1 Biologie cellulaire des homéoprotéines, CNRS UMR8542 Ecole Normale Supérieure 46 rue dUlm 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 dUlm F-75005 Paris, France
*Author for correspondence (e-mail: joliot{at}biologie.ens.fr)
Accepted 30 April 2002
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SUMMARY |
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Key words: Engrailed, Homeoprotein, Unconventional secretion, Phosphorylation, Regulation
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INTRODUCTION |
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Protein phosphorylation is widely used to regulate their subcellular distribution, both in nucleo-cytoplasmic exchanges and in membrane targeting. It is a common post-translational modification found in homeoproteins. Among homeoprotein kinases identified, the serine/threonine protein kinase 2 (CK2) is ubiquitously expressed and phosphorylates murine Cut (Coqueret et al., 1998), Hox-b6 (Fienberg et al., 1999
) and NKX2.5 (Kasahara et al., 1998
), as well as Drosophila melanogaster Antennapedia (Jaffe et al., 1997
), Engrailed (Bourbon et al., 1995
) and Even-skipped (Li and Manley, 1999
) homeoproteins. CK2 is a holoenzyme composed of two catalytic subunits (
and/or
') and two regulatory subunits (ß), which adopt tetrameric
'/ß2 or
2/ß2 structures. CK2 is essential for cell viability (Padmanabha et al., 1990
) and is involved in multiple cellular functions (reviewed by Allende and Allende, 1995
). We demonstrate a crucial role for CK2 activity in the regulation of the intercellular transfer of cEN2. CK2-dependent phosphorylation of a serine-rich domain in cEN2 drastically impairs cEN2 secretion. These results establish for the first time that intercellular transfer of homeoproteins is a regulated event.
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MATERIALS AND METHODS |
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Cell culture and electroporation
COS-7 cells were cultured and transfected by electroporation as previously described (Joliot et al., 1998). Rat embryonic neurones were prepared and cultured as previously described (Chamak and Prochiantz, 1989
; Joliot et al., 1998
).
Immunofluorescence and microscopy
COS-7 cells (5x103) were co-cultured with freshly dissociated rat E16 cortical neurones (2x105) for 48 hours and fixed as previously described (Joliot et al., 1998). Rabbit anti-Engrailed serum (1/1000) and rat monoclonal anti HOXC8 antibody were kind gifts from S. Saule and N. Peyreiras, respectively. Detection of NCAM was performed by a mouse monoclonal antibody (OB11, Sigma). Following secondary antibodies were used: Cy3-conjugated goat anti-mouse antibody (1/500, Jackson), biotinylated goat anti-rabbit antibody (1/200, Vector) and biotinylated donkey anti-rat antibody plus a DTAF-conjugated streptavidin (1/200, Jackson). All images were obtained using a digital camera (Spot, Diagnostic Instrument). Confocal microscopy was performed on a Leica system. All pictures were acquired using constant integration parameters and processed using Adobe Photoshop.
Quantification of intercellular transfer
Confocal sections (low magnification) were processed with NIH Image 1.6.2 (density slice tool). Briefly, cEN2 staining was quantified (AreaxIntensity) in neurones and COS-7 cells. The ratio neurone/COS cEN2 staining was expressed as a percent of the condition showing maximum intercellular transfer.
cEN2 internalisation
Recombinant cEN2 was incubated with 10 units of CIP (New England Biolabs) for 30 minutes at 30°C or was phosphorylated (see below) by CK2 in 10 mM Tris pH 7.5 and 10 mM MgCl2. The protein was diluted to 1µM in culture medium and incubated with 2x105 neurones for 1 hour at 37°C.
GST co-purification experiments
COS-7 cells (2.5x105) co-transfected with a HA-tagged CK2 subunits and GST fusions proteins were cultured for 24 hours on polyornithine-coated 60 mm culture dishes. After washing with PBS, cells were lysed and GST fusion protein was purified as described (Chatton et al., 1995).
