Fig. 3. Residues 142 to 169 of cEN2 are required for CK2-induced inhibition of intercellular transfer and phosphorylation. (A-D) Residues 142 to 169 are required for CK2-induced inhibition of transfer. Intercellular transfer of deletion mutants of cEN2 co-transfected with CK2/ß was analysed as in Fig. 2. CK2/ß overexpression inhibits intercellular transfer of both cEN2 (A) and cEN2(1-142) (B), but does not affect intercellular transfer of cEN2(1-180) (C) or cEN2(146-169) (D). Arrowheads indicate transfected COS-7 cells. Scale bar: 30 µm. (E-G) Residues 142 to 169 of cEN2 are required for CK2 phosphorylation. (E,F) Extracts of COS-7 cells expressing cEN2(146-169) alone (E) or together with CK2/ß (F) were separated on 2D gels and immunoblotted with the anti-Engrailed serum. cEN2(146-169) 2D pattern is restricted to the three most basic spots (E) and no additional acidic spots are detected when CK2/ß is overexpressed (F). The asterisk indicates the position of the most basic isoform of wild-type cEN2. (G) Total cellular extracts of COS-7 cells co-expressing the indicated proteins were loaded on GST affinity columns. Bound material was analysed by western blot using an anti-HA antibody. cEN2 interaction with CK2 required the presence of both residues (146-169) and CK2ß subunit.

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