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Engrailed homeoprotein secretion is a regulated process

¹ Biologie cellulaire des homéoprotéines, CNRS UMR8542 Ecole Normale Supérieure 46 rue d’Ulm F-75005 Paris, France
² INSERM EMI 0104 DBMS CEA, 17 rue des Martyrs F-38054 Grenoble, France
³ Développement et Neuropharmacologie, CNRS UMR8542 Ecole Normale Supérieure 46 rue d’Ulm F-75005 Paris, France

View larger version (25K): [in a new window]   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 (+CK2K68A/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 CK2K68A/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

View larger version (73K): [in a new window]   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), CK2K68A/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).

View larger version (81K): [in a new window]   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.

View larger version (104K): [in a new window]   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 CK2K68A/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 CK2K68A/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.

View larger version (120K): [in a new window]   Fig. 5. SRD phosphorylation by endogenous CK2 inhibits homeoproteins intercellular transfer. (A) Extracts of COS-7 cells expressing cEN2(146-169)SRD alone were separated by 2D gel and immunoblotted with anti-Engrailed serum. The acidic spot characteristic of wild-type cEN2 hyperphosphorylation is found (arrowhead). The asterisk indicates the position of the most basic isoform of wild-type cEN2. (B-E) Intercellular transfer of cEN2(146-169)SRD (B) or cEN2SRD (C), cEN2SRD/5A (D), cEN2SRD/5E (E) expressed alone was analysed as in Fig. 2. Intercellular transfer of is inhibited independently of kinase overexpression (B). Ectopic addition of cEN2 SRD at the C terminus of wild-type cEN2 has the same inhibitory effect on intercellular transfer (C). SA substitutions within the ectopic SRD restores transfer (D) but SE substitutions does not (E). Arrowheads indicate transfected COS-7 cells. Scale bar: 30 µm.

View larger version (76K): [in a new window]   Fig. 6. Intercellular transfer of HOXC8 is insensitive to CK2. (A-C) Intercellular transfer of HOXC8 expressed alone (A), HOXC8 co-expressed with CK2/ß (B) or of HOX-C8SRD expressed alone (C) was analysed as in Fig. 3. Intercellular transfer of HOXC8 is not inhibited by kinase overexpression (B). Ectopic addition of cEN2 SRD at the C terminus of HOXC8 has a strong inhibitory effect on intercellular transfer (C). Arrowheads indicate transfected COS-7 cells. Scale bar: 30 µm.