of June 13, 2013.
This information is current as
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; ;
, 17 of which you can access for free at:
cites 50 articles
is online at:
The Journal of Immunology
Information about subscribing to
Submit copyright permission requests at:
Receive free email-alerts when new articles cite this article. Sign up at:
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Immunologists All rights reserved.
Copyright © 2007 by The American Association of
9650 Rockville Pike, Bethesda, MD 20814-3994.
The American Association of Immunologists, Inc.,
is published twice each month by
The Journal of Immunology
by guest on June 13, 2013
The Adaptor Molecule CIN85 Regulates Syk Tyrosine Kinase
Level by Activating the Ubiquitin-Proteasome Degradation
Giovanna Peruzzi,2Rosa Molfetta,2Francesca Gasparrini, Laura Vian,3Stefania Morrone,
Mario Piccoli, Luigi Frati, Angela Santoni, and Rossella Paolini4
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?RI-
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?RI-induced tyrosine
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.
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
Fc?RI is composed by an IgE-binding ?-chain, a four-trans-
membrane-spanning ? subunit, and two identical disulfide-linked
? subunits (1). The ?- and ?-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 Fc?RI cross-linking, the
?-chain-associated Src family tyrosine kinase (PTK)5Lyn be-
timulation of mast cells by the aggregation of the high-
affinity receptor for IgE, Fc?RI, initiates a cascade of
biochemical events that result in degranulation and re-
comes activated and phosphorylates the ?- and ?-chain ITAMs.
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 Fc?RI 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 Fc?RI 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 Fc?RI ?- and ?-chains,
and have suggested a role for this modification in receptor down-
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.
1This 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.
2G.P. and R.M. contributed equally to this work.
3Current address: Department of Histology and Medical Embryology, University “La
Sapienza,” Rome, Italy.
4Address 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
5Abbreviations 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;
[Ca2?]i, 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, 2013
Syk to the proteasome for degradation, thus providing another
molecular mechanism for attenuating Fc?RI-mediated positive
More recently, we have shown that Cbl could promote Fc?RI
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 Fc?RI-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 Fc?RI-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-Fc?RI ?-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)?1
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
107/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 ?
106/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
Na3VO4, 50 mM NaF, 5 mM N-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) for 1 h at 4°C and then 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 ? 106/ml)
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% CO2. After two washes, cells were resuspended
at the concentration of 20 ? 106/ml. Aliquots of 1 ? 106cells were
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-
lute [Ca2?]iwas performed by the method of Kao et al. (26), using the
formula [Ca2?]i? Kd[(F ? Fmin)/(Fmax? F)]. Kd? 400 nM represents
the dissociation constant for Ca2?-bound Fluo 3.
Fmaxwas obtained by rendering the cells permeable to Ca2?in 1 mM
Ca2?-containing medium with 5 ?g/ml ionomycin (Sigma-Aldrich). To
obtain Fmin, 2 mM MnCl2was added to ionomycin-treated cells. Mn2?
displaces Ca2?from Fluo-3, forming a complex one-fifth as fluorescent as
the Ca2?-Fluo-3 complex. Therefore, Fminis calculated as follows: Fmin?
[Fmax? (Fmax? FMnCl2)] ? 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 ?2-microglobulin was amplified using a com-
mercial TaqMan assay reagent (Applied Biosystems).
PCR were performed on an ABI Prism 7700 Sequence Detection System
(Applied Biosystems) according to the manufacturer’s instructions.
For each amplification run, a standard curve was generated using five
serial dilutions of total cDNA. All amplification reactions were performed
in triplicate, and the averages of the threshold cycles were used to inter-
polate standard curves and to calculate the transcript amount in samples
using SDS version 1.7a software (Applied Biosystems).
Relative Syk mRNA amount of each transfectant, normalized with ?2-
microglobulin, was expressed as arbitrary units and referred to empty vec-
tor-transfected cells considered as calibrator.
