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 activit
Trang 1HIP/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 Demaugre1, Yannick Philippe1, Sokavuth Sar1, Bernard Pileire2, Laurence Christa1,
Chantal Lasserre1and Christian Brechot1
1
INSERM U370 CHU Necker Enfants Malades, Paris, France;2Laboratory 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 The biological
rele-vance of the interaction between these proteins was
established by demonstrating, 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 we showed that a fraction of HIP/PAP escaped the secretory apparatus and was recovered in the cytosol Basal PKA activity 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 protein containing only one
carbohydrate-binding domain (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 fetal marker Indeed, 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 under the influence of growth hormone releasing hormone and oestradiol, respectively [9,10], and by motor neurones 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/PAP was reported 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 the proteins interacting with HIP/PAP liver should help to understand the function(s) of HIP/PAP during hepatic carcinogenesis
In this study we have identified the RIIa regulatory subunit of cAMP-dependent protein kinase (PKA) as being
Correspondence to F Demaugre, INSERM U370 CHU 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, hepatocellular 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 July 2004)
Trang 2a 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 at the EcoRI site in the bacterial expression
plasmid pAR(deltaRI)[59/60] [17] This plasmid 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 complete HIP/PAP(1–175) sequence, amplified using
the same template, was subcloned at EcoRI and XhoI sites
in pcDNA3.1, and in pcDNA3.1/myc-His (Invitrogen) The
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 was 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 lysed at 4C with 10 lgÆmL)1
lysozyme in 50 mM Tris pH 8.0, 2 mM EDTA, 300 mM
KCl, 0.2% (v/v) Triton X-100 and 0.1 lgÆmL)1
phenyl-methylsulfonyl fluoride, and centrifuged Chimeric HIP/
PAP was purified from the supernatant using affinity
chromatography with monoclonal M2 anti-Flag agarose
(Sigma) Chimeric HIP/PAP was labelled using
[32P]ATP[cP] and the catalytic subunit of PKA as described
[17] and cleared from unincorporated [32P]ATP[cP] using
Sephadex G25 chromatography
Screening of a human HCC cDNA kgt11 library with
[32P]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 4C with32P-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 inserts amplified by PCR using Advantage cDNA polymerase and the kgt11 insert screening amplimer set (Clontech) were directly sequenced
Cell culture and transfection Chang cells (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· 106cells per 100 mm diameter dish were transfected with appropriate vectors (20 lg ADN) using the calcium 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 weeks with 800 lgÆmL)1neomycin 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 lMbrefeldin A was added to the culture medium At the end of incubation, cells lysed in buffer A (10 mMKH2PO4 pH 7.4, 150 mM NaCl, 10 mM EDTA, 1% (v/v) Triton X-100 and
2 lgÆmL)1 aprotinin, 1 lgÆmL)1 pepsatin, 2 lgÆmL)1 leu-peptin, 0.1 lgÆmL)1phenylmethylsulfonyl fluoride, 10 mM sodium fluoride, 2 mMsodium orthovanadate, 1 lM 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] The blots were revealed using an enhanced chemiluminecence system, according to 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 lg pCaEV encoding for the catalytic subunit of PKA [20] when indicated, cells were lysed with buffer A Cellular lysates (100 lg protein) were incubated overnight at 4C with 2 lg 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] Sarco/endoplasmic reticulum Ca2+ATPase 2 (SERCA 2), an integral protein of the endoplasmic reticulum [22], calreticulin, a protein of the endoplasmic reticulum lumen [23], HIP/PAP, the RIIa and the Ca subunits of PKA were checked by immunoblotting in both the 100 000 g pellet solubilized with buffer A and the supernatant using
Trang 3anti-HIP/PAP, anti-RIIa and anti-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 mM Tris pH 7.5, 2.5 mMMgCl2, 10 mM KCl, 0.5 mM
dithiothreitol, 0.05% (v/v) NP40, and protease and
phos-phatase inhibitors (see above) Extracts (400 lg protein)
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 mM KCl and 0.1% (v/v) NP40 Proteins
were released from beads using 50 lL of Laemmli buffer
One sample (45 lL) 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 4C They were
then incubated with anti-RIIa mAb and polyclonal
anti-(WAP-HIP/PAP) [5] for 1 h at room temperature
Immunodetection was 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, Paris, France) associated with
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 30C in 80 lL,
with 100 lM [32P]ATP[cP] (specific activity, 15 000 cpmÆ
pmol)1) and 25 units of bovine heart PKA in 20 mMTris
pH 7.5, 100 mMNaCl, 12 mMMgCl2.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 then washed in phosphoric acid and dried
as described [24] Radioactivity was measured by liquid
scintillation with Econofluor Incubation mixtures (2 lL)
