HIP/PAP, a C-type lectin overexpressed in hepatocellular carcinoma, binds the RIIa regulatory subunit of cAMP-dependent protein kinase and alters the cAMP-dependent protein kinase signalling France Demaugre 1 , Yannick Philippe 1 , Sokavuth Sar 1 , Bernard Pileire 2 , Laurence Christa 1 , Chantal Lasserre 1 and Christian Brechot 1 1 INSERM U370 CHU Necker Enfants Malades, Paris, France; 2 Laboratory of Biochemistry, CHU Antilles-Guyane Point a ` Pitre, Guadeloupe, France HIP/PAP is a C-type lectin overexpressed in hepatocel- lular carcinoma (HCC). Pleiotropic biological activities have been ascribed to this protein, but little is known about the function of HIP/PAP in the liver. In this study, therefore, we searched for proteins interacting with HIP/PAP by screening a HCC cDNA expression library. We have identified the RIIa regulatory subunit of cAMP-dependent protein kinase (PKA) as a partner of HIP/PAP. HIP/PAP and RIIa were coimmunoprecipi- tated in HIP/PAP expressing cells. T he biological rele- vance of the interaction between these proteins was established by d emonstrating, using fractionation meth- ods, that they are located in a same subcellular com- partment. Indeed, though HIP/PAP is a protein secreted via the Golgi apparatus w e showed that a fraction of HIP/PAP escaped the secretory apparatus and was recovered in the cytosol. Basal PKA activi ty was in- creased in HIP/PAP expressing cells, suggesting that HIP/PAP may alter PKA signalling. Indeed, we showed, using a thymidine kinase-luciferase reporter plasmid in which a cAMP responsive element was inserted upstream of the thymidine kinase promoter, that luciferase activity was enhanced in HIP/PAP expressing cells. Thus our findings suggest a novel mechanism for the biological activity of the HIP/PAP lectin. Keywords: C-type lectin; HIP/PAP; PKA; phosphorylation; liver. The HIP/PAP-encoding gene has been shown to be overexpressed in human hepatocellular carcinoma (HCC) [1] and in the pancreas during acute pancreatitis [2]. HIP/ PAP has been characterized as a protein belonging to the group 7 of C-type lectins [3,4]. HIP/PAP cDNA encodes a 175 amino acid p rotein containing only one carbohydrate- binding d omain (CRD) linked to an N-terminal sequence, part of which is cleaved during its maturation and secretion [5]. In humans, HIP/PAP protein is not expressed in normal liver but is overexpressed in 75% of HCC, in cholangio- carcinoma and in reactive ductular cells in nonmalignant liver [6]. HIP/PAP expression in HCC does not result from the re-expression of a f etal marker. Ind eed, analysis of mouse embryos has revealed that HIP/PAP is not expressed in the liver during development [7]. HIP/PAP has also been detected in the pancreas and in a subset of cells (Paneth cells) in the intestine [8]. Moreover in rats, the HIP/PAP homologue (PAP 1/peptide 23/Reg 2), is expressed in pituitary and uterine cells un der the influence of growth hormone releasing hormone and oestradiol, respectively [9,10], and by motor n eurones in vivo during their regener- ation and in vitro when incubated with ciliary neurotrophic factor-related cytokines [11,12]. Little is known about the physiopathological significance of HIP/PAP expression. In the pancreas, there is evidence that HIP/PAP may participate in the antiapoptotic pro- gramme developed by acinar cells during acute pancreatitis [13];indeed,HIP/PAPwasreported to protect pancreatic AR4–2 J cells against apoptosis induced by oxidative stress [14]. In pituitary cells, PAP1/peptide 23 was reported to act as a growth factor [10,15] and it has been shown that PAP1 (referred to as Reg 2) is an important neurotrophic factor for motor neurones in vitro and in vivo in the rat [11,12]. In liver recombinant HIP/PAP has been shown to promote the adhesion of rat hepatocytes and to bind elements of the extracellular matrix [8]. Moreover HIP/PAP has been recently reported to combine mitogenic and antiapoptotic functions regarding hepatocytes and to enhance liver regeneration [16]. Nothing is known concerning the possible role of HIP/PAP during liver carcinogenesis. Thus, identi- fication of t he pro teins interacting w ith H IP/PAP liver should help to understand t he function(s ) of HIP/PAP during hepatic carcinogenesis. In this study we have identified the RII a regulatory subunit of cAMP-dependent protein kinase (PKA) as being Correspondence to F. De maugre, INSERM U3 70 C HU Necker Enfants Malades, 156 rue de Vaugirard, 75015 Paris, France. Fax: +33 1 40615581, Tel.: + 33 1 40615343, E-mail: demaugre@necker.fr Abbreviations: CRD, carbohydrate-binding domain; CRE, cAMP response element; HCC, he patocellular carcinoma; HMK peptide, peptide phosphorylatable by heart muscle kinase; PKA, cAMP- dependent protein kinase; SERCA 2, sarco/endoplasmic reticulum Ca 2+ ATPase 2. (Received 19 March 2004, revised 9 July 2004 , accepted 23 J uly 2004) Eur. J. Biochem. 