2090DOWN-REGULATION OF Syk EXPRESSION BY CIN85
by guest on June 13, 2013
In vitro ubiquitination assay
Cells (5 ? 107/ml) were lysed in a buffer (pH 8) containing 1% Triton-
X-100, 0.1% SDS, 200 mM boric acid, 160 mM NaCl, 5 mM EDTA, 1 mM
PMSF, and 5 ?g/ml each of aprotinin, leupeptin, and pepstatin as previ-
ously described (27). c-Cbl was immunoprecipitated from cells transfected
with empty vector or with WT CIN85 and used as E3 ligase; Syk was
immunoprecipitated from untransfected RBL-2H3 cells and used as sub-
strate. The immunoprecipitates were washed separately five times with
lysis buffer and then mixed before performing the assay. After an addi-
tional wash with 1? ubiquitination buffer (50 mM Tris (pH 7.5), 0.5 mM
MgCl2, 0.1 mM ATP, 0.1 mM DTT, 1 mM creatine phosphate), the beads
were incubated in 40 ?l of the same buffer supplemented with 70% (v/v)
rabbit reticulocyte lysates, 10 U of creatine phosphokinase, and 10 ?g of
Ub for 2 h at 30°C. The samples were washed three times with lysis buffer,
eluted with SDS-sample buffer, resolved by SDS-PAGE, and transferred
electrophoretically to nitrocellulose filters.
Small interfering RNA (siRNA)
CIN85 siRNAs (siGenome SMART pool rat CIN85 (L-080145-01), a mix-
ture of four different siRNAs, those that proved to be theoretically and/or
empirically effective in gene knockdown) and a control siRNA (siCON
TROL NON-Targeting siRNA#2, 5?-UAAGGCUAUGAAGAGAUACU
UTT-3?) were purchased from Dharmacon. siRNA duplexes were resus-
pended at 100 ?M in 1? siRNA Universal Buffer.
CIN85 knockdown was achieved by transfecting RBL-2H3 cells with
CIN85 siRNA duplexes. The transfection was performed by electropora-
tion (310 V, 960 ?F) incubating 10 ? 106cells with 2.5 ?M siRNA in 500
?l of serum-free MEM. Controls included mock transfection in the absence
of siRNA as well as using the nontargeting siRNA.
After 24 and 48 h, total RNA was isolated with RNeasy Mini Kit (Qia-
gen), and CIN85 mRNA expression was analyzed by RT-Q-PCR using a
commercial TaqMan assay reagent (Applied Biosystems), as above
After 48 h, the cells were harvested, and cell extracts were processed on
Western blots or used for immunoprecipitation experiments.
CIN85 overexpression inhibits the Fc?RI-induced increase in
intracellular calcium and PLC? tyrosine phosphorylation
Fc?RI-mediated activation of mast cells results in the release of
preformed mediators from cytoplasmic granules (3, 5). We have
previously observed in RBL-2H3 cells a substantial inhibition of
multivalent Ag-induced degranulation by overexpression of WT
but not mutant forms of CIN85 interfering with membrane recep-
tor endocytic processes (17). Similar results were also obtained
upon stimulation of RBL cells with an anti-Fc?RI ?-chain mAb
(BC4) (data not shown).
Mast cell degranulation requires a calcium response that in-
volves both the release of calcium from intracellular stores and
calcium influx from the medium through channels in the plasma
membrane (3–6). Therefore, we analyzed the Fc?RI-induced tran-
sient rise in free intracellular [Ca2?]iconcentrations in cells over-
expressing CIN85. Cells were labeled with Fluo-3 and subjected to
Fc?RI clustering by addition of the anti-Fc?RI ?-chain BC4 mAb.
The induced [Ca2?]ichanges were monitored by flow cytometry.
The rapid response to BC4 was suppressed by 40% after overex-
pression of the WT, but not a mutant form of CIN85 only con-
taining the PCc domain (Fig. 1, A and B). A similar result was
obtained when RBL-2H3 clones generated by limiting dilution
from the polyclonal population of CIN85 transfectants were ana-
lyzed (data not shown).
Stimulation of all transfectants with ionomycin resulted in com-
parable levels of Ca2?mobilization, indicating that they were able
to mobilize Ca2?to similar extents (data not shown). These results
suggest that overexpression of CIN85 affects an early stage of
Fc?RI-induced cell activation.
