The Adaptor Molecule CIN85 Regulates Syk Tyrosine Kinase Level by Activating the Ubiquitin-Proteasome Degradation Pathway
Sapienza University of Rome, Roma, Latium, ItalyThe Journal of Immunology (Impact Factor: 4.92). 09/2007; 179(4):2089-96. DOI: 10.4049/jimmunol.179.4.2089
Triggering of mast cells and basophils by IgE and Ag initiates a cascade of biochemical events that lead to cell degranulation and the release of allergic mediators. Receptor aggregation also induces a series of biochemical events capable of limiting FcepsilonRI-triggered signals and functional responses. Relevant to this, we have recently demonstrated that Cbl-interacting 85-kDa protein (CIN85), a multiadaptor protein mainly involved in the process of endocytosis and vesicle trafficking, regulates the Ag-dependent endocytosis of the IgE receptor, with consequent impairment of FcepsilonRI-mediated cell degranulation. The purpose of this study was to further investigate whether CIN85 could alter the FcepsilonRI-mediated signaling by affecting the activity and/or expression of molecules directly implicated in signal propagation. We found that CIN85 overexpression inhibits the FcepsilonRI-induced tyrosine phosphorylation of phospholipase Cgamma, thus altering calcium mobilization. This functional defect is associated with a substantial decrease of Syk protein levels, which are restored by the use of selective proteasome inhibitors, and it is mainly due to the action of the ubiquitin ligase c-Cbl. Furthermore, coimmunoprecipitation experiments demonstrate that CIN85 overexpression limits the ability of Cbl to bind suppressor of TCR signaling 1 (Sts1), a negative regulator of Cbl functions, while CIN85 knockdown favors the formation of Cbl/Sts1 complexes. Altogether, our findings support a new role for CIN85 in regulating Syk protein levels in RBL-2H3 cells through the activation of the ubiquitin-proteasome pathway and provide a mechanism for this regulation involving c-Cbl ligase activity.
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Ubiquitin-Proteasome Degradation Pathway
Tyrosine Kinase Level by Activating the
The Adaptor Molecule CIN85 Regulates Syk
Angela Santoni and Rossella Paolini
Laura Vian, Stefania Morrone, Mario Piccoli, Luigi Frati,
Giovanna Peruzzi, Rosa Molfetta, Francesca Gasparrini,
2007; 179:2089-2096; ;J Immunol
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The Adaptor Molecule CIN85 Regulates Syk Tyrosine Kinase
Level by Activating the Ubiquitin-Proteasome Degradation
Francesca Gasparrini, Laura Vian,
Mario Piccoli, Luigi Frati, Angela Santoni, and Rossella Paolini
Triggering of mast cells and basophils by IgE and Ag initiates a cascade of biochemical events that lead to cell degranulation and
the release of allergic mediators. Receptor aggregation also induces a series of biochemical events capable of limiting Fc
triggered signals and functional responses. Relevant to this, we have recently demonstrated that Cbl-interacting 85-kDa protein
(CIN85), a multiadaptor protein mainly involved in the process of endocytosis and vesicle trafﬁcking, regulates the Ag-dependent
endocytosis of the IgE receptor, with consequent impairment of Fc
RI-mediated cell degranulation. The purpose of this study was
to further investigate whether CIN85 could alter the Fc
RI-mediated signaling by affecting the activity and/or expression of
molecules directly implicated in signal propagation. We found that CIN85 overexpression inhibits the Fc
phosphorylation of phospholipase C
, thus altering calcium mobilization. This functional defect is associated with a substantial
decrease of Syk protein levels, which are restored by the use of selective proteasome inhibitors, and it is mainly due to the action
of the ubiquitin ligase c-Cbl. Furthermore, coimmunoprecipitation experiments demonstrate that CIN85 overexpression limits the
ability of Cbl to bind suppressor of TCR signaling 1 (Sts1), a negative regulator of Cbl functions, while CIN85 knockdown favors
the formation of Cbl/Sts1 complexes. Altogether, our ﬁndings support a new role for CIN85 in regulating Syk protein levels in
RBL-2H3 cells through the activation of the ubiquitin-proteasome pathway and provide a mechanism for this regulation involving
c-Cbl ligase activity. The Journal of Immunology, 2007, 179: 2089 –2096.
