Tài liệu Báo cáo khoa học: Insulin-dependent phosphorylation of DPP IV in liver Evidence for a role of compartmentalized c-Src ppt

Insulin-dependent phosphorylation of DPP IV in liver
Evidence for a role of compartmentalized c-Src
Nicolas Bilodeau
1
, Annie Fiset
1
, Guy G. Poirier
2
, Suzanne Fortier
1
, Marie-Claude Gingras
3
,
Jose
´
e N. Lavoie
3
and Robert L. Faure
1
1 Pediatric Research Unit, CRCHUL ⁄ CHUQ, Faculty of Medicine, Laval University, Que
´
bec, Canada
2 Quebec Proteomic Center, CRCHUL ⁄ CHUQ, Faculty of Medicine, Laval University, Que
´
bec, Canada
3 Cancer Research Center, CRHDQ ⁄ CHUQ, Faculty of Medicine, Laval University, Que
´
bec, Canada
Dipeptidyl peptidase IV (DPP IV, CD26, EC 3.4.14.5)
is a type II membrane glycoprotein that is expressed in
a variety of cell types [1]. DPP IV belongs to a serine

class of proteases exhibiting a restricted substrate spe-
cificity which favours release of Xaa–Pro or Xaa–Ala
dipeptides from the N terminus of proteins [2,3].
Within a cell, DPP IV is transported with high preci-
sion [4] and is synthesized with an uncleaved signal
sequence that functions as a membrane-anchoring
domain [5]. It has been shown that cysteine residues
and conformational changes are important compo-
nents that facilitate sorting [6]. Glycosylation is crucial
[7,8] and recent data have highlighted the importance
of both glycosylation and the lipid microenvironment
[9]. Among the proteins DPP IV may bind are: adeno-
sine deaminase [10], the kidney Na
+
⁄ H
+
exchanger
[11], the protein-tyrosine phosphatase (PTP) CD45 [12]
and the tyrosine kinase of the cellular Src (c-Src) fam-
ily p56
lck
[13]. In hepatocarcinoma cells, kinase activity
was detected in DPP IV immunoprecipitates [14].
In liver parenchyma, immunohistochemistry studies
have shown that DPP IV is located mainly in the bile
canalicular membrane [1]. In the renal brush border,
DPP IV is located in the microvilli and not in the
Keywords
c-Src; DPP IV; endosomes; tyrosine
phosphorylation, subcellular fractionation

Correspondence
R.L. Faure, Pediatric Research Unit (Cell
Biology Laboratory), Room 9800, CHUL
Medical Research Center, 2705 Laurier
Boulevard, Que
´
bec, QC, G1V 4G2, Canada
Fax: +1 418 654 2753
Tel: +1 418 656 4141, extn 48263
E-mail:
(Received 16 November 2005, revised 23
December 2005, accepted 3 January 2006)
doi:10.1111/j.1742-4658.2006.05125.x
Dipeptidyl peptidase IV (DPP IV, CD26, EC 3.4.14.5) serves as a model
aimed at elucidating protein sorting signals. We identify here, by MS, sev-
eral tyrosine-phosphorylated proteins in a rat liver Golgi ⁄ endosome (G ⁄ E)
fraction including DPP IV. We show that a pool of DPP IV is tyrosine-
phosphorylated. Maximal phosphorylation was observed after 2 min fol-
lowing intravenous insulin injection. DPP IV coimmunoprecipitated with
the cellular tyrosine kinase Src (c-Src) with maximal association also
observed after 2 min following insulin injection. DPP IV was found phos-
phorylated after incubation of nonsolubilized G ⁄ E membranes with
[c-
32
P]ATP. The c-Src inhibitor PP2 inhibited DPP IV phosphorylation.
Oriented proteolysis experiments indicate that a large pool of c-Src is pro-
tected in G ⁄ E fractions. Following injection of the protein-tyrosine phos-
phatase inhibitor bpV(phen), DPP IV levels markedly decreased by 40%
both in plasma membrane and G ⁄ E fractions. In the fraction designated
Lh, DPP IV levels decreased by 50% 15 min following insulin injection.

Therefore, a pool of DPP IV is tyrosine-phosphorylated in an insulin-
dependent manner. The results suggest the presence of a yet to be
characterized signalling mechanism whereby DPP IV has access to c-Src-
containing signalling platforms.
Abbreviations
bpV(phen), bisperoxovanadium 1,10-phenanthroline; c-Src, cellular tyrosine kinase Src; Cyt, cytosol; DPP IV, dipeptidyl peptidase IV; ER,
endoplasmic reticulum; G ⁄ E, Golgi ⁄ endosome; Gi and Gh, Golgi intermediate and heavy endosomes; GLP-1, glucagon-like peptide-1;
IR, insulin receptor; Li and Lh, light intermediate and heavy endosomes; PM, plasma membrane; PTP, protein-tyrosine phosphatase;
PY, phosphotyrosine; WGL, wheat germ lectin.
992 FEBS Journal 273 (2006) 992–1003 ª 2006 The Authors Journal compilation ª 2006 FEBS
coated pit microdomain [15]. In hepatocytes, DPP IV
is transported rapidly from the basolateral membrane
to the apical membrane by endocytosis [16]. In
Madin–Darby canine kidney (MDCK) cells, a study of
chimeric forms of DPP IV has shown that the luminal
domain of DPP IV carries dominant apical sorting
information while the short cytoplasmic tail and the
transmembrane domain contain competing basolateral
sorting information [17]. From one cell type to
another, DPP IV is sorted by different mechanisms.
Hence in hepatocytes, DPP IV reaches the apical mem-
brane by transcytosis; while in MDCK cells, apical
and basolateral proteins are segregated from each
other in the trans-Golgi network [18].
DPP IV exopeptidase activity is involved in a variety
of regulatory processes including chemokine regulation
[19] and maintenance of physiological glucose homeos-
tasis [20]. Knockout mice lacking the gene for DPP IV
show enhanced insulin secretion and accelerated clear-
ance of blood glucose coincident with increased endo-