The aggregation of Fc?RI results in tyrosine phosphorylation
and activation of both PLC?1 and PLC?2, which generate inositol
1,4,5-triphosphate that in turn mediates the increase in intracellular
calcium (6). To compare the phosphorylation status of PLC? in the
different CIN85 transfectants, adherent cells were incubated over-
night with anti-DNP IgE mAb and stimulated (or not) with the
multivalent Ag DNP-HSA for 1 min. Cell lysates were subjected
to immunoprecipitation with anti-PLC? Abs, separated by SDS-
PAGE, and analyzed by immunoblotting with anti-pTyr mAb. As
a consequence of WT CIN85 overexpression, the extent of tyrosine
phosphorylation of both PLC?1 and PLC?2 upon receptor engage-
ment was lower than in cells transfected with the empty vector, or
the mutant form of CIN85 (Fig. 1C). The membranes were re-
probed for PLC?1 and PLC?2, respectively, to verify an equal
loading of proteins. These results suggest that CIN85-mediated
inhibition of PLC? phosphorylation lowers inositol 1,4,5-triphos-
phate production and hence reduces the amplitude of the transient
PLC? tyrosine phosphorylation. A, RBL-2H3 cells transfected with empty
vector or with constructs for FLAG-tagged WT CIN85 or CIN85-PCc mu-
tant were loaded with Fluo-3 and stimulated with an anti-Fc?RI ?-chain
mAb (BC4; 0.5 ?g/106cells). Kinetic analysis of [Ca2?]iwas performed as
described in Materials and Methods. B, The histogram represents the
means ? SD of maximum calcium concentrations released upon BC4 stim-
ulation calculated as described in Materials and Methods from three inde-
pendent experiments performed as in A. C, RBL-2H3 transfectants were
loaded with anti-DNP IgE and stimulated or not with 1 ?g/ml DNP-HSA
(Ag) for 1 min at 37°C. Cell lysates (3 ? 107/sample) were immunopre-
cipitated with anti-PLC?1 or anti-PLC?2 polyclonal Ab, resolved by 7.5%
SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-
pTyr mAb. Anti-PLC?1 and anti-PLC?2 blots were used to verify equal
protein loading. The results represent one of three independent
CIN85 inhibits Ag-induced intracellular calcium flux and
2091The Journal of Immunology
by guest on June 13, 2013
Overexpression of CIN85 affects the expression level of Syk
The decrease of Ag-induced PLC? tyrosine phosphorylation ob-
served after WT CIN85 overexpression suggests a possible al-
teration in activity and/or expression of PTKs, and in particular
of Syk which is required for both PLC?1 and PLC?2 activation
To determine the phosphorylation status of this kinase in RBL-
2H3 transfectants, cell lysates obtained before and after Fc?RI
stimulation were immunoprecipitated with anti-Syk Ab and the
immunoprecipitates were resolved by SDS-PAGE, transferred to
nitrocellulose and immunoblotted as indicated (Fig. 2, A–C). The
level of Syk tyrosine phosphorylation was markedly reduced upon
WT CIN85 overexpression; however, the amount of Syk precipi-
tated from cells overexpressing WT CIN85 was much lower than
that in control cells (Fig. 2A). This decrease was already observed
in absence of receptor stimulation, suggesting that overexpression
of WT CIN85 was implicated in the down-regulation of Syk pro-
Therefore, we extended our analysis comparing the basal level
of Syk in the cell lysates obtained by the different transfectants.
The immunoblotting with the anti-CIN85 mAb that detects both
the FLAG-tagged human CIN85 overexpressed forms and the en-
dogenous rat CIN85 isoform demonstrated a 5-fold increase of
CIN85 expression, whereas the immunoblotting with the anti-
FLAG mAb showed a comparable level of overexpressed CIN85
proteins (Fig. 2B). A decrease of Syk expression (?70%) was
observed only in the transfectants overexpressing WT CIN85
when compared with cells transfected with empty vector or the
mutant form of CIN85 (Fig. 2C). The same membranes were se-
quentially probed with Abs specific to other molecules known to
be involved in the Fc?RI-induced secretory response. The expres-
sion levels of none of them were affected (Fig. 2C and data not
shown). Similar results were also obtained when RBL-2H3-trans-
fected clones were analyzed (data not shown).
We next examined the effect of CIN85 overexpression on Syk
mRNA levels. We compare the Syk mRNA amount in the different
transfectants by RT-Q-PCR using a Syk-deficient RBL-2H3 clone
as negative control. The results showed that Syk mRNA was
slightly decreased in the presence of WT CIN85 overexpression
and increased when PCc mutant was expressed (Fig. 2D). These
alterations do not correlate with the amount of Syk protein levels.
The substantial decrease in Syk protein level observed upon WT
CIN85 overexpression suggests that a posttranslational mechanism
is involved in Syk degradation.