271, 3812–3820 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04302.x a partner of HIP/PAP, and we have demonstrated that PKA activity is enhanced in HIP/PAP expressing cells. Materials and methods Plasmid constructs The HIP/PAP(29–175) coding sequence amplified by PCR using human HIP/PAP cDNA as a template [1] was subcloned a t the EcoRI site in the bacterial expression plasmid p AR(deltaRI)[59/60] [17]. This p lasmid allowed the production of HIP/PAP in fusion at the N-terminal extremity, with Flag and heart muscle kinase (HMK) peptides which allowed, respectively, the purification of chimeric HIP/PAP and its phosphorylation by bovine heart PKA. The sense primer (5¢-GTCGAATTCCAAGGTG AAGAACCCCAG-3¢) was located at nucleotides 63–90 of the coding sequence, and the antisense primer (5¢-TG CTGAATTCCCTCCCTCCTGCACTAGTCAG-3¢)over- lapped the stop codon. DNA sequencing confirmed the restored open reading frame of the fusion construct. The c omplete H IP/PAP(1–175) sequence, amplified using the same template, was subcloned at EcoRI a nd XhoIsites in pcDNA3.1, and in pcDNA3.1/myc-His (Invitrogen). T he QuickChange Site-directed Mutagenesis Kit (Stratagene) was used to switch serines 73 and 138 and threonine 153 of the HIP/PAP protein for alanines. Oligonucleotides cas- settes containing the desired mutations were inserted into pcDNA3-HIP/PAPmyc-His as indicated by the manufac- turer. Direct sequencing confirmed the sequence of the inserts. Production, purification and labelling of Flag-HMK- HIP/PAP(29–175) Chimeric HIP/PAP w as produced in BL21 (DE3) Escheri- chia coli transformed with pAR(deltaRI)[59/60]-HIP/ PAP(29–175) using conventional methods. At the end of the culture the bacteria were l ysed at 4 °Cwith10lgÆmL )1 lysozyme in 50 m M Tris pH 8.0, 2 m M EDTA, 300 m M KCl, 0.2% (v/v) T riton X-100 and 0.1 lgÆmL )1 phenyl- methylsulfonyl fluoride, and centrifuged. Chimeric H IP/ PAP was purified from the supernatant using affinity chromatography with monoclonal M2 anti-Flag agarose (Sigma). Chimeric HIP/PAP was labelled using [ 32 P]ATP[cP] and the catalytic subunit of PKA as described [17] and cleared from unincorporated [ 32 P]ATP[cP] using Sephadex G25 chromatography. Screening of a human HCC cDNA kgt11 library with [ 32 P]Flag-HMK- HIP/PAP(29–175) An amplified human HCC cDNA library, inserted in kgt11 (provided by C. Lasserre), was plated with Y1090 E. coli and induced with isopropyl thio-b- D -galactoside, as des- cribed previously [18]. At the end of culture, nitrocellulose filters subjected to a denaturation-renaturation cycle [19] were hybridized overnight at 4 °Cwith 32 P-labelled chimeric HIP/PAP at a final concentration of 100 000–300 000 cpmÆmL )1 as described [17]. Plaques hybridized with the probe were grown until they were purified. Phage DNA was purified using the kgt11 DNA purification kit (Stratagene). The i nserts amplified by PCR using Advantage cDNA polymerase and the kgt11 insert screening amplimer set (Clontech) were directly sequenced. Cell culture and transfection Chang c ells (CCL13, ATCC) seeded in 100 mm Petri dish were maintained in DMEM supplemented with 7% (v/v) fetal bovine serum, 100 lgÆmL )1 streptomycin and 100 lgÆmL )1 penicillin. Cells plated at a density of 1.5 · 10 6 cells per 100 mm diameter dish were transfected with appropriate vectors (20 lgADN)usingthecalcium precipitation method, and further cultured for 48 h unless indicated. For the isolation of stable transformants Chang, cells transfected with pcDNA-HIP/PAP were cultured for 4 w eeks with 800 lgÆmL )1 neomycin and screened for HIP/ PAP by immunoblot. Proteins were quantified using the BioRad protein