genous levels of both glucagon-like peptide-1 (GLP-1)
and glucose-dependent insulinotropic polypeptide [21].
Pharmacological inhibition of DPP IV activity increases
insulin production and improves glucose control in dia-
betic animals [20,22–24] as well as in humans [25]. Apart
from its proteolytic activity, DPP IV is also engaged in
multiple functions depending on its ability to bind to
extracellular matrix [26]. Hence, DPP IV may be
involved in normal tissue architecture and growth pat-
terns [27]. DPP IV binding to type 1 collagen and fibro-
nectin has been demonstrated [28,29] and DPP IV can
be considered as a cell surface adhesion receptor for
fibronectin [30] with possible implications in cell migra-
tion and metastasis [27,30,31]. Also, DPP IV functions
in triggering the immune response [19,32].
Previously, we reported the presence of a series of
tyrosine-phosphorylated proteins in a wheat germ lec-
tin (WGL) subfraction prepared from a hepatic endo-
somal fraction [33]. Using MS, we identified the most
abundant tyrosine-phosphorylated proteins first. We
show here that one of these proteins, DPP IV, is tyro-
sine-phosphorylated in a ligand-dependent manner.
Results
MS analysis of major proteins purified
by antiphosphotyrosine (PY) affinity column
chromatography
We have reported previously the presence of several
tyrosine-phosphorylated proteins in WGL affinity col-
umn chromatography eluates prepared from a com-
bined fraction of endosomes and Golgi elements (G ⁄ E)

isolated from liver parenchyma [33]. Identification, by
finger printing MS, of the nine major proteins purified
by anti-PY affinity column chromatography, stained
with Coomassie blue, indicates that of these proteins,
four [insulin receptor (IR), LAR, ER-60, SAPAP-3]
are related to signalling events (Table 1). Identification
of the IR was expected, as it is readily concentrated by
the WGL affinity column chromatography step [34].
The PTP LAR is less well characterized. However pre-
viously, it was reported as being a regulator of the IR
[35–37]. The cysteine protease ER-60 was purified first
from the endoplasmic reticulum (ER) of rat liver [38].
ER-60 was found to be regulated by insulin and
PTP-1B [39]. SAPAP-3 is a signalling protein found
associated with tyrosine phosphorylation events in
membrane subdomains [40–43]. The other major
proteins identified were: the chaperone BIP, the
Table 1. MS analysis of major endosomal proteins purified by anti-PY affinity column chromatography. Endosomal glycoproteins were eluted
from anti-PY affinity column. Major proteins of: 220, 180, 117, 110, 106, 79, 61, 60, 38 kDa stained with Coomassie blue were excised from
gels after SDS ⁄ PAGE and subjected to proteolysis and MALDI-TOF analysis. Data were analysed using
MASCOT; accession numbers for each
scored protein in the NCBI nonredundant databank are listed; Sequence coverage indicates the percentage of the identified protein covered
by the sequences of identified peptides; m indicates the molecular mass of each protein predicted from the sequence (pred.) or experiment-
ally observed in the gel (expt.)
Protein
Sequence coverage (%) m
pred. ⁄ expt
(Da)Accession no Name
NP_062122 Protein-tyrosine phosphatase, receptor-type, F: LAR 4 198 640 ⁄ 180 000
NP_058767 Insulin receptor (precursor) 19 159 420 ⁄ 220 700