Proteasome inhibitors restore Syk protein levels in
Work by many research groups including our own, implicates pro-
teasome-dependent mechanisms in the regulation of Syk tyrosine
kinase expression levels in both resting and activated human ba-
sophils and RBL-2H3 cells (16, 24, 25). To investigate whether
CIN85 overexpression could affect the steady state protein level of
Syk by promoting proteasome degradation, cells were treated with
cell-permeable protease inhibitors or with a corresponding volume
of the vehicle DMSO as control and analyzed after 8 h for the
expression of Syk by Western blotting on whole cell lysates. In-
cubation with epoxomicin, a selective and irreversible inhibitor of
the proteasome proteolytic activities, restores Syk protein expres-
sion in the transfectants overexpressing WT CIN85 (Fig. 3A).
Syk. A, RBL-2H3 transfectants were loaded with anti-DNP IgE and stim-
ulated or not with 1 ?g/ml DNP-HSA (Ag) for 1 min at 37°C. Cell lysates
(3 ? 107/sample) were immunoprecipitated with anti-Syk polyclonal Ab,
resolved by 7.5% SDS-PAGE, transferred to nitrocellulose and immuno-
blotted with anti-pTyr and anti-Syk Abs, as indicated. The position of m.w.
markers is indicated on the left. B and C, Total cell lysates (TCL) from
each transfectant were resolved by 10% SDS-PAGE, transferred to nitro-
cellulose and immunoblotted with the indicated Abs. The position of m.w.
markers is indicated. Results are representative of one of four independent
experiments. D, Two micrograms of total RNA obtained from the different
transfectants were reverse transcribed, and rat Syk mRNA expression was
analyzed by RT-Q-PCR. The endogenous gene rat ?2-microglobulin was
also amplified to normalize each sample. A Syk-deficient cell line (Syk?)
was used as negative control. Relative Syk mRNA amount of each trans-
fectant, normalized with ?2-microglobulin, was expressed as arbitrary units
and referred to empty vector-transfected cells considered as calibrator.
Data are expressed as the mean ? SD obtained from three independent
CIN85 regulates the expression level of the tyrosine kinase
cells. RBL-2H3 cells transfected with empty vector or WT CIN85 were
pretreated for 8 h with DMSO (control), 10 ?M epoxomicin or 25 ?M
PI-116 (A), and for 12 h with DMSO (control) or 25 ?M PI-116 (B). Cells
were then directly lysed with hot Laemmli buffer, and total cell lysates
were resolved by SDS-PAGE, transferred to nitrocellulose, and immuno-
blotted with anti-Syk Ab (top). The membranes were stripped and reprobed
with anti-actin (bottom) to verify equal protein loading. Results are repre-
sentative of one of three independent experiments.
Syk proteasome degradation in CIN85-overexpressing
2092 DOWN-REGULATION OF Syk EXPRESSION BY CIN85
by guest on June 13, 2013
Another specific proteasome inhibitor, PI-116, caused only a mod-
est increase of Syk protein level. However, when the pretreatment
in the presence of the last inhibitor was prolonged (12 h), a total
restoration of Syk expression was induced (Fig. 3B). Caspase and
calpain inhibitors as well as ammonium chloride known to inhibit
lysosome function had no detectable effects on Syk levels (data not
shown). The membranes were reprobed for actin to verify an equal
loading of proteins (Fig. 3, bottom). These results suggest that
overexpression of CIN85 affects the expression level of Syk
mainly by promoting its proteasome degradation.
CIN85 overexpression increases c-Cbl ligase activity and
promotes a constitutive c-Cbl/CIN85 association
The finding that CIN85 exerts a regulatory effect on Syk ex-
pression by promoting its proteasome-dependent degradation
prompted us to analyze whether CIN85 overexpression can ac-
tivate ligase(s) able to ubiquitinate Syk.
We decided to focus our attention on c-Cbl, because we have
previously demonstrated that it acts as E3 ligase mediating Ag-
induced ubiquitination of Syk on RBL-2H3 cells (16).
We have compared c-Cbl ligase activity in empty vector and
WT CIN85-transfected cells by immunoprecipitating the enzyme
from total cell extracts and performing an in vitro ubiquitination
assay (Fig. 4A). We have used rabbit reticulocyte lysates as source
of E1 and E2 enzymes and Syk immunoprecipitated from unstimu-
lated RBL-2H3 cells as substrate, because we could never detect in
vivo Syk ubiquitination in resting cells (16).