Assay. Analysis of HIP/PAP in transiently HIP/PAP expressing Chang cells Effect of brefeldin A. Twenty-four hours post transfection with pcDNA-HIP/PAP, cells were seeded in 60-mm Petri dishes and further grown for 24 h before 10 l M brefeldin A was added to t he culture m edium. At the e nd of incubation, cells lysed in buffer A (10 m M KH 2 PO 4 pH 7.4, 150 m M NaCl, 10 m M EDTA, 1% (v/v) Triton X-100 and 2 lgÆmL )1 aprotinin, 1 lgÆmL )1 pepsatin, 2 lgÆmL )1 leu- peptin, 0.1 lgÆmL )1 phenylmethylsulfonyl fluoride, 10 m M sodium fluoride, 2 m M sodium orthovanadate, 1 l M oka- daic acid) and the culture medium were resolved in 13% SDS/PAGE and analyzed for HIP/PAP by Western blotting using anti-HIP/PAP Ig [4]. T he blots were revealed using a n enhanced chemiluminecence system, according t o the manufacturer’s instructions (Amersham Life Science). Effect of PKA overexpression. Forty hours post transfec- tion with 18 lg of either the wild or mutated forms of pcDNA-HIP/PAPmyc and 2 lgpCaEV encoding for the catalytic subunit of PKA [20] when indicated, cells were lysed with buffer A. C ellular l ysates (100 lgprotein)were incubated overnight at 4 °Cwith2lg monoclonal anti-myc and then for 2 h with 10 lL protein G Sepharose beads (Amersham Life Science). Immune complexes washed with buffer A were released from beads using Laemmli buffer and analyzed by Western blotting for HIP/PAP using polyclonal antibody anti-HIP/PAP and for phosphorylated serine using polyclonal anti-phosphoserine (Zymed Labor- atories). Cell fractionation HIP/PAP expressing and control Chang cells were fraction- ated between soluble and particulate fractions as described [21]. S arco/endoplasmic r eticulum Ca 2+ ATPase 2 (SERCA 2), an i ntegral protein of the endoplasmic reticulum [22], calreticulin, a protein of the endoplasmic reticulum lumen[23],HIP/PAP,theRIIa and the C a subunits of PKA were checked by immunoblotting in both the 100 000 g pellet solubilized with buffer A and t he supernatant using Ó FEBS 2004 HIP/PAP alters PKA signalling (Eur. J. Biochem. 271) 3813 anti-HIP/P AP, anti-RII a and a nti-Ca (Transduction Laboratories, Lexington, KY, USA), anti-(SERCA 2) (clone IID8; Tebu, Paris, France) and anti-calreticulin (ABR Golden Co.) Igs. Co-immunoprecipitation experiments Forty-eight hours post transfection with either pcDNA- HIP/PAP or the empty vector Chang cells were lysed in 10 m M Tris pH 7.5, 2.5 m M MgCl 2 ,10m M KCl, 0.5 m M dithiothreitol, 0.05% (v/v) NP40, and protease and phos- phatase inhibitors (see above). Extracts (400 lgprotein) clarified by centrifugation at 6000 g, were incubated over- night with 2 lg of either polyclonal anti-RIIa (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or control serum, in lysis buffer. The immune complexes were recovered with 10 lL of protein G Sepharose, washed with lysis buffer adjusted to 100 m M KCl and 0.1% (v/v) NP40. Proteins were released from beads using 50 lL of Laemmli buffer. Onesample(45lL) was analyzed by Western blotting for HIP/PAP by 13% (w/v) SDS/PAGE and the other (5 lL) for RIIa by 9% (w/v) SDS/PAGE, using anti-RIIa mAb (Transduction Laboratories). Immunofluorescence and confocal analysis After transfection with pcDNA-HIP/PAP, cells grown on glass coverslips were fixed with 4% (v/v) paraformalde- hyde and permeabilized with methanol at 4 °C. They were then incubated with anti-RIIa mAb and polyclonal anti-(WAP-HIP/PAP) [5] for 1 h at room temperature. Immunodetection w as carried out using fluorescein iso- thiocyanate-conjugated anti-rabbit Ig for HIP/PAP and/or cyanin-5 conjugated anti-mouse Ig for RIIa detection. Monoclonal antibody CTR433 (a gift from M. Bornens, Curie Institute, P aris, France) associated w ith cyanin-5- conjugated anti-mouse Ig was used for labelling of median Golgi. The coverslips were analyzed using laser confocal scanning microscopy. Fluorochrome-conjugated secon- dary antibodies were from Jackson (West Grove, PA, USA). Phosphorylation of recombinant HIP/PAP by PKA Recombinant HIP/PAP [4] was incubated at 30 °Cin80 lL, with 100 l M [ 32 P]ATP[cP] (specific activ ity, 15 000 cpmÆ pmol )1 ) and 25 units of bovine heart PKA in 2 0 m M Tris pH 7.5, 100 m M NaCl, 12 m M MgCl 2. Control incubations performed without recombinant HIP/PAP were conducted in