were also analyzed using SDS/PAGE, and [32P]HIP/PAP
was detected by autoradiography of the wet gel
Protein kinase assays
Two independent clones of stably expressing HIP/PAP
Chang cells (HIP 9 and HIP 4) and two independent control
clones (PC4 and PC8) stably transfected with the empty
vector were seeded at a density of 2· 106cells per 100 mm Petri dish 30 h before the assays They were lysed in 20 mM Tris, pH 7.5, containing 1 mMEDTA, 1 mMdithiothreitol, 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 seeded at a density of 2· 105cells per
35 mm diameter dish were transfected with 5 lg of total DNA including either 2 lg of TK-LUC reporter plasmid or
2 lg of CRE-TK-LUC reporter plasmid [25] and when indicated 0.5 lg of pCa EV [20] Cells were lysed 48 h post-transfection Luciferase activity was measured by a standard assay with a Lumat LB9501 luminometer (Fisher Bioblock 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 [32P]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 their 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 pattern was observed in HIP/PAP-expressing Chang cells (Fig 1A) HIP/PAP was recovered 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
Trang 4previously 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 2B) a marker of median Golgi [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 locations of HIP/PAP and RIIa were further
analyzed using a fractionation method (Fig 1B) The
regulatory RIIa and the catalytic Ca subunits of PKA were
detected in the 100 000 g ultracentrifugation pellet and in
the supernatant indicating their presence 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 pellet confirming its presence in the secretory
apparatus, but also in the supernatant (23 and 28% of
total HIP/PAP in two independent experiments) Presence
of HIP/PAP in the soluble fraction did not result from a
significant contamination of this fraction with elements of
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 Thus, using
polyclonal anti-RIIa, we tested whether HIP/PAP could be
coimmunoprecipitated with RIIa (Fig 1C) HIP/PAP was
recovered in the precipitate if the experiment was performed
with anti-RIIa Ig, but not with a control serum We did not
detect any protein with an electrophoretic mobility similar
to that of HIP/PAP when experiments were conducted with control cells (results not shown)
HIP/PAP is phosphorylated by PKA Analysis of the HIP/PAP protein sequence revealed the presence of three potential PKA phosphorylation sites (serines 73 and 138, and threonine 153) In vitro, recombinant HIP/PAP was phosphorylated by PKA (Fig 3A) It has been determined that phosphorylation was more efficient at
30C than at lower or higher temperature (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
32PO4 was bound to 1 mol of recombinant HIP/PAP (Fig 3B) Whether HIP/PAP expressed 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 were further analyzed by Western blot using first polyclonal anti-HIP/PAP and then anti-phosphoserine Ig, after stripping of the membrane (Fig 3C,D) HIP/PAP was detected by anti-HIP/PAP as a single band When PKA was overexpressed, this antibody labelled two faint additional bands with reduced electrophoretic mobility Anti-phospho-serine Ig labelled one protein with electrophoretic migration similar to that of the upper one detected by anti-HIP/PAP In contrast, no extra 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 wild or the mutated forms of HIP/PAPmyc (results not shown)
Fig 1 HIP/PAP expression in Chang cells Experiments were performed with Chang cells transiently expressing HIP/PAP (A) Effect of brefeldin
A on HIP/PAP distribution in cell culture After incubation for 2 h with or without 10 l M brefeldin A, lysed cells and culture media were analyzed for HIP/PAP by Western blotting (B) Fractionation experiments Pellets and supernatants recovered 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 protein) were incubated overnight with control serum (1), polyclonal anti-RIIa (2) or without serum (3) The resulting immune complexes recovered with protein G Sepharose, were analyzed for HIP/PAP and RIIa by Western blot using polyclonal anti-HIP/PAP and mAb anti-RIIa.
Trang 5PKA 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 inhibiactiva-tor 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) For the sake of
convenience (see Material and 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 lM
8-bromo-Fig 2 Immunofluorescence analysis of RIIa and HIP/PAP subcellular
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 cyanin-5 (b) Part (c) depicts a phase
con-trast image of the analyzed cells (B) Colocalization of HIP/PAP with a
marker of median Golgi (CTR433) Cells were processed for double
immunofluorescence using antibodies against HIP/PAP labelled with
FITC (green; a) and CTR433, labelled with cyanin-5 (b)
Colocaliza-tion of HIP/PAP and CTR433 is visible as yellow staining (c) when the
colour images merge (C) Colocalization of HIP/PAP with RIIa Cells
were processed for double immunofluorescence 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 merge and the
cytofluorogramme (d) demonstrate the colocalization of HIP/PAP
with RIIa Staining was analyzed by confocal laser scanning
micros-copy Image is an optical section of 0.3 lm along the z-axis.
