Fig. 1. cEN2 is phosphorylated at multiple sites both in COS-7 cells and is a substrate for CK2. (A) COS-7 cells were transiently transfected with control plasmid (pTL) or cEN2 expression plasmid (cEN2). Twenty-four hours later, cells were labelled with ³²Pi for 30 minutes and the cellular content was immunoprecipitated using an anti-Engrailed serum. Immunoprecipitated fractions were analysed by SDS-PAGE and autoradiography. (B) Crude cell extracts from COS-7 cells transiently expressing cEN2 were either non-treated or treated with CIP (CIP). Proteins were separated on 2D gels and subjected to immunoblotting with the anti-Engrailed anti serum. cEN2 is resolved into six isoforms (1-6). Arrowheads indicate isoforms selectively lost upon CIP treatment. (C) Crude cell extracts from E18.5 mouse mesencephalon were separated on 2D-gels and subjected to immunoblotting with an anti-Engrailed anti serum. EN2 is resolved in many isoforms. Arrowheads indicate isoforms selectively lost upon CIP treatment of the extracts (not shown). Two dimensional gels have been aligned using an invariant standard (bacterially expressed GST-cEN2). The asterisk indicates the position of the most basic isoform of cEN2 and mouse EN2; NEPHGE, non equilibrium pH gradient electrophoresis. (D) cEN2 is phosphorylated by CK2. Recombinant GST or GST-cEN2 were incubated with [-³²P]ATP in the presence of CK2 holoenzyme (lanes 1, 2) or of recombinant CK2 alone (lane 3). Phosphoproteins were resolved by SDS-PAGE and autoradiographed. GST is not phosphorylated and phosphorylation of cEN2 by CK2 is increased fourfold in presence of CK2ß. (E) cEN2 phosphorylation is increased by CK2 transfection in COS-7 cells. COS-7 cells co-transfected with cEN2 and either wild-type CK2 (+CK2/ß) or a catalytically deficient mutant (+CK2^K68A/ß) were incubated with [³²P] H₃PO₄. [³²P] incorporation in cEN2 was quantified following anti-Engrailed immunoprecipitation. The relative phosphorylation of cEN2 upon CK2/ß versus CK2^K68A/ß co-transfection is 2.15±0.17 (mean±s.e.m, n=4). The lower panel indicates that equal amounts of cEN2 are immunoprecipitated. (F,G) cell extracts of COS-7 cells expressing the indicated combination of proteins were resolved by 2D gel and probed with anti-Engrailed antiserum. A dramatic increase in the hyperphosphorylated isoform (black arrowhead) is observed only upon CK2/ß overexpression. The asterisk (*) indicates the position of the most basic isoform of wild-type cEN2