parallel. At indicated times, 5 lL of incubation mixtures were spotted on phosphocellulose filters (Whatman P81) which were t hen w ashed i n phosphoric acid and dried as described [24]. Radioactivity wa s measured by liquid scintillation with Econofluor. Incubation mixtures (2 lL) were also analyzed using SDS/PAGE, and [ 32 P]HIP/PAP was detected by autoradiography of the wet gel. Protein kinase assays Two independent clones of stably expressing HIP/PAP Chang c ells (HIP 9 and HIP 4) and two independent control clones (PC4 and PC8) stably transfected with the empty vector were see ded at a density of 2 · 10 6 cells per 100 mm Petri dish 30 h before the assays. They were lysed in 20 m M Tris, pH 7.5, containing 1 m M EDTA, 1 m M dithiothreitol, and protease and phosphatase inhibitors (see above), and centrifuged at 3000 g. Supernatants were assayed immedi- ately for kinase activity as described previously [24]. Reporter gene assays HIP 9 and PC8 clones s eeded at a density of 2 · 10 5 cells per 35 mm diameter dish were transfected with 5 lgoftotal DNA including either 2 lg o f TK-LUC reporter p lasmid o r 2 lg of CRE-TK-LUC reporter plasmid [25] and when indicated 0.5 lgofpCa EV [20]. Cells were lysed 4 8 h post- transfection. Luciferase activity was measured b y a standard assay with a Lumat LB9501 luminometer (Fisher B ioblock Scientific, Illkirch, Cedex, France). Statistical analysis Using the nonparametric Kolmogorov–Smirnov test and the Levene test, it was established that the distribution of data obtained with different clones was normal. Student’s t-test was used to compare mean values of enzymatic activities measured under different conditions. Similar levels of statistical significance were obtained when HIP/PAP effects were analyzed in individual control and HIP/PAP clones or in pooled clones. Results Identification of the RIIa regulatory subunit of PKA as a partner of HIP/PAP In order to assess the biological consequences of HIP/PAP expression in hepatocellular carcinoma, we looked for proteins capable of interacting with this protein by screening a human HCC cDNA expression library in kgt11 using [ 32 P]chimeric HIP/PAP as a probe. For this purpose, we cloned HIP/PAP(29–175) in the pAR[DRI] vector. Of the 750 000 plaques analyzed, two of them hybridized with the probe. The sequences of the inserted cDNA were identical. In frame with the kgt11 Lac Z coding sequence they contained 1500 bp DNA, 1120 bp of which encoded for the C-terminal portion of the RIIa regulatory subunit of PKA. No hepatic cell line expressing HIP/PAP was available. Thus we have established hepatic cell models expressing HIP/PAP through t heir transfection with pcDNA-HIP/ PAP in order to validate HIP/PAP–RIIa interaction. HIP/ PAP was expressed more efficiently in Chang cells. Experi- ments were therefore performed using this cell line. HIP/ PAP was recovered in the serum of patients with hepato- cellular carcinoma which suggested that, in an in vivo setting, HIP/PAP was secreted by liver cells [6]. A similar patternwasobservedinHIP/PAP-expressing Chang cells (Fig. 1 A). HIP/PAP was r ecovered in the cells and the culture medium, and brefeldin A, an inhibitor of protein secretion [26], reduced HIP/PAP expression in the culture medium which indicated that HIP/PAP was secreted via a pathway involving the Golgi apparatus. Expression of HIP/PAP and RIIa in Chang cells was analyzed using immunofluorescence methods (Fig. 2). As 3814 F. Demaugre et al. (Eur. J. Biochem. 271) Ó FEBS 2004 previously observed in other HIP/PAP expressing cell lines [12,27] the immunostaining generated by anti-HIP/PAP Ig was cytoplasmic and mostly present in the juxta nuclear area (Fig. 2Aa). It partially colocalized with CTR433 (Fig. 2 B) a marker of m edian G olgi [28]. Immunostaining generated by anti RIIa antibody was not altered in HIP/ PAP expressing cells. As observed in other cell lines [29], it was mostly juxta nuclear in control and in HIP/PAP expressing cells. Detailed confocal analysis (Fig. 2C) showed that these proteins partly colocalized, suggesting their presence in a same subcellular compartment. The l ocations of HIP/PAP a nd RIIa were further analyzed using a fractionation method (Fig. 1B). The regulatory RIIa a nd the catalytic C a subunits of PKA