timulation of mast cells by the aggregation of the high-
afﬁnity receptor for IgE, FcRI, initiates a cascade of
biochemical events that result in degranulation and re-
lease of inﬂammatory mediators (1–5). Details of the IgE-me-
diated signaling pathway have been established primarily in the
RBL-2H3 mast cell model and in mouse bone-marrow derived
FcRI is composed by an IgE-binding
-chain, a four-trans-
subunit, and two identical disulﬁde-linked
subunits (1). The
-chains each contain a conserved
ITAM within their cytoplasmic tails and mediate the signal trans-
duction of this receptor (1– 4).
It is generally accepted that upon FcRI cross-linking, the
-chain-associated Src family tyrosine kinase (PTK)
comes activated and phosphorylates the
Phosphorylated ITAMs in the
-chain recruit and activate another
key PTK, Syk, which ultimately triggers various mast cell re-
sponses (1– 6). Syk is broadly distributed throught hemopoietic
lineages, and it is also found in endothelial, epithelial, and other
cell types (7). In hemopoietic cells, Syk is recruited not only to the
activated FcRI but also to activated Fc
Rs, BCRs, TCRs, and
platelet receptors (8).
To ensure that mast cells are not inappropriately activated, sig-
naling pathways downstream of the FcRI are subjected to multi-
ple levels of positive and negative regulation (1– 6).
Recent studies have identiﬁed a new class of negative regula-
tors, namely Cbl family ubiquitin (Ub) ligases that control the
intensity and duration of receptor-generated signals by speciﬁc Ub
modiﬁcation of activated receptors, associated PTKs, and down-
stream signaling proteins (9 –11). Ubiquitination is a posttransla-
tional modiﬁcation whereby Ub, a small and highly conserved pep-
tide, is bound to target proteins through the action of Ub ligases
(E3 enzymes; Ref. 12). Polyubiquitination, a modiﬁcation in which
a chain of Ub is added to the substrate, drives targeting for pro-
teasomal degradation (12, 13).
We and others have demonstrated that c-Cbl is the E3 Ub ligase
responsible for the ubiquitination of different immunoreceptor sub-
units, including the TCR
-chain and the FcRI
and have suggested a role for this modiﬁcation in receptor down-
modulation (14 –16).
Moreover, we have demonstrated that Cbl-mediated ubiquiti-
nation of Syk on mast cells is responsible for targeting activated
Department of Experimental Medicine, Institute Pasteur-Fondazione Cenci Bolo-
gnetti, University “La Sapienza,” Rome, Italy
Received for publication September 19, 2006. Accepted for publication June 1, 2007.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
This work was partially supported by grants from Italian Association for Cancer
Research, Ministero dell’Istruzione, dell’Universita` e della Ricerca and the Centre of
Excellence in Molecular Biology and Medicine.
G.P. and R.M. contributed equally to this work.
Current address: Department of Histology and Medical Embryology, University “La
Sapienza,” Rome, Italy.
Address correspondence and reprint requests to Dr. Rossella Paolini, Department of
Experimental Medicine, University “La Sapienza,” Viale Regina Elena 324, Rome,
Italy. E-mail address: email@example.com
Abbreviations used in this paper: PTK, protein tyrosine kinase; anti-pTyr, anti-
phosphotyrosine; Ub, ubiquitin; CIN85, Cbl-interacting 85-kDa protein; SH3, Src
homology 3; WT, wild type; PLC, phospholipase C; HSA, human serum albumin;
, intracellular calcium ion concentration; RT-Q-PCR, real-time quantitative
PCR; siRNA, small interfering RNA; PCc, C-terminal proline-rich and coiled coil;
Sts, suppressor of TCR signaling.
Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00
The Journal of Immunology
by guest on June 13, 2013http://www.jimmunol.org/Downloaded from
Syk to the proteasome for degradation, thus providing another
molecular mechanism for attenuating FcRI-mediated positive
More recently, we have shown that Cbl could promote FcRI
internalization via a pathway that is functionally separable from its
Ub ligase activity and is dependent on Cbl interaction with a mul-
tidomain protein, Cbl-interacting 85-kDa protein (CIN85; Ref. 17).