Fig 3 HIP/PAP is a substrate for PKA (A) Recombinant HIP/PAP was incubated for 30 min at 30 C with the catalytic subunit of PKA and 100 l M [ 32 P]ATP[cP] in 80 lL as described in the Materials and methods Aliquots of incubation mixtures (2 lL) were analyzed by SDS/PAGE [32P]HIP/PAP was detected by autoradiography (1 h at room temperature) of 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 C with PKA and
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 Materials and methods The incorporated radioactivity was determined by scintillation counting (C) and (D) Chang cells were cotransfected with 18 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 expressing vector when indicated Forty-eight hours post-transfection, cells were lysed 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 phosphorylated protein using polyclonal anti-phosphoserine (D) Molecular masses indicated on the right of the figures are deduced from the electrophoretic migration of molecular mass markers run in parallel with the samples.
Trang 6cAMP (optimal concentration to activate PKA in both
groups of cells, data not shown) or with 100 lM PKI,
inhibitor of PKA [31] On the other hand
phosphotrans-ferase activity assayed without any effector of PKA was
increased by about 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 disclosed 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 HIP/PAP expressing cells we examined the
effects of HIP/PAP upon the expression of a gene whose
promoter is under the control of PKA The cAMP response
element (CRE) present in the promoter of cyclin A2 has
been shown to respond to PKA [25] Thus using a thymidine
kinase-luciferase reporter plasmid (TK-LUC) in which one
copy of the cyclin A2 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% when 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 pancreatic, pituitary and motor neurone viability and proliferation, has led to the hypothesis 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 being 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 subcellular distribution of HIP/PAP had not been studied and thus it was considered that HIP/PAP, protein secreted via the Golgi apparatus, was probably exclusively expressed in the luminal compartment of the secretory apparatus We showed, using immunofluorescence studies
Fig 5 Reporter gene assays HIP9 and PC4 clones were transfected with 5 lg DNA including TK-LUC (2 lg) or CRE-TK-LUC (2 lg) and 0.5 lg CaEV (0.5 lg) when indicated Luciferase activity was assayed 48 h post-transfection In each experiment, transfections were performed in triplicate for the different studied conditions Results are expressed as mean ± SEM of four independent experiments Student’s t-test was used to compare mean values activities determined 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 cAMP and 100 l M
PKI, in two clones of Chang cells stably expressing HIP/PAP (called
HIP9 and HIP4) and two clones of Chang cells stably transfected with
the empty vector (control clones called PC4 and PC8) Each assay was
performed in triplicate Data were obtained from eight independent
experiments (A) Protein kinase activities measured in the different
conditions (B) PKA activities: data obtained in presence of PKI were
subtracted from the kinase activities measured without effector (basal
PKA activity) or with 8-bromo-cAMP (overall PKA activity) Results
are expressed as mean ± SEM Student’s t-test was used to compare
mean values of enzymatic activities measured under different
condi-tions NS, not statistically significant.
Trang 7and fractionation experiments, that a fraction of the cellular
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) present in the probe used to screen the cDNA library
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 of HIP/PAP In vitro PKA was shown to
phosphorylate recombinant HIP/PAP Analysis of HIP/
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 is no
evidence that PKA may phosphorylate proteins present in
the luminal compartment of the secretory pathway Thus it
is likely that PKA phosphorylates the fraction of HIP/PAP
escaping the secretory pathway Whether phosphorylation
alters HIP/PAP properties remains to be investigated It has
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 the regulatory subunit-dimer Thus, we
examined whether PKA activity was altered in cells
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 catalytic, and the
RIa and RIIa regulatory subunits of PKA, well represented
in liver [37], is not altered in HIP/PAP expressing cells (results not shown) Thus, the enhanced native 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 rat [12] Thus 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 activity
On the other hand, HIP/PAP via its interaction with RIIa might impair the association of PKA catalytic subunits with the RIIa dimer, thus increasing PKA native activity without altering overall PKA activity
Whether the 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 involve cAMP and therefore probably, PKA-dependent 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] Recently, it was shown that, in transgenic mice expressing human HIP/ PAP in the liver, HIP/PAP enhances liver regeneration and acts as a hepatic cytokine that combines mitogenic and anti-apoptotic functions using pathways involving PKA [16]
In conclusion, our findings lead us to propose PKA as a target for HIP/PAP, a C-type lectin and thus offer a novel mechanism for its biological activity
Acknowledgements
We are grateful to Dr Michael Blanar for generously providing the pAR(DRI)[59/60] plasmid We thank D Kremsdorf and P Soussan for helpful discussions This work was supported by a grant from ARC number 5156 (France).
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