Phosphorylation assays
Recombinant oligomeric CK2 or its isolated monomeric CK2 subunit was expressed in Sf9 cells and purified to homogeneity (Filhol et al., 1991
). CK2 assays were performed using a specific CK2 peptide substrate as previously described (Bojanowski et al., 1993
). GST or GST-cEN2 (3 µg) was incubated at 22°C with [
-32P] ATP or 10 µM ATP, and 10 mM MgCl2 in the presence of 0.3 µg of either oligomeric CK2 or 1.0 µg monomeric CK2
subunit. After 15 minutes, the phosphorylated proteins were analysed by SDS electrophoresis and autoradiography.
Metabolic labelling and immunoprecipitation
COS-7 cells (2.5x105) expressing indicated constructs, were cultured for 24 hours on polyornithine-coated 60 mm culture dishes. After washes with phosphate-free medium (ICN), cells were incubated for 30 minutes in the same medium containing 250 µCi of [32P] H3PO4 inorganic phosphate (HCl free, ICN biomedicals), and 10% (v/v) foetal calf serum dialysed overnight against TBS. Cells were lysed in boiling 2% SDS, scraped, boiled for 5 minutes and centrifuged for 15 minutes at 4°C. The supernatant was pre-cleared on protein A-sepharose for 30 minutes at room temperature and immunoprecipitated by an anti-Engrailed serum as described (Rousselet et al., 1988). Radioactivity was quantified by phosphoimaging.
Preparation of cell extracts for bi-dimensional electrophoresis
COS-7 cells (2.5x105) co-expressing indicated combination of constructs were cultured for 24 hours on polyornithine-coated 60 mm culture dishes. Cells were washed twice in PBS, scraped in 500 µl of PBS containing a protease inhibitor cocktail (Complete, Boehringer) and 1 mM sodium orthovanadate (except for CIP treatment) and sonicated. Extracts were incubated in 100 mM Tris pH 9.2 with 10 U of CIP (New England Biolabs) for 30 minutes at 30°C with or without 200 mM NaH2PO4 as a competitor. Rat mesencephalon was immediately resuspended in 8 M urea after dissection.
Gel electrophoresis and western blotting
SDS-PAGE was performed according to classical methods. Two-dimensional PAGE was performed according to OFarrel (OFarrell et al., 1977) and Larcher (Larcher et al., 1992
) with minor modifications. First dimension separations were performed at basic non-equilibrium pH gradient electrophoresis (NEPHGE) for 1.5 hours at 500 V in 4% polyacrylamide gel containing 2% ampholine (1.6% pH 3.5-10.0 and 0.4% pH 5.0-8.0). Western blots were performed as described previously (Joliot et al., 1998
). Rabbit polyclonal anti-Engrailed (1/25000) or AF24 rabbit polyclonal anti-Engrailed (1/1000) were used for Engrailed detection. Rat monoclonal anti-HA antibody (3F10, Boehringer) was used at 1/100 dilution.
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RESULTS |
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CK2-dependent cEN2 phosphorylation was then analysed in a cellular context. As its 2D pattern indicates, the protein was poorly phosphorylated by endogenous kinases in COS-7 cells (Fig. 1B). cEN2 phosphorylation was increased by co-transfection with CK2 expression vectors. COS-7 cells co-transfected with vectors coding cEN2, CK2ß and either CK2 or a kinase inactive mutant (CK2
K68A) were incubated with 32Pi and cell extracts were immunoprecipitated with the anti-Engrailed antibody. cEN2 phosphorylation was increased by twofold in CK2
/ß compared to CK2
K68A/ß co-transfected cells (Fig. 1E, upper panel), although cEN2 was expressed at similar levels (lower panel). The 2D pattern of cEN2 was shifted toward the most acidic isoform by CK2
/ß co-transfection (Fig. 1F). Co-transfection with CK2
alone (Fig. 1G) or CK2
K68A/ß (not shown) did not modify cEN2 phosphorylation pattern.