Immunoblotting with anti-Syk and anti-Ub Abs revealed the
presence of Syk molecular species modified by a few Ub mole-
cules together with a smear in the high-m.w. region, characteristic
of polyubiquitination. An increase of Syk ubiquitination was ob-
served when c-Cbl ligase was immunoprecipitated from WT
CIN85-overexpressed cell lysates (Fig. 4; compare lanes 4 and 6).
Anti-Cbl (lane 1) or a mix of anti-Cbl and anti-Syk (lane 2) protein
A-Sepharose conjugated beads were used in the absence of cell
extracts as negative control. This result demonstrates that CIN85
overexpression increases c-Cbl E3 ligase activity promoting a
more robust in vitro Syk ubiquitination.
To investigate the mechanism that regulates c-Cbl ligase activ-
ity, we first decided to examine whether overexpression of CIN85
can affect the formation of c-Cbl/CIN85 complexes. We have pre-
viously reported the presence of constitutive c-Cbl/CIN85 com-
plexes in RBL-2H3 cells, and we have also demonstrated that the
level of c-Cbl/CIN85 association correlates with that of c-Cbl ty-
rosine phosphorylation induced upon Fc?RI engagement (17).
Cell lysates obtained before and after Fc?RI stimulation were
subjected to immunoprecipitation with anti-CIN85 mAb to precip-
itate the endogenous rat CIN85 or with anti-FLAG mAb to pre-
cipitate only the overexpressed FLAG-tagged forms of human
CIN85, separated by SDS-PAGE, and analyzed by immunoblot-
ting with anti-Cbl polyclonal Ab (Fig. 4B). An Ag-inducible as-
sociation of endogenous CIN85 with c-Cbl was observed in RBL-
2H3 cells transfected with empty vector, confirming our previous
finding (17). WT CIN85 overexpression caused the formation of
additional c-Cbl/CIN85 complexes in resting cells, and this asso-
ciation was increased upon receptor aggregation. The mutant form
of CIN85 (CIN85-PCc) failed to interact with endogenous c-Cbl,
confirming the requirement of CIN85 SH3 domains to bind Cbl.
Similar results were obtained when anti-Cbl immunoprecipitation
and anti-CIN85 immunoblotting was performed (data not shown).
These results demonstrate that CIN85 overexpression favors the
formation of additional c-Cbl/CIN85 complexes in resting cells.
CIN85 affects the formation of Cbl/Sts1 complexes
It has been recently suggested that the Cbl-interacting proteins
belonging to the Sts family, Sts1 and Sts2, act as modulators of
(5 ? 107/sample) transfected with empty vector (lanes 3 and 4) or with WT CIN85 (lanes 5 and 6) was used in an in vitro ubiquitination assay performed
as described in Materials and Methods. Syk immunoprecipitated from unstimulated RBL-2H3 cells was used as substrate (lanes 4 and 6). Protein
A-Sepharose beads conjugated with anti-Cbl (lane 1) or anti-Cbl and anti-Syk (lane 2) Abs were used in the absence of cell extracts as negative control.
After the reaction proteins were eluted and resolved by 7.5% SDS-PAGE, transferred to nitrocellulose, and immunoblotted with the indicated Abs. B,
RBL-2H3 cells transfected with empty vector, WT CIN85, or CIN85 PCc mutant were loaded with anti-DNP IgE and left stimulated or not with 1 ?g/ml
DNP-HSA (Ag) for 1 min at 37°C. Cell lysates (3 ? 107/sample) were immunoprecipitated with anti-CIN85 or anti-FLAG mAbs, resolved by 7.5%
SDS-PAGE, transferred to nitrocellulose and immunoblotted with anti-Cbl (top) and anti-CIN85 CT (bottom) polyclonal Abs. The position of m.w. markers
is indicated on the left. Results represent one of three independent experiments.
c-Cbl ligase activity and constitutive CIN85/Cbl complexes increase upon CIN85 overexpression. A, c-Cbl immunoprecipitated from cells
2093 The Journal of Immunology
by guest on June 13, 2013
biological responses elicited by TCR and receptor tyrosine kinases,
by regulating Cbl functions (29, 30). Interaction between Cbl and
Sts is independent on Cbl tyrosine phosphorylation and is medi-
ated by the SH3 domains of Sts binding to the proline-rich region
of Cbl (30). CIN85 is also composed of SH3 domains that are
involved in interaction with Cbl (19). Therefore, we investigated
whether exogenous overexpressed CIN85 could compete with Sts1
in c-Cbl binding.