NP_019369 Inter-alpha-inhibitor H4 heavy-chain 9 103 930 ⁄ 117 400
P97838 SAPAP-3 7 106 970 ⁄ 110 000
A39914 DPP IV, membrane-bound form precursor 21 91 650 ⁄ 106 400
P06761 BIP 14 72 500 ⁄ 79 000
NP_059015 ER-60 protease 29 57 030 ⁄ 61 100
NP_445770 Hemopexin 12 52 010 ⁄ 60 000
AAF31764 Beta-1 adducin 24 20 990 ⁄ 38 000
N. Bilodeau et al. Regulation of DPP IV trafficking
FEBS Journal 273 (2006) 992–1003 ª 2006 The Authors Journal compilation ª 2006 FEBS 993
protease inhibitor inter-alpha-inhibitor H4, the trans-
porter hemopexin and beta-1 adducin—a component
of the cytoskeleton.
DPP IV phosphorylation in the G/E fraction
Assessment of DPP IV distribution in our hepatic frac-
tions, using anti-DPP IV (26C), showed that  80% of
the amount of DPP IV detected was located in the
G ⁄ E fraction. No DPP IV signal was observed in the
cytosol (Cyt). The remaining portion ( 20%) was
present in the plasma membrane (PM) fraction
(Fig. 1). We then verified DPP IV phosphorylation in
the G ⁄ E fraction by use of an anti-PY IgG (4G10).
Following insulin injection (1.5 lgÆ100 g
)1
body
weight), the IR was readily internalized as originally
described [44] (Fig. 2A, upper panel). The IR tyrosine
phosphorylation and autophosphorylation activity
were both maximal before 15 min postinsulin injection.
Under these circumstances, analysis of DPP IV
immuno-complexes revealed a signal detected by the

anti-PY IgG. This signal increased after 2 min post-
injection (Fig. 2A, lower panel). Sequence analysis
(LC-MS ⁄ MS) of the excised immunoprecipitated
110-kDa band (SYPRO Ruby staining) confirmed
unambiguously that this protein was DPP IV
(Fig. 2B). Previously, DPP IV was thought to be asso-
ciated with c-Src-like kinases in lymphocytes [45]. In
addition, another study has shown that c-Src is present
in endosomes of fibroblasts [46]. We detected c-Src in
DPP IV immunocomplexes, with an increased signal
observed 15 min following insulin injection (Fig. 2A,
lower panel).
We then verified DPP IV phosphorylation in vitro.
Following incubation of the nonsolubilized G ⁄ E
fraction in the presence of [c-
32
P]ATP, and immuno-
precipitation with anti-DPP IV IgG, a phosphorylated
protein of appropriate apparent molecular mass
(110 kDa) was observed (Fig. 3A). DPP IV
32
P
phosphorylation was alkali resistant. Insulin injection
(1.5 lgÆ100 g
)1
body weight) also increased DPP IV
phosphorylation by twofold above basal levels by
15 min. When the PTP inhibitor bisperoxovanadium
1,10-phenanthroline [bpV(phen)] was added to the
incubation medium, DPP IV phosphorylation was

enhanced at 0 (control) and 2 min, but not at 15 min
postinsulin injection (Fig. 3A). In order to link this
phosphorylation event with c-Src catalytic activity,
samples were incubated either in the presence or
absence of the c-Src inhibitor PP2 [47] prior to
DPP IV immunoprecipitation. The results show that
DPP IV phosphorylation was readily abolished when
PP2 was added to the incubation medium. Also, we
note the presence of an associated band around
56 kDa, presumably c-Src itself or a putative substrate
(Fig. 3B).
Localization of c-Src in G/E fractions
We assessed further c-Src localization in our fractions.
Permeabilization of the G⁄ E membranes with Triton
X-100 resulted in loss of several proteins, most notably
albumin (66 kDa), indicating that during permeabiliza-
tion, soluble luminal proteins were washed out
(Fig. 4B). A number of proteins ‘disappeared’ fol-
lowing treatment with proteinase K alone while the
66-kDa albumin was protected. The 66-kDa albumin
band almost completely disappeared when permeabi-
lized membranes were treated with proteinase K along
with other bands including the 110-kDa band (presum-
ably DPP IV) (Fig. 4B). The quality of the permeabili-
zation step was also assessed by electron microscopy.
The G ⁄ E fraction mainly contains typical lipoprotein-
filled tubulovesicular elements as well as 70–400-nm
diameter vesicles [48] (Fig. 4C). The permeabilization
step resulted in empty vesicular elements (Fig. 4D).
Therefore, while partial solubilization of membrane

Fig. 1. Distribution of DPP IV in hepatic subcellular fractions. The
Cyt, PM and G ⁄ E fractions were submitted directly to immunoblot
analysis (80 lg protein; 7.5% resolving gel) using the anti-DPP IV
(26C) IgG. Results are also presented as a percentage of the total
amount of DPP IV detected, calculated from the yields measured
for each fraction (see Experimental procedures). This experiment
was repeated three times with similar results; mean ± SD are
shown.
Regulation of DPP IV trafficking N. Bilodeau et al.
994 FEBS Journal 273 (2006) 992–1003 ª 2006 The Authors Journal compilation ª 2006 FEBS
elements by 0.1% Triton X-100 is possible, we were still
in a position to address the question of the orientation
of c-Src. We used the endosomal fraction G ⁄ E as well
as the Golgi intermediate and heavy endosomes
(Gi ⁄ Gh) and the light intermediate and heavy endo-
somes (Li ⁄ Lh) fractions [49]. Li is a homogeneous
fraction containing late endosomes and a negligible
amount of the marker enzymes sialyl transferase and
galactosyl transferase. The Gi fraction representing
early endosomes is contaminated ( 50%) by Golgi
elements. The other fractions (Lh, Gh) contain less
characterized endosomes and are rich (80%) in Golgi
elements. As above, the fractions were subjected to
proteinase K digestion after a membrane permeabiliza-
tion step (Triton X-100 0.1%). In the G ⁄ E, Lh and Gh
fractions, c-Src was detected easily in all conditions
(control, Triton 0.1%, proteinase K) except for the per-
meabilized membranes subjected to proteolysis (Triton
0.1% + proteinase K) (Fig. 4A). In contrast, c-Src was
not protected from proteinase K degradation in the