CK2 overexpression inhibits cEN2 secretion and intercellular transfer
The influence of cEN2 phosphorylation on its intercellular transfer was then investigated. COS-7 cells co-transfected with cEN2 and either wild-type CK2/ß or the catalytic deficient mutant CK2
K68A/ß expression vectors, were co-cultured with primary neurones (Joliot et al., 1998
). After 48 hours, cells were fixed and immunodetection of cEN2 was performed. When expressed alone, cEN2 was transferred from the producing COS-7 cells to the recipient neuronal cells, as illustrated by the presence of cEN2-positive nuclei in neuronal cells (characterised by NCAM staining) (Fig. 2A). When co-transfected with wild type CK2
/ß, cEN2 intercellular transfer was dramatically impaired as shown by the reduced number of cEN2-positive neuronal nuclei localised in the vicinity of producing COS cells (Fig. 2B). Co-transfection of the catalytic-defective holoenzyme CK2
K68A/ß (Fig. 2C) or CK2ß subunit alone (not shown) had no effect on cEN2 intercellular transfer. The kinase activity of CK2 is therefore absolutely required for the inhibition of cEN2 intercellular transfer. In addition, the co-transfection of CK2
alone, which poorly phosphorylates cEN2, had no effect on the intercellular transfer of the protein (Fig. 2D). Intercellular transfer was quantified by measurement of cEN2 staining in neurones normalised by the staining measured in producing COS-7 cells (
15 COS cells analysed for each condition). Co-transfection with CK2
/ß resulted in a 75% reduction of cEN2 intercellular transfer compared to CK2
K68A/ß (Fig. 2E).
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Fragment 146-169 of cEN2 is necessary for both phosphorylation and CK2-induced inhibition of intercellular transfer
To determine which part of the protein was involved in this inhibition, nested N-terminal deletions of cEN2 were constructed and tested for their sensitivity to CK2/ß co-transfection in the co-culture assay. Deletion of the 142 N-terminal amino acids did not affect CK2-induced intercellular transfer inhibition (compare Fig. 3A with 3B). By contrast, the transfer of cEN2
(1-180) (lacking the 180-first amino-acids) was not inhibited by CK2
/ß co-transfection (Fig. 3C). Fragment 142-180, was further subdivided and it was found that deleting amino acids 146-169 [cEN2
(146-169)] was sufficient to confer resistance to CK2-induced intercellular transfer inhibition (Fig. 3D). When transfected alone, all the deleted proteins were efficiently transferred between cells (not shown).
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Phosphorylation state of cEN2 serine rich domain controls intercellular transfer
The N-terminal part of cEN2 fragment 146-169 contains a domain highly enriched in serine residues (seven serines, SGAELSVSSDSDSSQAG). To verify that CK2-induced effects were mediated through this serine-rich domain, we performed SA and S
E substitutions in the SSDSDSS sequence. These mutations should mimic unphosphorylated and phosphorylated serine residues, respectively. First, five serine to alanine substitutions were introduced in the sequence. The resulting protein (cEN2/5A) was co-expressed either with the active or the inactive CK2 holoenzyme and its 2D pattern was analysed. When co-transfected with CK2
/ß, cEN2/5A pattern was reduced to the most basic isoforms (Fig. 4B), and was almost identical to cEN2
(146-169) pattern (Fig. 3E). As expected, the same pattern was observed in extracts from cells co-transfected with the inactive CK2
K68A/ß (Fig. 4A). Concomitantly, intercellular transfer of cEN2/5A became insensitive to CK2 co-transfection (Figs. 4C,D). When the five serine residues were then substituted by glutamates, cEN2/5E intercellular transfer was constitutively inhibited (Fig. 4E). Intercellular transfer of both cEN2/5A and cEN25E becomes independent of the presence of active CK2 (Fig. 4F). S
E substitution resulted in a 85% inhibition of cEN2 intercellular transfer compared with S
A substitution. The opposite effects of alanine and glutamate substitutions clearly demonstrate that the phosphorylation of these serines is directly responsible for the CK2-induced inhibition of intercellular transfer.
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Specificity of CK2-induced intercellular transfer inhibition
Although SE substitutions within the native SRD efficiently inhibit cEN2 intercellular transfer in the absence of overexpressed CK2, we could not ruled out an indirect effect of CK2 overexpression on the intercellular transfer of homeoproteins. Similar to cEN2, HOXC8 homeoprotein is transferred between cells (Fig. 6A) but is not a CK2 substrate (Fienberg et al., 1999
). It was therefore a good candidate to investigate the specificity of CK2-induced intercellular transfer inhibition. As shown in Fig. 6A,B intercellular transfer of HOXC8 was insensitive to CK2 overexpression. However, grafting SRD to the C terminus of the protein led to constitutive inhibition of intercellular transfer (Fig. 6C), strongly suggesting that the effects of CK2 (overexpressed or not) on intercellular transfer absolutely requires the presence of CK2 sites within the homeoprotein.