To analyze the presence of Sts proteins in mast cells, lysates
from RBL-2H3 cells were immunoprecipitated with anti-Sts1 or
anti-Sts2 Abs or normal rabbit serum as control. Immonoblotting
revealed the presence of a 70-kDa specific form detected after
anti-Sts1 but not anti-Sts2 immunoprecipitation and on total cell
lysates (Fig. 5A). To investigate whether Sts1 could interact with
c-Cbl, lysates obtained from cells transfected with empty vector or
CIN85 proteins were subjected to immunoprecipitation with a rab-
bit anti-Cbl polyclonal Ab, separated by SDS-PAGE, and analyzed
by immunoblotting with anti-Sts1 Ab (Fig. 5B, right). We found
that Sts1 constitutively interacts with c-Cbl on RBL-2H3 cells
(data not shown) and on cells transfected with empty vector. Fol-
lowing overexpression of WT CIN85, we observed a decrease of
c-Cbl/Sts1 complexes, whereas the mutant form of CIN85 un-
able to bind Cbl did not alter the c-Cbl/Sts1 complex formation
(Fig. 5, B and C).
The expression level of Sts1 was not affected by CIN85 over-
expression (Fig. 5B, left). These results suggest that overexpressed
CIN85 competes with endogenous Sts1 for binding to c-Cbl.
We next assessed the role of endogenous CIN85 in limiting the
formation of c-Cbl/Sts1 complexes by performing siRNA-medi-
ated knockdown of CIN85 expression. We found that CIN85 pro-
tein expression cannot be completely suppressed in RBL-2H3 cells
(we reproducibly observed ?60% inhibition; Fig. 6A). However,
siRNA-mediated reduction of CIN85 increased the amount of c-
Cbl/Sts1 complexes (Fig. 6B), suggesting that endogenous CIN85
can compete with Sts1 to bind c-Cbl.
Because of its identification as a Cbl-interacting protein (18),
CIN85 has been found to interact with several adaptor molecules
mainly implicated in the process of endocytosis and vesicle traf-
ficking, which is the central mechanism for receptor down-regu-
lation (21, 22, 31–33).
In this respect, we have recently proposed a role for CIN85 in
controlling the clearence of Fc?RI engaged receptor complexes
from the cell surface of mast cells, thus providing a mechanism to
attenuate the intracellular signaling initiated by IgE receptors (17).
More recent evidence suggest that in addition to promote clath-
rin-mediated receptor internalization, CIN85 can also regulate the
activity of several enzymes responsible for signal propagation
The impairment in Ca2?mobilization and PLC? tyrosine phos-
phorylation observed upon stable overexpression of WT CIN85 in
RBL-2H3 cells (Fig. 1) strongly indicates that CIN85 interferes
with early signaling components in Fc?RI signal transduction. In-
deed, we found a reduction of Syk expression level in WT CIN85
overexpressing cells when compared with control cells (Fig. 2).
In a rodent model, the use of Syk-specific inhibitors and Syk-
negative mast cell lines has demonstrated an obligatory role for
this kinase in Fc?RI-mediated signaling (28, 38–41). In humans,
inhibited upon CIN85 overexpression. A, Cell lysates
from RBL-2H3 cells (3 ? 107/sample) were immuno-
precipitated with anti-Sts1 or anti-Sts2 polyclonal Abs,
resolved by 7.5% SDS-PAGE, transferred to nitrocellu-
lose, and immunoblotted with the indicated Abs. Nor-
mal rabbit serum (NRS) was used as negative control.
Total cell lysates (TCL) were also loaded (80 ?g) as a
positive control. The position of m.w. markers is indi-
cated. B, Lysates obtained from the different RBL-2H3
transfectants (3 ? 107/sample) were immunoprecipi-
tated with anti-Cbl polyclonal Ab, resolved by 7.5%
SDS-PAGE, and immunoblotted with anti-Sts1 and anti-
Cbl Abs (right). TCL were immunoblotted with anti-
Sts1 Ab to verify the presence of equal amount of pro-
tein in the different transfectants (left). C, Results
represent the mean ? SD of three independent experi-
ments performed as in B. The amount of Sts1 protein
associated with c-Cbl, expressed as arbitrary units, was
normalized with the band intensity of c-Cbl and referred
to empty vector.