PM fraction for both conditions (nonpermeabilized or
permeabilized) (Fig. 4A). Using the same assay, we also
determined that DPP IV signal disappeared only when
permeabilized membranes (G ⁄ E) were submitted to
proteolysis (Fig. 4A). Therefore, the results indicate
that in endosomal fractions, c-Src is largely protected
from exogenously added proteinase K.
DPP IV levels following stimulation with insulin
and bpV(phen)
In order to examine changes in DPP IV levels follow-
ing insulin stimulation, rats were injected with the PTP
inhibitor bpV(phen) 16 h and 30 min prior to insulin
injection and isolation of the G ⁄ E and PM fractions.
No effect on DPP IV level was detected following
insulin injection. However, DPP IV levels, as detected
by immunoblotting (26C), decreased by  40% when
bpV(phen) was injected (Figs 5A and C). Such a
A
B
Fig. 2. Insulin-dependent tyrosine phosphorylation of DPP IV and
its association with c-Src in the G ⁄ E fraction. (A) Rats were injected
with insulin [1.5 lgÆ100 g
)1
body weight (bw)]. The G ⁄ E fraction
was isolated at the indicated times postinjection. (Upper panels)
Proteins were separated by SDS ⁄ PAGE (80 lg, 7.5% resolving
gel); the IR was detected by using either the anti-IR b-subunit IgG
or the anti-PY IgG (4G10). Autophosphorylation of the IR (95 kDa
32
P panel) was achieved by incubating aliquots (30 lg protein) with

[c-
32
P]ATP. Following centrifugation, the pellet was solubilized and
proteins immunoprecipitated using the anti-IR b-subunit IgG. Immu-
noprecipitates were separated by SDS ⁄ PAGE and gels were sub-
jected to alkali treatment and autoradiography. (Lower panels)
DPP IV immunoprecipitation: Aliquots of G ⁄ E fraction (200 lg pro-
tein) were immunoprecipitated using an anti-DPP IV IgG (MA-2607).
Immunoprecipitated proteins were separated by SDS ⁄ PAGE (10%
resolving gel). Membranes were incubated with anti-DPP IV (26C),
anti-PY (4G10) or anti-c-Src IgG (pieces of the same membrane).
The signals were submitted to densitometric analysis, and the
results were expressed as a percentage of the maximum signal.
Each value represents the mean ± SD of three independent experi-
ments. (B) Amino acid sequence of rat DPP IV (NCBI accession
number NP_36921, Swiss-Prot P14740). The immunoprecipitated
110-kDa band, stained with SYPRO Ruby (left panel), was excised
and subjected to proteolysis. Rat DPP IV peptide sequences that
were identified by LC-MS ⁄ MS are boxed. Hashed boxes indicate
that a common sequence is present in two different peptides.
Results are representative of three independent experiments.
N. Bilodeau et al. Regulation of DPP IV trafficking
FEBS Journal 273 (2006) 992–1003 ª 2006 The Authors Journal compilation ª 2006 FEBS 995
decrease was also observed for the PM fraction
(Figs 5B and C). In order to assess whether this effect
on DPP IV level was coincident with a phosphoryla-
tion event, we used an antibody which reconizes
residues phosphorylated by Src kinases (aPY-42
antibody). The results revealed the coincident hyper-
phosphorylation of a 100-kDa band. This is consistent

with the view that a c-Src-dependent phosphorylation
event had occurred (Fig. 5A).
Further fractionation was then used to refine our
assessment of DPP IV and c-Src distribution. Follow-
ing insulin injection, IR accumulation and tyrosine
phosphorylation was observed for all examined frac-
tions, most evidently for the Lh, Gi and Gh fractions
(Fig. 6A). The results show that both c-Src and
DPP IV are also located mainly in the Lh and Gh
fractions. No changes in DPP IV levels are observed
following insulin injection, except at 15 min postinjec-
tion where the signal declines significantly by more
than 50% in the Lh fraction (n ¼ 4; P < 0.001)
(Fig. 6B). No significant changes in c-Src levels were
observed.
Discussion
Previously, we have reported the presence of a series
of tyrosine-phosphorylated proteins partially purified
from hepatic endosomes [33]. Following anti-PY affin-
ity column chromatography, a systematic identification
performed first on the more abundant protein species
reveals here that one of these is DPP IV (Table 1).
DPP IV is well represented in the G ⁄ E fraction where
it is found even more abundantly than in the PM frac-
tion (Fig. 1). At the cell surface, DPP IV is located
mainly in the bile canalicular domain. This relative
abundance in the G⁄ E fraction may be explained by
the diverse representation of the three major domains
(sinusoidal, lateral, bile canalicular) of the hepatocytes
present in the PM fraction [50].

