Fig. 4. cEN2 SRD phosphorylation controls intercellular transfer. (A-B) Serine to alanine substitutions within SRD abolishes CK2-induced phosphorylation. Extracts of COS-7 cells expressing cEN2/5A together with CK2^K68A/ß (A) or CK2/ß (B) were separated on 2D gel and immunoblotted with anti-Engrailed serum. cEN2/5A 2D pattern is restricted to the three most basic spots (A) and no additional acidic spots are detected when functional CK2 is expressed (B). The asterisk indicates the position of the most basic isoform of wild-type cEN2. (C-F) Serines substitution within SRD can mimic CK2-induced inhibition of transfer. Intercellular transfer of 5S5A (C,D) or 5S5E (E) substitution mutants of cEN2 co-expressed with either CK2^K68A/ß (C,E) or CK2/ß (D) was analysed as in Fig. 2. Although both mutants are insensitive to CK2 overexpression, cEN2/5A transfers between cells (C,D) and cEN2/5E does not transfer (E). (F) Quantification of cEN2 intercellular transfer. Arrowheads indicate transfected COS-7 cells. Scale bar: 30 µm.

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