The formation of Cbl/Sts1 complex is
siRNA-mediated knockdown of CIN85. A, Total cell lysates (TCL) derived
from untreated RBL-2H3 cells and cells transfected with control (Ctrl)
siRNA or with CIN85 siRNA as described in Material and Methods were
immunoblotted with anti-CIN85 and anti-actin mAbs. B, Lysates obtained
from RBL-2H3 cells (5 ? 107/sample) treated as in A were immunopre-
cipitated with anti-Cbl polyclonal Ab, resolved by 7.5% SDS-PAGE and
immunoblotted with anti-Sts1 and anti-Cbl Abs. The relative Sts1 protein
amount, normalized with the band intensity of c-Cbl, was referred to the
untreated sample and indicated at the bottom. Results are representative of
one of three independent experiments.
The formation of Cbl/Sts1 complexes is enhanced upon
2094 DOWN-REGULATION OF Syk EXPRESSION BY CIN85
by guest on June 13, 2013
a minority of normal blood donors contain basophils that fail to
degranulate, and these nonreleaser basophils express normal level
of Fc?RI but contain very low levels of Syk protein (42, 43). Thus,
it is very likely that the reduction of Syk protein level observed
upon CIN85 overexpression may account for the impairment of
Fc?RI-induced functional responses.
Despite the basal low level of Syk present in CIN85-overex-
pressing cells, the kinase is tyrosine phosphorylated upon receptor
engagement (Fig. 2A). This result suggests that the enzymes acting
upstream to Syk are not affected by CIN85 overexpression. In
support of this conclusion, we found no alteration in the expression
level of Lyn and Fyn (Fig. 2C). Furthermore, the ligand-induced
tyrosine phosphorylation of Fc?RI subunits was not affected by
CIN85 overexpression (data not shown), indicating a normal ac-
tivity of Lyn.
After CIN85 overexpression, we have observed an alteration of
Syk mRNA levels that does not correlate with the strong reduction
of Syk protein levels (Fig. 2; compare D and C), evocating the
action of a posttranslational mechanism mainly responsible for
Evidence from several laboratories has demonstrated that Syk is
highly susceptible to the Ub proteasome-mediated proteolysis in
both resting and activated hemopoietic cell types (16, 24, 25, 27).
In the present investigation, we have found that proteasome inhib-
itors restored Syk expression in CIN85-overexpressing cells (Fig.
3), strongly implicating the Ub-proteasome pathway in the regu-
lation of Syk stability. However, we fail to observe a concurrent
restoration of Fc?RI-induced degranulation (data not shown). The
explanation very likely lies in additional effect(s) of proteasome
inhibitors occurring upstream and/or downstream to Syk. Relevant
to this, Youssef et al. (24) have reported a dramatic impairment of
receptor phosphorylation on human basophils treated with protea-
some inhibitor I.
The Syk binding Ub ligase c-Cbl has been implicated in Syk
degradation both in RBL-2H3 cells and B cells (10, 16, 44). In
particular, we have demonstrated that upon Fc?RI engagement,
c-Cbl mediates Syk ubiquitination and marks the kinase for pro-
teasome degradation (16).
c-Cbl expression levels were not affected upon CIN85 overex-
pression (Fig. 2C); however, we found a more robust in vitro Syk
ubiquitination when c-Cbl was immunoprecipitated from cells
transfected with WT CIN85 than from control cells (Fig. 4A), sug-
gesting that c-Cbl ligase activity contributes to the instability of
Syk protein levels.
It has been recently demostrated that the ligase activity of Cbl
proteins can be negatively regulated by different families of scaf-
fold proteins, including Sts adaptors (45, 29, 30).
We have found that Sts1 constitutively associates with c-Cbl on
RBL-2H3 cells and that this association decreases upon CIN85
overexpression and increases after CIN85 knockdown (Figs. 5B
and 6B, respectively). Both Sts1 and CIN85 contain SH3 domains
directly involved in the interaction with c-Cbl proline-rich region
(20, 30); thus, it is likely that the two adaptors compete to bind
Cbl. In agreement with this hypothesis, we found an enhanced
formation of CIN85/Cbl complexes upon CIN85 overexpression
(Fig. 4B), likely affecting the action of Sts1 as negative regulator
of c-Cbl ligase activity.
Although CIN85 knockdown favors the formation of c-Cbl/Sts1
complexes, we were unable to appreciate any increase of Syk pro-
tein levels (data not shown). Thus, it remains possible that other
mechanisms operate to control Syk expression in resting RBL-2H3
cells. In this respect, Siegel et al. (46) have recently described a
new mechanism regulating Syk protein stability on B cells that
implicates a direct interaction between unphosphorylated forms of
Syk and the transcriptional factor OCA-B.