To the best of our knowledge, tyrosine phosphoryla-
tion of DPP IV has not been reported before. In addi-
tion, the results show that DPP IV phosphorylation is
regulated, thus defining a new insulin-dependent effect.
The observation that maximal DPP IV phosphoryla-
tion (after 2 min postinsulin injection) does not corres-
pond with maximal IR tyrosine phosphorylation is
consistent with the fact that DPP IV is not phosphor-
ylated by the IR.
Previous studies performed with immune cells have
shown that DPP IV is associated with the c-Src related
tyrosine kinase p56
lck
[13], despite a short (6 residues)
cytoplasmic tail. Investigation here for a role of c-Src
in DPP IV phosphorylation indeed reveals that not
only is c-Src associated with DPP IV, but its associ-
ation is dependent of insulin with maximal association
coincident with maximal tyrosine phosphorylation of
DPP IV in vivo (Fig. 2). Insulin-dependent DPP IV
phosphorylation is also readily detected in vitro and is
furthermore inhibited by the c-Src inhibitor PP2. This
confirms that DPP IV is tyrosine-phosphorylated and
further supports the idea that c-Src is involved in
DPP IV phosphorylation.
A
B
Fig. 3. In vitro phosphorylation of DPP IV.
(A) Rats were injected with insulin
(1.5 lgÆ100 g

)1
body weight). The G ⁄ E frac-
tion was isolated at the indicated times
postinjection and aliquots (100 lg protein)
were incubated with [c-
32
P]ATP in the pres-
ence or absence of 100 l
M bpV(phen). They
were solubilized and proteins immunoprecip-
itated using the anti-DPP IV IgG (MA-2607).
The immunoprecipitates were separated by
SDS ⁄ PAGE (10% resolving gel) and gels
were subjected to autoradiography before
and after alkali treatment. (B) The G ⁄ E
fraction was isolated 15 min following
insulin injection and was subjected to
phosphorylation, in the presence or absence
of the c-Src inhibitor PP2 (10 l
M), and then
immunoprecipitated as above. Results are
representative of three independent
experiments.
Regulation of DPP IV trafficking N. Bilodeau et al.
996 FEBS Journal 273 (2006) 992–1003 ª 2006 The Authors Journal compilation ª 2006 FEBS
A
B
D
C
Fig. 4. Oriented proteolysis: The tyrosine kinase c-Src is protected from exogenously added protease in endosomal fractions. (A, upper pan-

els) The G ⁄ E, Lh, Gh and PM fractions (100 lg protein) were incubated in the presence or absence of Triton X-100 and proteinase K before
immunoblotting with the anti-c-Src IgG. The same experiment (lower panel) was performed using the G ⁄ E fraction and the anti-DPP IV (26C)
IgG. Results shown are typical of three independent experiments. (B) The G ⁄ E fraction (100 lg protein) was incubated in the presence or
absence of Triton X-100 (0.1%) and proteinase K before staining with SYPRO Ruby. The 66-kDa (albumin) and 110-kDa bands are shown
with arrows. (C) Electron microscopy of the G ⁄ E fraction that was purified and processed as described in Experimental procedures. Note
the presence of typical tubulovesicular structures as well as lipoprotein-filled vesicles (70–400 nm) (Scale bar ¼ 200 nm). (D) Electron micros-
copy of the G ⁄ E fraction treated with 0.1% Triton X-100 as described in Experimental procedures. Note the absence of typical lipoprotein-
filled (dark) vesicles. (Scale bar ¼ 200 nm).
N. Bilodeau et al. Regulation of DPP IV trafficking
FEBS Journal 273 (2006) 992–1003 ª 2006 The Authors Journal compilation ª 2006 FEBS 997
c-Src is known to be located in endosomes [46] and
Golgi elements [51] where it is thought to regulate
retrograde transport [52]. In lipid raft compartments,
c-Src plays a role in signalling events [53]. It is also
clear, either by immunofluorescence microscopy of
endogenous or transfected c-Src (data not shown) or
by immunoblotting of endosomal fractions (G ⁄ E, Lh,
Gh and Gi, Li) (Figs 2, 4 and 6), that c-Src is distri-
buted in the vacuolar system. Our results also provide
the first evidence that c-Src has access to the lumen.
Indeed, oriented proteolysis indicates that a pool of
c-Src is protected from exogenous proteolysis. There
are no known translocation motifs in c-Src, and the
mechanism of c-Src translocation is yet to be charac-
terized. However, the dynamic role of the translocons
[54,55] and membrane restructuring enzymes [56] in
protein targeting is beginning to be perceived. For
instance, one striking example of c-Src location inside
one organelle has been reported for the inner mem-
brane of mitochondria of osteoclasts [57]. Moreover,