CIN85 is a member of a newly discovered subfamily of broadly
expressed adaptor proteins that share the presence of several do-
mains able to promote multiple protein-protein interactions (18 –
20). CIN85 binding to Cbl is mediated by its Src homology 3
(SH3) domains and is largely dependent on the tyrosine phosphor-
ylation of Cbl, whereas the proline-rich region of CIN85 acts as an
interaction module for additional SH3 domain-containing proteins
(21, 22). We have generated transfectants stably overexpressing
CIN85 using the RBL-2H3 rat mast cell line, and demonstrated
that CIN85 overexpression accelerates the redistribution of en-
gaged receptor complexes, their sorting in early endosomes, and
their delivery to a lysosomal compartment for degradation (17).
RBL transfectants were also impaired in their ability to degranu-
late after Ag stimulation, suggesting that the accelerated down-
regulation of activated receptors contributes to dampen the func-
The purpose of the present study was to further evaluate the
function of CIN85 as a negative regulator of FcRI-mediated de-
granulation. In particular, we analyzed whether exogenous CIN85
overexpression could affect the activity and/or expression of mol-
ecules directly implicated in Ag-mediated signaling.
We found that wild-type (WT) CIN85 overexpression reduces
Syk protein levels, thus affecting the FcRI-mediated functional
responses. Our results support previous evidence for proteasome-
dependent pathways in the regulation of Syk tyrosine kinase ex-
pression (16, 23–25) and provide a mechanism for this regulation
involving the action of CIN85 and Cbl proteins.
Materials and Methods
Chemical reagents and Abs
All chemical and drugs were obtained from Sigma-Aldrich, unless other-
The rabbit polyclonal anti-CIN85 (raised against the C terminus), anti-
suppressor of TCR signaling (Sts) 1 and anti-Sts2 Abs were a gift from Dr.
I. Dikic (Goethe University Medical School, Frankfurt, Germany); the
mouse monoclonal anti-FcRI
-chain (BC4) was purchased from BD
Biosciences; the mouse monoclonal anti-CIN85 (clone 84) and anti-
phosphotyrosine (anti-pTyr) 4G10 Abs were purchased from UBI; rabbit
anti-Cbl C-15, anti-Syk N-19, anti-Lyn 44, anti-phospholipase C (PLC)
530, and anti-PLC
2 Q-20 polyclonal Abs, and the anti-Fyn 15 mAb were
purchased from Santa Cruz Biotechnology; anti-FLAG M2 and anti-
actin AC15 mAbs, and monoclonal anti-DNP-speciﬁc mouse IgE (clone
SPE-7) were purchased from Sigma-Aldrich. The proteasome inhibitors
epoxomicin and PI-116 and the mouse monoclonal anti-Ub FK2 (PW8810)
were purchased from Afﬁnity Research Products. G418 was from Invitro-
gen Life Technologies. Fluo 3-AM and Pluronic F-127 were obtained from
Molecular Probes. Rabbit reticulocyte lysates (L415/1-3) were purchased
Cell culture and stimulation
The RBL-2H3 mast cell line was cultured in monolayers as described pre-
viously (14). The Syk-negative variant of RBL-2H3 cells was kindly pro-
vided by Drs. J. Zhang and R. P. Siraganian (National Institutes of Health,
Bethesda, MD; Ref. 16).
Stable transfectants overexpressing FLAG-tagged human WT CIN85 or
CIN85-C-terminal proline-rich and coiled coil (PCc) mutant were gener-
ated as described previously (17), established as polyclonal cell lines by
culture in the presence of 700
g/ml G418 (Invitrogen Life Technologies),
and used in all the experiments presented. Transfected cell clones were also
generated by limiting dilution.
Adherent cells were incubated with 0.5
g/ml monomeric anti-DNP
mouse IgE for 12 h at 37°C. The cells were then harvested, resuspended at
/ml in prewarmed EMEM, and stimulated by adding DNP coupled to
human serum albumin (HSA; l
g/ml) for the indicated lengths of time.
