C-terminus-truncated inv and the ankyrin repeat-deleted inv mRNA. (b) Cardiac reversal in embryos injected with the full-length inv mRNA and mutant inv mRNA lacking the region encoding the IQ1 and/or IQ2 sites. The full-length inv mRNA and mutant inv mRNA injected are shown in the left lane. The side of blastomere injection is indicated in the middle lane. Percentages of embryos with cardiac reversal are shown in the right lane. The number of embryos with cardiac reversal/number of embryos examined is shown in parentheses. No difference from the group whose right blastomere was injected with full-length mouse inv mRNA. *Statistically different compared with the group whose right blastomere was injected with full-length mouse inv mRNA.
	Fig. 8. Model for inv activation by Ca2+–calmodulin. In the absence of Ca2+, the inv protein binds to calmodulin at the IQ2 site and is active (left). When intracellular Ca2+ is elevated, calmodulin is unable to bind to inv and inv becomes inactivated (right). *Ca2+ dependency of calmodulin binding to the IQ1 site has not been determined.