there are 50 tyrosine residues in the sequence of rat
DPP IV, all of which are located in the lumen.
A
B
C
Fig. 5. Effect of the PTP inhibitor bpV(phen) on DPP IV levels in
G ⁄ E and PM fractions. (A) bpV(phen) was injected (0.3 mgÆ100 g
)1
body weight) 16 h and 30 min before the injection of insulin
(1.5 lgÆ100 g
)1
body weight). Endosomes (G ⁄ E) were isolated at
the noted times and were submitted directly to immunoblot analy-
sis (100 lg protein; 7.5% resolving gel) using the anti-DPP IV (26C)
IgG or the aPY-42 antibody. (B) bpV(phen) was injected into rats
16 h and 30 min before liver excision. The PM fraction was pre-
pared as described and immunoblotted as in (A). (C) DPP IV signals
obtained in (A) and (B) were submitted to densitometric analysis,
and the results were expressed as a percentage of the maximum
signal, respectively. Means ± SD are shown (n ¼ 11 in G ⁄ E frac-
tion, n ¼ 4 in PM fraction).
A
B
Fig. 6. DPP IV level is decreased by insulin in the Lh subfraction.
(A) Following insulin injection (1.5 lgÆ100 g
)1
body weight), frac-
tions (Li, Lh, Gi and Gh) were isolated at the indicated times. Aliqu-
ots were immunoblotted (40 lg protein; 7.5% resolving gel) using
an anti-IR b-subunit IgG (95 kDa, b -subunit panel) or an anti-PY IgG

(95 kDa aPY panel). (B) Immunoblot analysis of c-Src and DPP IV
(80 lg protein; 7.5% resolving gel) using anti-c-Src and anti-DPP IV
(26C) IgG (pieces of the same membrane). DPP IV signals are
expressed as a percentage of the maximal value; means ± SD are
shown (DPP IV: *Lh: P < 0.01, 0 min vs. 15 min, n ¼ 4; Student’s
t-test).
Regulation of DPP IV trafficking N. Bilodeau et al.
998 FEBS Journal 273 (2006) 992–1003 ª 2006 The Authors Journal compilation ª 2006 FEBS
The results show also that DPP IV levels are affec-
ted following stimulation with a potent PTP inhibitor
[bpV(phen)]. The observation that this effect coincides
with the hyperphosphorylation of a 110-kDa band
revealed by the aPY-42 antibody, supports the idea
that DPP IV tyrosine phosphorylation is related to
DPP IV levels (Fig. 5). Hence, in the presence of
bpV(phen) a relatively small pool of DPP IV can be
continuously diverted towards lysosomal compart-
ments for degradation. Alternatively, a soluble form of
DPP IV can be released from the cell surface. In this
regard, an increased circulating concentration of the
soluble form of DPP IV may result in an increased
proteolysis of GLP-1 peptides and thus decreased insu-
lin secretion [20]. The insulin-dependent phosphoryla-
tion of DPP IV observed here may thus provide a
regulatory loop among a target organ (liver) and the
insulin secreting cells. Deregulation of this DPP IV
phosphorylation mechanism may have implications for
the homeostasis of circulating GLP-1 levels and diabe-
tes. However, we did not detect significant changes in
circulating DPP IV activity, using Gly-Pro-p-nitro-

anilide as a substrate in our acute conditions of sti-
mulation (insulin dose: 15 lgÆ100 g
)1
, control: 0.122 ±
0.013 UÆmL
)1
, n ¼ 25; bpV(phen): 0.124 ± 0.0045
UÆmL
)1
, n ¼ 27; U ¼ amount of enzyme which
hydrolyses 1 lmol substrateÆmin
)1
). It remains possible
that more chronic alteration of circulating insulin
results in significant changes of circulating DPP IV.
Indeed, further fractionation demonstrated the pres-
ence of a significant decrease in DPP IV levels in the
Lh fraction thus revealing that DPP IV is subject to
ligand control in a precise microenvironment. This is
also consistent with the fact that the IR, DPP IV and
c-Src are present mainly in the same fractions (Lh and
Gh). This therefore points to the importance of sub-
jecting these fractions to further purification and bio-
chemical characterization in order to gain a more
detailed understanding of this process.
In conclusion, the results presented here demonstrate
that DPP IV is tyrosine-phosphorylated in an insulin-
dependent manner in hepatic endosomal fractions. The
possible involvement of luminal c-Src in this process
suggests the presence of a mechanism whereby DPP IV

en route along with the endocytosed IR can reach
compartments where c-Src is present.
Experimental procedures
Reagents and antibodies
Porcine insulin was from Sigma (St. Louis, MO). The anti-
body directed against the b-subunit of the IR was from BD
Transduction Laboratories (rabbit polyclonal, 188430). The
hybridoma (26C, clone 287) expressing the monoclonal
antibody against DPP IV was kindly provided by
M.G. Farquhar (University of California, San Diego, CA)
and was used for immunoblotting experiments. The anti-
DPP IV used for immunoprecipitation experiments was
from Endogen (Woburn, MA). A mixture of antibodies
against c-Src 1 : 1 (N-16, sc-19 and SRC2, sc-18, Santa
Cruz Biotechnologies, Inc., Santa Cruz, CA) was used for
immunoblots. The monoclonal anti-PY IgG (4G10) used to
detect IR b-subunit phosphorylation was purchased from
Upstate (Lake Placid, NY). The antiphospho-E4orf4 (Y42)
42-2 was produced by injecting rabbits with a chemically
synthesized peptide comprising phosphorylated Tyr42
(HEGVY[PO
3
H
2
]IEPEARGRLC) coupled to mcKLH
(Imject Mariculture Keyhole Limpet Hemocyanin, Pierce
Biotechnology Inc., Rockford, IL), following recommenda-
tions of the manufacturer. This phosphosite is the major
residue phosphorylated by Src kinases on the adenoviral
protein E4orf4 [58,59]. The serum was absorbed on immo-

