Fig. 2. CK2 inhibits intercellular transfer of cEN2. COS-7 cells transfected with cEN2 alone (A), or co-transfected with cEN2 and CK2/ß (B), CK2^K68A/ß (C) or CK2 (D) were co-cultured with rat embryonic neurones for 48 hours. After fixation cEN2 (green) and NCAM (red) were immunodetected. cEN2 transfer from COS-7 cells (open arrowheads, NCAM negative) to recipient neurones (NCAM positive) was monitored. cEN2 accumulation in recipient neurones (A-D, white arrowheads) is specifically inhibited upon CK2/ß overexpression (B, broken arrows). (E) Quantification of cEN2 intercellular transfer shown in A-D. (F,G) Bacterially produced cEN2 was phosphorylated by recombinant CK2 holoenzyme (G) or treated with CIP (F) and incubated with cultured rat embryonic neurones for 1 hour at 37°C. In both cases, cEN2 (green) is immunodetected on confocal sections within the nucleus of neurones (NCAM positive, red). Scale bar: 30 µm. (H) Bacterially produced cEN2 was phosphorylated by recombinant CK2 holoenzyme in presence of [-³²P]ATP (lane 1) and incubated with neurones for one hour at 37°C. Half of the internalisation medium was collected and immunoprecipitated with the anti-Engrailed serum (lane 2). The remaining medium (lane 3) and neurones (lane 4) were incubated with CIP for 15 minutes at 37°C and immunoprecipitated. Immunoprecipitates were resolved on SDS-PAGE and detected by autoradiography. ³²P-cEN2 is immunoprecipitated from CIP-treated cell extracts (lane 4), but not from CIP-treated medium (lane 3).

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