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

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.

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)

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

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

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

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].

)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-

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

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

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

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

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

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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N. Bilodeau et al. Regulation of DPP IV trafficking
FEBS Journal 273 (2006) 992–1003 ª 2006 The Authors Journal compilation ª 2006 FEBS 1003