w ere detected in the 100 00 0 g ultracentrifugation pellet and in the supernatant ind icating their p resence in both soluble and particulate forms in Chang cells as reported for other cell lines [30]. HIP/PAP was recovered associated to membranes in the p ellet confirming its presence in t he secretory apparatus, but also in the supernatant (23 and 28% of total HIP/PAP in two i ndependent experiments). Presence of HIP/PAP in the soluble fraction did not result from a significant contamination of this fraction with e lements o f the endoplasmic reticulum, as SERCA 2, an integral protein of endoplasmic reticulum, and calreticulin, protein of the reticulum lumen, were only detected in the centrifugation pellet. The antibodies we raised against HIP/PAP [4,5] are not suitable for immunoprecipitation experiments. T hus, using polyclonal anti-RIIa, we tested whether HIP/PAP could be coimmunoprecipitated with RIIa (Fig.1C).HIP/PAPwas recovered i n t he precipitate if the experiment was performed with anti-RIIa I g, but n ot with a co ntrol serum. W e did not detect any protein with an electrop horetic mobility similar to that of HIP/PAP when experiments were conducted w ith control cells (results not shown). HIP/PAP is phosphorylated by PKA Analysis of the HIP/PAP protein seq uence revealed the presence of three potential PKA phosphorylation sites (serines 73 and138, and threonine 153). In vitro, r ecombinant HIP/PAP was phosphorylated by PKA (Fig. 3A). It has been determined that phosphorylation w as more efficient at 30 °C than at lower or higher temper ature (results not shown). Thus time course of recombinant HIP/PAP phos- phorylation by PKA was studied at this temperature. HIP/ PAP phosphorylation increased with the incubation time and reached a plateau. After a 2 h incubation, 0.75 mol of 32 PO 4 was bound to 1 mol of recombinant HIP/PAP (Fig. 3 B). Whether HIP/PAP e xpressed in Chang cells might be phosphorylated by PKA was studied in cells transfected with pcDNA-HIP/PAPmyc. Cellular lysates were immuno- precipitated with monoclonal anti-myc Ig and the precipi- tates w ere further analyzed by Western blot u sing first polyclonal anti-HIP/PAP and then anti-phosphoserine Ig, after stripping of the m embrane (Fig. 3C,D). HIP/PAP was detected by anti-HIP/PAP as a single b and. When PKA was overexpressed, this antibody labelled two faint additional bands with re duced electrophoretic mobility. Anti-phospho- serine Ig labelled one protein with e lectrophoretic migration similar to t hat o f the upper one detected by anti-HIP/PAP. In contrast, no e xtra band was detected in cells expressing the mutated form of HIP/PAPmyc where the three potential PKA phosphorylation sites were mutated to alanine. Anti- phosphothreonine did not detect any band labelled by anti- HIP/PAP in cells expressing either the w ild or the mutated forms of HIP/PAPmyc (results not shown). Fig. 1. HIP/PAP expression in Chang c ells. Experiments w ere performed with Ch ang cells transiently expressing HIP/PAP. (A) E ffect of brefeldin A on H IP/PAP distribution in cell culture. After incubation f or 2 h with or without 10 l M brefeldin A, lysed cells and c ultu re media were analyzed for HIP/PAP by Western b lotting. (B) F ractionation exp eriments. Pellets and s upernatants r ecovered after centrifugation at 100 000 g of homo- genates from control (Neo) and HIP/PAP-expressing cells were analyzed by Western blotting for HIP/PAP [13% (w/v) SDS/PAGE] and, for SERCA 2, RIIa and Ca subunits of PKA, and calreticulin [9% (w/v) SDS/PAGE]. (C) Co-immunoprecipitation of HIP/PAP with RIIa.Cell lysates (400 lg prot ein) were incubated overnight with control serum (1), polyclon al anti-RIIa (2) or w ithout serum ( 3). The resulting immune complexes recovered with protein G Se pharose, were analyzed for H IP/PAP a nd RIIa by Western blot u sing polyclonal anti -HIP/PAP and mAb anti-RIIa. Ó FEBS 2004 HIP/PAP alters PKA signalling (Eur. J. Biochem. 