Stimulation was stopped on ice by addition of cold PBS, and cells (25 ⫻
/ml) were lysed in a buffer (pH 8) containing 0.5% Triton-X-100, 200
mM boric acid, 160 mM NaCl, 5 mM EDTA, 1 mM PMSF, 1 mM
,50mMNaF,5mMN-ethylmaleimide, and 5
g/ml each of
aprotinin, leupeptin, and pepstatin as previously described (16). Lysates
were cleared of debris by centrifugation at 15,000 ⫻ g for 20 min; the
protein concentration was determined using the Bradford protein assay
(Bio-Rad) with BSA (Amresco) as standard, and the normalized samples
were used as whole cell lysates or for immunoprecipitation.
For experiments requiring inhibition of proteasome degradation, cells
were pretreated with 10
M epoxomicin or 25
M PI-116 for 8 or 12 h as
speciﬁed, washed in cold PBS, and directly lysed in hot Laemmli buffer (75
mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, and 1% 2-ME).
Immunoprecipitation, electrophoresis, and immunoblotting
For immunoprecipitation, postnuclear supernatants were ﬁrst precleared by
mixing with protein G- (Sigma-Aldrich), or protein A-Sepharose beads
(Amersham Pharmacia Biotech Italia) for1hat4°Candthen immuno-
precipitated with the indicated Abs prebound to protein G- or protein A-
Sepharose beads. After gentle rotation at 4°C for 2–12 h, the beads were
washed ﬁve times with lysis buffer, and bound proteins were eluted with
Laemmli buffer, resolved by SDS-PAGE on precasted minigels (7.5 or
10% Tris-HCl; Bio-Rad), and transferred electrophoretically to nitrocellu-
lose ﬁlters. After blocking nonspeciﬁc reactivity, ﬁlters were probed with
speciﬁc Abs diluted in 20 mM Tris-HCl pH 8, 150 mM NaCl and 0.05%
Tween 20 (TBS-T). After extensive washing in TBS-T, the membranes
were incubated with HRP-labeled goat anti-mouse Ig or goat anti-rabbit Ig
Abs (Amersham Biosciences), and immunoreactivity was visualized by
using the ECL system (Amersham Biosciences).
Densitometric analysis of the ﬁlms was performed using the NIH Image
RBL-2H3 cells were washed once in RPMI 1640 containing 1% FCS. This
medium was used during the entire procedure. The cells (20 ⫻ 10
were loaded with 7
M Fluo 3-AM and 1
g/ml Pluronic F-127 in the dark
for 45 min at 37°C and 5% CO
. After two washes, cells were resuspended
at the concentration of 20 ⫻ 10
/ml. Aliquots of 1 ⫻ 10
warmed to 37°C for 5 min, stimulated by adding 0.5
g of BC4, and
immediately analyzed by ﬂow cytometry with a FACScan (FACSCalibur;
BD Biosciences). The green ﬂuorescence emission was measured on a
logarithmic scale every 3 s for kinetic study as indicated. Unstimulated
cells were analyzed for 2 min to establish baseline ﬂuorescence levels.
Calibration procedure to convert arbitrary ﬂuorescence units into abso-
was performed by the method of Kao et al. (26), using the
[(F ⫺ F
⫺ F)]. K
⫽ 400 nM represents
the dissociation constant for Ca
-bound Fluo 3.
was obtained by rendering the cells permeable to Ca
-containing medium with 5
g/ml ionomycin (Sigma-Aldrich). To
, 2 mM MnCl
was added to ionomycin-treated cells. Mn
from Fluo-3, forming a complex one-ﬁfth as ﬂuorescent as
-Fluo-3 complex. Therefore, F
is calculated as follows: F
)] ⫻ 1.25.
mRNA expression analysis
Total RNA was isolated with the RNeasy Mini Kit (Qiagen). Two micro-
grams of total RNA were reverse transcribed with murine leukemia virus
reverse transcriptase and random hexamers (Applied Biosystems). Rat Syk
mRNA expression was analyzed by real-time quantitative PCR (RT-Q-
PCR) using a commercial TaqMan assay reagent (Applied Biosystems).
The endogenous gene rat
-microglobulin was ampliﬁed using a com
mercial TaqMan assay reagent (Applied Biosystem