Sequence comparison of mNkd and the Drosophila Nkd. (A) Protein sequence alignment of mNkd and Drosophila Nkd. Deduced protein sequences of mNkd and Nkd (17) are compared with the Macvector clusterw program. Identical residues are highlighted in dark gray; conserved changes are highlighted in light gray. The EF-hand motifs and the surrounding amino acids in both proteins are underlined. (B) Alignment of the underlined amino acids of mNkd, Nkd (17), human Recoverin (41), and Drosophila Frequenin (42). Residues that are identical in more than two proteins are highlighted in dark gray; conserved changes are highlighted in light gray. Consensus residues in the EF-hand that bind calcium with high affinity are indicated by shaded dots above these residues (17, 20).

mNkd interacts with Dishevelled. (A) Lysates were prepared from Cos7 cells expressing mNkd, mNkd no EF-hand, or GFP constructs. Total cell lysates were immunoprecipitated with monoclonal antibodies 1, 2, and 3 to Dishevelled (Santa Cruz Biotechnology); after SDS/PAGE and membrane transfer, the membrane was blotted with Xpress antibody (Invitrogen). (B) mNkd mutations in the EF-hand do not have a significant effect on the binding of mNkd to Dishevelled. Cell lysates prepared from HEK293 cells expressing Myc-tagged Dishevelled and Xpress-tagged mNkd proteins were immunoprecipitated with Myc antibody (Roche Molecular Biochemicals); after SDS/PAGE and membrane transfer, the membrane was blotted with Xpress antibody. (C) Summary of mutations in the EF-hand of mNkd. The mNkd mutant m2 contains amino acid changes D144V and D146V. The mNkd mutant m3 contains amino acid change G149W. The mNkd mutant m4 contains amino acid change V151K. –, identical amino acids as the wild type; … . . , deleted amino acids in the mNkd no EF-hand mutant. (D) mNkd binds to the conserved middle region of Dishevelled. (I) Schematic diagram of GST-fusion proteins containing different regions of Drosophila Dishevelled (Dsh). (III) Myc-tagged mNkd protein, labeled with ³⁵S, was immunoprecipitated with Myc antibody or precipitated by GST-fusion proteins. The immunocomplexes were separated by SDS/PAGE and detected by autoradiography. (II) Equal amounts of GST-fusion proteins used in III were separated by SDS/PAGE and detected by Coomassie blue stain.

mNkd activates JNK in mammalian cell culture and alters convergent extension movements in Xenopus laevis. (A) The JNK assay was carried out as described (15). NIH 3T3 cells were transfected with plasmids expressing c-Jun and mNkd or c-Jun and mDvl3. Cell lysates were separated by SDS/PAGE and transferred onto a nitrocellulose membrane. The membrane was blotted with PhosphoPlus c-Jun (Ser-63) II antibody (New England Biolabs). The same membrane was then stripped and blotted with Xpress antibody (Invitrogen) to detect the amount of Xpress-tagged mNkd or blotted with Myc antibody to detect Dishevelled. (B) Dorsal expression of mNkd in developing Xenopus embryos inhibited the normal elongation and straightening of the anteroposterior axis (Left; control embryo at top, mNkd-injected embryos below). (Center Upper) Control open-face Keller explants of the dorsal marginal zone elongated and changed shape significantly. (Right Upper) Explants expressing mNkd failed to elongate or to change shape. (Center Lower) Overexpression of wild-type Xdsh mimicked overexpression of mNkd. (Right Lower) Downstream activation of the canonical Wnt pathway by coexpression of dominant-negative GSK-3β did not attenuate the inhibitory effect of mNkd on convergent extension. Expression of dominant-negative GSK-3β alone had no effect on convergent extension (data not shown).

mNkd expression. (A) Result of mouse-embryo Northern blotting. (B) Result of mouse multiple-tissue Northern blotting. Actin was used as an internal standard for RNA loading.

mNkd inhibits the canonical Wnt pathway and mNkd-mRNA level is up-regulated by Wnt. (A) HEK293 cells were transfected with expression constructs that included 0.02 μg of LEF-1, 0.2 μg of luciferase reporter (25), 0.02 μg of pRL-TK (Promega), 0.1 μg of pCGWnt-1, and 0.2 μg of GFP, mNkd, or mNkd-mutant derivatives. LEF-1 luciferase reporter activities were determined. (B) mNkd did not inhibit the β-catenin-activated Lef-1 reporter. HEK293 cells were transfected with expression constructs that included 0.02 μg of LEF-1, 0.2 μg of luciferase reporter (25), 0.02 μg of pRL-TK (Promega), 0.1 μg of β-catenin, and 0.2 μg of vector or mNkd. (C) mNkd inhibited Xwnt-8-induced secondary axes formation in Xenopus. Control Xenopus embryos are shown at Upper Left. Embryos were injected ventrally with 5–10 pg of Xwnt-8 RNA and developed with secondary axes (Upper Right). Secondary axes were scored as complete (arrows) or partial (arrowheads) based on development of anterior structures such as eyes and cement glands. Coexpression of mNkd with Xwnt-8 decreased the frequency of secondary axes formation as well as the percentage of secondary axes that contained anterior structures (Lower Left and Lower Right). This effect was dose-dependent. (D) BALB/c LI mouse liver epithelial cells were treated with either Wnt-3a or Neo-conditioned medium (34) for indicated hours. PCR was carried out by using primer pairs 5′-TGTGAACCATTCCCCCACATCAA-3′ and 5′-AAATGGGGTGTCAAGGAGGTGGAA-3′. The PCR products were separated by agarose gel electrophoresis. GAPDH, glyceraldehyde-3-phosphate control.