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DISCUSSION |
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The inhibitory effect of CK2 on cEN2 intercellular transfer tightly correlates with cEN2 phosphorylation. Both phenomena require the presence of the regulatory CK2ß subunit and intercellular transfer of HOXC8 (which is not phosphorylated by CK2) is insensitive to CK2 overexpression. Three different approaches have identified a cluster of five serine residues (amino acids 150-156 of cEN2) as the main phosphorylated target. First, the deletion of residues (146-169) in the cEN2 homeoprotein or SA substitutions in the serine cluster, abolishes both CK2-induced cEN2 intercellular transfer inhibition and cEN2 phosphorylation. Second, S
E substitution of the same serine residues efficiently mimics the effect of CK2 on intercellular transfer. Third, shifting SRD (143-159) to the C terminus of cEN2 strongly increases phosphorylation of the serine cluster by endogenous kinases and provokes a constitutive inhibition of cEN2 intercellular transfer. It can be noticed that the serine cluster characterised in cEN2 is conserved in all vertebrate Engrailed 2 orthologues, both in term of sequence and position relative to the homeodomain.
cEN2 is poorly phosphorylated in COS-7 cells and the inhibition of its intercellular transfer is observed only when CK2 is overexpressed. Using a highly sensitive test for CK2 interaction, we have shown that cEN2 specifically interacts with endogenous CK2, requiring the presence of cEN2 SRD (Table 1). It should be mentioned that the deletion of the SRD as well as the SA substitution impairs endogenous phosphorylation of cEN2 (compare Fig. 1B with Fig. 3E, Fig. 4B). SRD shift at the C terminus of the protein greatly enhances an interaction with endogenous CK2 (6,7x) that correlates with its phosphorylation in the absence of overexpressed CK2. These results strongly suggest that, in its native position, SRD accessibility to CK2 is downregulated, possibly by protein conformation or protein/protein interactions. Masking/unmasking the SRD and making it accessible to phosphorylation would thus be a primary event in intercellular transfer regulation.
Our study raises the question of the molecular mechanism of cEN2 intercellular transfer inhibition. SRD phosphorylation may promote interactions with regulators of intercellular transfer, either through direct interactions (supported by the autonomous activity of ectopic SRD) or through indirect effects on other domains of the protein. In the latter case, the homeodomain would be a potent candidate as it is the major determinant of homeoprotein subcellular distribution (Derossi et al., 1994; Joliot et al., 1998
; Maizel et al., 1999
). Indeed, the cEN2 homeodomain is efficiently transferred between cells (A. M. and A. J., unpublished), indicating that it contains all the sequences required for this process. In addition, HOXC8, which shares only the homeodomain in common with cEN2, becomes unable of intercellular transfer upon addition of cEN2 SRD.
Regulation of homeoprotein intercellular transfer could have important consequences on their physiological functions, as it could restrict this process spatially and temporally and limit it to well defined situations. The predominance of phosphorylated forms of Engrailed in physiological situation (Fig. 1C), may indicate that most of the protein is kept in an incompetent state for intercellular transfer. Such regulation could be specific to each class of homeoproteins, as the regulatory domain identified in cEN2 localised in a portion poorly conserved among homeoproteins.
Protein intercellular transfer is not a unique feature of homeoproteins. Indeed, several other proteins, although unrelated both in sequence and function, present a similar behaviour (reviewed by Prochiantz, 2000). The mechanisms involved in this process could be partly similar. In particular, intercellular transfer does not provoke irreversible modifications of these proteins, such as cleavage of a secretion signal sequence. In all cases, the transfer of these proteins between cells necessitates their accumulation in distinct subcellular compartments. Phosphorylation status may represent an important mechanism co-ordinating and/or regulating protein movements. Interestingly, the phosphorylation of HSV-1 VP22 protein has been proposed as a possible regulator of its intercellular transfer (Elliott et al., 1999
; Pomeranz and Blaho, 1999
).
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ACKNOWLEDGMENTS |
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