Fig. 1. Interaction between DOC1R and MAPK; alignments of DOC1R sequences. (A) Two-hybrid interaction between DOC1R and MAPK. DOC1R interacts with ERK2WT and ERK2KD but not with the negative control Su(Fu). Yeast strains transformed with LexAERK2WT, LexA-ERK2KD (Waskiewicz et al., 1997), LexA-53 (positive control), LexASu(Fu) or LexA alone were mated with yeast strains transformed respectively with the B42-DOC1R, B42-AD-T (positive control), or empty B42. The diploids obtained were tested for transactivation of both the ß-galactosidase and the LEU2 reporter genes on glucose (Glu)- or galactose (Gal/Raf)-containing mediums. The B42 constructs are under the control of the galactose promoter. The B42-DOC1R fusion protein clearly interacts both with LexA fusions of ERK2WT and ERK2KD as strongly as the positive control, whereas it does not interact with the negative control Su(Fu). (B) DOC1R co-immunoprecipitates with endogenous p42^mapk (ERK2) from immature Xenopus oocyte extracts. Lanes 1 and 2: total immature Xenopus oocyte extracts expressing either MYC-WNT11 (Xenopus WNT11, a negative control, lane 1) or MYC-DOC1R mRNA (lane 2). Lanes 3 and 4: anti-p42^mapk (ERK2) immunoprecipitates prepared from the MYC-WNT11 (lane 3) and MYC-DOC1R (lane 4) expressing oocyte lysates. All samples were analysed by immunoblotting using the anti-MYC antibody. This experiment was repeated twice. (C) Amino acid sequence alignments of the DOC1R protein from different vertebrate species. DOC1R is rich in proline in its N-terminal end and contains one potential MAPK phosphorylation site (blue), one CDK2 binding site (red) and one cyclin/CDK-binding site (green). (D) Percentage of identities (I) and similarities (S) between the amino acid sequences of DOC1R from different vertebrate species (h, human; m, mouse; xt, Xenopus tropicalis; xl, Xenopus laevis).

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