bilized phosphorylated peptide using a SulfoLink Kit (Bio-
Lynx, Brockville, ON) and blocked against an excess of
nonphosphorylated peptide during immune detection. The
specificity of the purified 42-2 antibody was tested by west-
ern blot analysis of E4orf4 immune complexes and total cell
lysates from cells transfected with wild type Flag-E4orf4 as
compared to mutant Flag-E4orf4 (Y42F) alone, or together
with c-Src or v-Src to induce maximum tyrosine phos-
phorylation of Ad2 E4orf4 and of Src substrates as well.
The antibody reacted specifically with wild-type Ad2
E4orf4 but not with mutant E4orf4 (Y42F) and the signal
was proportional to the level of tyrosine phosphorylation.
This antibody does not react with the tyrosine-phosphoryl-
ated IR but it does react against other PY proteins that are
selectively modulated by E4orf4 and whose phosphoryla-
tion is modulated by Src (data not shown). E4orf4 is itself
a Src substrate, which acts as a modifier of Src-dependent
phosphorylation [59,60]. For western blot studies we used
the enhanced chemiluminescence kit Western Plus (Perkin
Elmer Life Sciences Inc., Boston, MA) and Immobilon-P
transfer membrane (Millipore, Bedford, MA). [c-
32
P]ATP
(1000–3000 CiÆmmol
)1
) was from New England Nuclear
Radiochemicals (Lachine, QC). The c-Src inhibitor PP2
was from EMD Biosciences (La Jolla, CA). Reagents for
SDS ⁄ PAGE were obtained from Bio-Rad (Mississauga,
ON). bpV(phen) was synthesized as described [61]. All

other chemicals were of analytical grade and were pur-
chased from either Fisher (Sainte-Foy, QC) or Roche
Laboratories (Laval, QC).
Subcellular fractionation
Sprague–Dawley rats (female, 140–150 g body weight) were
purchased from Charles River Ltd (St. Constant, QC).
Work was conducted with the approval of the Laval
N. Bilodeau et al. Regulation of DPP IV trafficking
FEBS Journal 273 (2006) 992–1003 ª 2006 The Authors Journal compilation ª 2006 FEBS 999
University Animal Care committee. Rats were fasted over-
night, anesthetized with Nembutal (6.7 mgÆ100 g
)1
body
weight) and injected with insulin via the left jugular vein
(1.5 lgÆ100 g
)1
body weight). When bpV(phen) was used in
vivo, a peritoneal injection (0.3 mgÆ100 g
)1
body weight)
was made 16 h and 30 min before insulin injection. Livers
were excised rapidly at the noted times postinjection and
minced in ice-cold homogenization buffer (250 mm sucrose,
50 mm Hepes pH 7.4, 40 mm sodium fluoride, 1 mm
MgCl
2
,1mm benzamidine, 1 mm phenlymethylsulphonyl
fluoride). The G ⁄ E fraction was prepared immediately as
described previously [44]. This fraction has been character-
ized by transmission electron microscopy, enzyme markers,

silver staining and receptor-mediated endocytosis. SYPRO
Ruby (Eugene, OR) staining of SDS ⁄ PAGE 1-D gels is
also shown here (Fig. 4B). The endosomal fractions previ-
ously designated Li ⁄ Lh and Gi ⁄ Gh were prepared from the
parent light mitochondrial (L) and microsomal (P) frac-
tions, respectively, by a flotation method as originally des-
cribed elsewhere [62]. The yield of the G ⁄ E fraction was
0.38 ± 0.015 mg proteinÆg
)1
liver weight (n ¼ 36). The
yields from the other fractions were: Li, 0.18 ± 0.036 mg
proteinÆg
)1
liver weight; Lh, 0.09 ± 0.01 mg proteinÆg
)1
liver weight; Gi, 0.015 ± 0.004 mg proteinÆg
)1
liver weight;
Gh, 0.034 ± 0.005 mg proteinÆg
)1
liver weight, n ¼ 36.
The PM fraction was prepared according to the method
of Hubbard with modifications [44] and used directly. A
yield of 1.18 ± 0.61 mg proteinÆg
)1
liver weight (n ¼ 22)
was obtained. The Cyt fraction was generated by centrifu-
ging the homogenate at 100 000 g for 1 h and the
supernatant was collected. Protein content of fractions
was determined by a modification of the Bradford method