271) 3815 PKA activity in Chang cells expressing HIP/PAP We investigated PKA activity in two clones isolated from a Chang cell line stably expressing HIP/PAP (HIP9 and HIP4 clones), and in two clones of Chang cells stably transfected with the empty vector as controls (PC4 and PC8 clones). Protein kinase activity assayed with kemptide, a specific substrate of PKA was measured with or without 8-bromo-cAMP and PKI, respectively, activa- tor and inhibitor of PKA in order to estimate basal and overall PKA activities. Endogenous phosphotransferase activity measured without kemptide did not differ between the two groups of cells (data not shown). F or the sak e of convenience (see Material a nd methods), pooled data from the two groups of cells are presented in Fig. 4. No difference was observed between the two groups of cells when the assays were conducted with 2 l M 8-bromo- Fig. 2. Immunofluorescence analysis o f RIIa and HIP/PAP sub cellular location in HIP/PAP expressing Chang cells. (A) Transiently HIP/PAP expressing cells were processed for immunofluorescence using the antibody against HIP/PAP labelled with FITC (a) or antibodies against RIIa labelled with c yanin-5 (b). Part (c) depicts a phase con- trast image of the analyzed cells. (B) Colocalization o f HIP/PAP with a marker of median Golgi (CTR433). Cells were proc essed for double immunofluorescence using antibo dies against HIP/PAP labelled w ith FITC (green; a) and CTR433, labelled w ith cyanin-5 (b). Colocaliza- tion of HIP/PAP and CTR433 is visi ble as yellow staining (c) when the colour images merge. (C) Colocalization of H IP/PAP with RII a. C ells were processed for double im munofluoresce nce using anti HIP/PAP Ig labelled with FITC (a) and anti-RIIa Ig labelled with cyanin-5 (b). The yellow staining (c) observed when the colour images m erge and the cytofluorogramme (d) demonstrate the colocalization of HIP/PAP with RII a. Staining was analyzed by confocal laser s canning micros- copy. Image is an optical section of 0.3 lmalongthez-axis. Fig. 3. HIP/PAP is a s ubstrate for PKA. (A)RecombinantHIP/PAP was incubated for 30 min at 30 °C with the catalytic subunit of P KA and 100 l M [ 32 P]ATP[cP] in 80 lL as described in the Materials and methods. Aliquots of i ncubation mixture s (2 lL) were analyzed by SDS/PAGE. [ 32 P]HIP/PAP was detected by autoradiography (1 h at room temperature) o f the gel. T, control reaction performed without HIP/PAP. (B) Time course of HIP/PAP phosphorylation. Recom- binant HIP/PAP (60 pmol) was incubated at 30 °CwithPKAand 100 l M [ 32 P]ATP[cP] in 80 lL as described in the Materials and methods. Control incubations were performed in parallel without recombinant HIP/PAP. At indicated times, 5 lL of incubation mix- tures were spotted on phosphocellulose filters, which were treated as indicated in M aterials and methods. The inco rporated radioactivity was determined by scintillation counting. (C) and (D) Chang cells were cotransfected with 1 8 lg of either the mutant or the wild type HIP/ PAPmyc expressing vector (empty vector called Neo was used in controls), and 2 lg of PKA expre ssing vector when indicated. Forty- eight hours post-transfection, cells were lyse d and immunoprecipitated with anti-myc mAb. Immune complexes recovered with protein G Sepharose were analyzed for by Western blotting for HIP/PAP using polyclonal anti-HIP/PAP (C) and for p hosphorylated protein using polyclonal anti-phosphoserine (D). Mole cular masses ind icated on t he right of the figures are ded uced from the e lectro phoretic migrat ion of molecular mass markers run in parallel with the samples. 3816 F. Demaugre et al. (Eur. J. Biochem. 271) Ó FEBS 2004 cAMP (optimal concentration to activate PKA in both groups of cells, data not shown) or with 100 l M PKI, inhibitor of PKA [31]. On the other hand phosphotrans- ferase activity assayed without any effector of PKA was increased by abo ut 20% in HIP/PAP-expressing cells suggesting that HIP/PAP expression did not alter overall PKA activity but enhanced basal PKA activity. This effect was better d isclosed when the phosphotransferase activities measured in presence of PKI, which may not be attributed to PKA, were subtracted from the data obtained in absence and presence of 8-bromo-cAMP. To further document the enhanced basal PKA activity observed in H IP/PAP expressing cells we examined the effects of HIP/PAP upon the expression of a gene whose promoter is under the control o f P KA. T he cAMP response element (CRE) present in the promoter of cyclin A2 has been shown t o respond to PKA [25]. Thus using a thymidine kinase-luciferase reporter plasmid (TK-LUC) in which one copy of the cyclin A 2 CRE was inserted upstream of the TK promoter (CRE-TK-LUC) we examined if the TK promo- ter was activated in HIP/PAP expressing cells. As shown in Fig. 5, expression of HIP/PAP did not alter luciferase activity in cells transfected with TK-LUC but increased luciferase activity by about 65% w hen cells were transfected with CRE-TK-LUC. That effect was no more observed when cells were cotransfected with CRE-TK-LUC and the pCaEV vector encoding for the catalytic subunit of PKA. Thus, taken together, these data indicated that HIP/PAP expression enhanced native PKA activity in Chang cells. Discussion HIP-encoding gene has been identified by our group as a gene over-expressed in tumourous but not in normal hepatocytes. The subsequent finding that this gene was identical to the PAP I/peptide 23/Reg2-encoding gene, which controls p ancreatic, pituitary and motor neurone viability a nd proliferation, has led to the h ypothesis that this C-type lectin may play an important physiological and/or physiopathological role. The biological function of this protein in the liver is unknown. To address this issue, we therefore looked for proteins capable of interacting with HIP/PAP in hepatocellular carcinoma cells. By screening a HCC cDNA library expressed in E. coli with [ 32 P]Flag- HMK-HIP/PAP(29–175) as a probe, we identified the regulatory RIIa subunit of PKA as b eing a partner of HIP/PAP. The demonstration of the biological relevance of the HIP/ PAP–RIIa interaction in HIP/PAP expressing cells required to establish that the two proteins may be located in a same subcellular compartment where they might interact. Indeed there was no evidence that the RIIa regulatory subunit of PKA is expressed anywhere other than the cytosol and the cytoplasmic surfaces of membranes [29]. On the other hand accurate su bcellular distribution of HIP/PAP had not been studied and thus it was considered that HIP/PAP, protein secreted via the Golgi a pparatus, was probably exclusively expressed in the luminal c ompartment of the s ecretory apparatus. We showed, using immunofluorescence studies Fig. 5. Reporter gene assay s. HIP9 and PC4 clones were transfected with 5 lgDNAincludingTK-LUC(2lg) or CRE- TK-LUC (2 lg) and 0.5 lgCaEV (0.5 lg) when indicated. Luciferase activity was assayed 4 8 h post-transfection. In each experiment, transfections were performed in triplicate for th e different s tudie d conditions. R esults are expressed as mean ± SE M of fou r independe nt experiments. Stu dent’s t-test was used to com pare mean values activities determ ined in PC4 and HIP9. Fig. 4. Protein kinase activity in HIP/PAP expressing Chang cells. Protein kinase activity was assayed with 50 l M kemptide as the sub- strate in the presence or absence of 2 l M 8-bromo c AMP a nd 100 l M PKI, in two clones of Chang cells stably expressing HIP/PAP (called HIP9 and H IP4) and two clones of Chang cells stably transfected with the empty vecto r (control clones called P C4 and P C8). Each as say was performed in triplicate. Data were obtained from eight independent experiments. (A) Protein kinase activities measured in the different conditions. (B) PKA activities: d at a o btained in presence of PKI were subtracted from the k inase a ctivitie s measured without effector (basal PKA a ctivity) or with 8-bromo-cAMP (overall PKA activity). Results are expres sed as mea n ± S EM. Stu dent’s t-test was used to compare mean values of enzymatic activities measured u nder different condi- tions. NS, not statistically sign ificant. Ó FEBS 2004 HIP/PAP alters PKA signalling (Eur. J. Biochem. 