using BSA as a standard. Statistical analysis was
performed with statview (Abacus Concepts Inc.,
Berkeley, CA).
Electron microscopy
The G ⁄ E fraction was immediately fixed with 2.5% glutar-
aldehyde, and 100 mm sodium cacodylate pH 7.4. Samples
were rinsed and postfixed in 1% ferrocyanide osmium tetr-
oxide, dehydrated in a graded series of ethanol and then
processed for embedding in EPON. Ultrathin sections of
each block were cut and placed on copper grids, stained
with uranyl acetate and lead citrate [48]. Sections were
examined with a Philips EM 301 electron microscope (Phi-
lips, Eindhoven, the Netherlands).
MS
Tyrosine-phosphorylated proteins were recovered from the
WGL subfraction of the G ⁄ E fraction prepared as des-
cribed previously [33]. The WGL subfraction was applied
to an anti-PY affinity column (PY-20-agarose) and phos-
phoproteins were eluted by re-suspension of each column
in 40 mm para-nitrophenylphosphate (pNPP) for 120 min
at room temperature. The columns were spun to yield the
eluates and protein contents in the fractions were deter-
mined as above. Eluates were then separated by
SDS ⁄ PAGE and the major bands, stained with Coomassie
blue, were excised and subjected to alkylation and diges-
tion procedures using lysyl endopeptidase C [63]. Digestion
products were spotted on a stainless steel MALDI plate
(Applied Biosystems, Foster City, CA). Analyses utilized
an automated acquisition procedure on a Voyager-DE
PRO MALDI-TOF mass spectrometer (Applied Biosys-

tems) operated in a delayed extraction mode. mascot
(Matrix Science Inc., Boston, MA) [64] was used for
searches in the nonredundant NCBI database. For identifi-
cation of the immunoprecipitated DPP IV, a 110-kDa
band, stained with SYPRO Ruby, was excised from the gel
and subjected to trypsin digestion [63]. The resulting pep-
tides were separated by a capillary HPLC reverse phase
C18 column (Picofrit BioBasic, 10 cm length, 0.075 mm
internal diameter New Objective, Woburn, MA) and ana-
lysed by tandem MS using a LC-MS ⁄ MS quadrupole ion
trap mass spectrometer (Finnigan LCQ Deca XP, Thermo
Electro Corporation, San Jose, CA). mascot was used for
searches in the nonredundant NCBI database [64].
Phosphorylation and immunoprecipitation assays
IR autophosphorylation and KOH treatment (hydrolysis of
phosphorylated serine and threonine residues) of gels were
conducted as reported previously [65] with minor modifica-
tions. Aliquots of intact endosomes (G ⁄ E fraction) were
incubated at 37 °C for 15 min in the kinase buffer (50 mm
Hepes pH 7.4, 3 mm benzamidine, 40 mm MgCl
2
,1mm
MnCl
2
, 0.05% Triton X-100), in the presence of [c-
32
P]ATP
(25 lm, 3000 CiÆmmol
)1
). When indicated, an inhibitor

[100 lm bpV(phen) or 10 lm PP2] was added. Samples
were then solubilized in 1% Triton X-100 for 60 min and
centrifuged at 250 000 g for 30 min. The resulting superna-
tants were immunoprecipitated for IR (1 lg affinity purified
antibodyÆml
)1
; 100 lg protein) or DPP IV (5 lg affinity
purified antibodyÆml
)1
; 500 l g protein). The resulting
immuno-complexes were separated by SDS ⁄ PAGE (7.5%
resolving gel) and gels were subjected to autoradiography.
Unlabelled DPP IV immuno-complexes were analysed
directly by immunoblotting using anti-DPP IV (26C), anti-
PY or anti-c-Src IgG.
Oriented proteolysis
Oriented proteolysis experiments were performed essen-
tially as described [33]. Endosomes (G ⁄ E, Lh and Gh
fractions) or PM fraction were incubated in 50 m m Hepes
pH 7.4, at 4 °C for 30 min in the presence or absence of
0.1% Triton X-100 and proteinase K (60 ngÆ100 l g
)1
pro-
tein of fractions). The membranes were then diluted
Regulation of DPP IV trafficking N. Bilodeau et al.
1000 FEBS Journal 273 (2006) 992–1003 ª 2006 The Authors Journal compilation ª 2006 FEBS
(1 : 10) in ice-cold buffer without Triton X-100 and
proteinase K and centrifuged for 30 min at 250 000 g
(TL-100, Beckman Coulter, Fullerton, CA). The resulting
pellet was analysed by transmission electron microscopy

or re-suspended in Laemmli sample buffer and subjected
to SDS ⁄ PAGE (10 lg protein). Gels were stained with
SYPRO Ruby for examination or immunoblotted with
anti-c-Src and anti-DPP IV (26C) IgG.
Acknowledgements
This work is supported by the Natural Sciences and
Engineering Research Council (NSERC) of Canada
(RF: OGPO157551), by the Canadian Diabetes Associ-
ation (CDA) and a grant from the Fondation pour la
Recherche sur les Maladies Infantiles (FRMI). JL is a
chercheur-boursier (Junior 2, FRSQ), NB and AF were
supported by Canadian Institutes of Health Research
(CIHR) scholarships. Dr Paul Khan (Laval University)
is greatly acknowledged for comments.
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