271) 3817 and fractionation experimen ts, that a fraction of the c ellular pool of HIP/PAP escaped the secretory pathway. Similar observations concerning the hepatitis C virus protein E2 have been recently reported [32]. E2 has previously been considered as a protein with an exclusive location in the endoplasmic reticulum [33], but in that study it was demonstrated that it also exists in the cytosol where it impairs cellular functions [32]. Thus, HIP/PAP and RIIa are both present as soluble forms in the cytosol of cells where they may interact. We have shown that they were coimmunoprecipitated in HIP/PAP-expressing cells. Thus our finding indicates that the location of HIP/PAP and RIIa is consistent with the relevance of their interaction. HIP/PAP has been classified in the group 7 of C-type lectins because it binds lactose and contains only one CRD [3,4]. The HIP/PAP sequence (the 146 C-terminal amino acids)presentintheprobeusedtoscreenthecDNAlibrary encompasses the CRD. E. coli does not express enzymes involved in glycosylation . Thus the interaction between HIP/PAP and RIIa is not dependent on sugar residues, suggesting that the CRD might bind both nonglycosylated and glycosylated proteins. HIP/PAP may be a target for PKA-dependent phos- phorylation. Three potential PKA phosphorylation sites (serines 73 and 138 and threonine 153) are detected in the sequence o f H IP/PAP. In vitro PKA was shown to phosphorylate recombinant H IP/PAP. Analysis of H IP/ PAP-expressing Chang cells has allowed us to determine that PKA phosphorylated a serine in the HIP/PAP protein. Indeed antiphosphoserine antibody recognized in PKA- overexpressing cells an HIP/PAP form when cells expressed wild HIP/PAP but not when PKA-phosphorylation sites of this protein were mutated to alanine. PKA-dependent phosphorylation of recombinant HIP/PAP did not alter its electrophoretic mobility (results not shown). On the other hand the antiphosphoserine antibody recognized in HIP/ PAP expressing cells, an HIP/PAP form whose electropho- retic migration was reduced, which suggests that HIP/PAP may be the target of additional post-translational modifi- cations altering its electrophoretic mobility. There i s no evidence that PKA may phosphorylate proteins present in the luminal compartment of the secretory p athway. Thus it is likely that PKA phosphorylates the fraction of HIP/PAP escaping the secretory pathway. Whether phosphorylation alters HIP/PAP p roperties r emains to be investigated. It h as to be noted that the PKA-dependent phosphorylation pattern remains unexplored and has to be determined to understand properties of HIP/PAP. However, as demon- strated for other lectins such as galectin 3 [34–36] HIP/PAP phosphorylation might alter its biological properties. PKA regulatory subunits control the release of catalytic subunits from the inactive tetramer complex upon binding of cAMP to th e regulatory subunit-dimer. Thus, we examined whether PKA activity w as altered in ce lls expressing HIP/PAP. Two independent methods were used to address this question: assay of PKA activity and study of the expression of a gene whose promoter contains a sequence responding to PKA. These approaches gave consistent results and allowed us to conclude that HIP/ PAP did not alter overall PKA activity but increased native PKA activity. The expression of the Ca cataly tic, and t he RIa and RIIa regulatory subunits of PKA, well represented in liver [37], is not altered in H IP/PAP expressing cells (results not shown). Thus, t he enhanced n ative PKA activity may result from the impaired association of catalytic and regulatory PKA subunits. PAP 1 (referred to as Reg 2) prevents neuronal cell death using both autocrine and paracrine ways in r at [12]. T hus two nonexclusive hypothesis may be put forward to explain the effects of HIP/PAP upon PKA. HIP/PAP has been reported to promote hepatocyte adhesion [8]. Thus through its interaction with a yet unidentified receptor, it could activate adenylcyclase and thus increase cellular cAMP levels and native PKA a ctivity. On the other hand, HIP/PAP via its interaction with RIIa might i mpair t he association of P KA catalytic subunits with the R IIa dimer, th us increasing PKA native activity without altering overall PKA activity. Whether t he biological functions of HIP/PAP results from its effects upon PKA remains to be established. It is noteworthy that links between HIP/PAP and the PKA-dependent pathways have already been suggested previously. In the rat the stimulatory effect of PAP 1 on Schwann cell proliferation was reported to i nvolve cAMP and therefore probably, PKA-depend ent pathways [11]. In liver, PKA is an important regulator of numerous metabolic functions. It has been involved in the protec- tion of hepatocytes against apoptosis [38–40] and in the control of their proliferation [41–44]. 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