A second member of Cbl mammalian protein able to act as an
E3 ligase, namely Cbl-b, is also expressed on RBL-2H3 cells and
has been reported to act, together with c-Cbl, as a negative regu-
lator of mast cell functions (47, 48). We found that upon CIN85
overexpression Cbl-b can bind to CIN85 (data not shown), thus
likely implying also its contribution to the instability of Syk
The interaction between c-Cbl and CIN85 increases upon Fc?RI
engagement (Fig. 4B). Furthermore, as a consequence of WT
CIN85 overexpression, the Ag-induced decrease of Syk protein
level is greater than in cells expressing the empty vector or the
mutant form of CIN85 unable to bind Cbl (data not shown). This
result suggests that overexpressed CIN85 in addition to control the
basal level of Syk can also contribute to limit the Fc?RI-mediated
signal by accelerating the Ub-proteasome degradation of Syk in-
duced upon receptor engagement.
In summary, our finding supports a new role for CIN85 in reg-
ulating Syk protein levels in RBL-2H3 cells through the activation
of the Ub-proteasome pathway involving the action of c-Cbl.
A regulated expression of Syk protein has been previously re-
ported in several hemopoietic cells, including T and B cells (49,
50). We have already mentioned the case of the nonreleaser ba-
sophils that express normal level of Fc?RI but contain low levels
of Syk proteins compared with releaser basophils (42, 43). It is
important to verify in the future whether there is a correlation
between the expression level of CIN85 and the integrity of the
molecular machinery that regulates Syk stability.
We are grateful to Dr. I. Dikic for generous access to anti-CIN85 and
anti-Sts polyclonal Abs and to Drs. J. Zhang and R. P. Siraganian for
providing the Syk-deficient cell line. We thank Dr. E. Ferretti for assistance
and helpful advice with real-time PCR and the laboratory of Dr. G. Macino
for helpful advice with siRNA. We also thank P. Birarelli, A. Bressan, and
B. Milana for technical assistance and R. Centi Colella and P. Di Russo for
The authors have no financial conflict of interest.
1. Nadler, M. J., S. A. Matthews, H. Turner, and J. P. Kinet. 2000. Signal trans-
duction by the high-affinity immunoglobulin E receptor Fc?RI: coupling form to
function. Adv. Immunol. 76: 325–355.
2. Kawakami, T., and S. J. Galli. 2002. Regulation of mast-cell and basophil func-
tion and survival by IgE. Nat. Rev. Immunol. 2: 773–786.
3. Siraganian, R. P. 2003. Mast cell signal transduction from the high-affinity IgE
receptor. Curr. Opin. Immunol. 15: 639–646.
4. Yamasaki, S., and T. Saito. 2005. Regulation of mast cell activation through
Fc?RI. Chem. Immunol. Allergy 87: 22–31.
5. Gilfillan, A. M., and C. Tkaczyk. 2006. Integrated pathways for mast-cell acti-
vation. Nat. Rev. Immunol. 6: 218–230.
6. Rivera, J. 2002. Molecular adapters in Fc?RI and the allergic response. Curr.
Opin. Immunol. 14: 688–693.
7. Yanagi, S., R. Inatome, T. Takano, and H. Yamamura. 2001. Syk expression and
novel function in a wide variety of tissues. Biochem. Biophys. Res. Commun. 288:
8. Turner, M., E. Schweighoffer, F. Colucci, J. P. Di Santo, and V. L. Tybulewicz.
2000. Tyrosine kinase SYK: essential functions for immunoreceptor. Immunol.
Today 21: 148–154.
9. Thien, C. B., and W. Y. Langdon. 2001. Cbl: many adaptations to regulate protein
tyrosine kinases. Nat. Rev. Mol. Cell Biol. 2: 294–307.
10. Rao, N., I. Dodge, and H. Band. 2002. The Cbl family of ubiquitin ligases: critical
negative regulators of tyrosine kinase signalling in the immune system. J. Leu-
kocyte Biol. 71: 753–763.
11. Dikic, I., I. Szymkiewicz, and P. Soubeyran. 2003. Cbl signalling networks in the
regulation of cell function. Cell. Mol. Life Sci. 60: 1805–1827.
12. Hochstrasser, M. 2006. Lingering mysteries of ubiquitin-chain assembly. Cell
2095The Journal of Immunology
by guest on June 13, 2013
13. Ciechanover, A., A. Orian, and A. L. Schwartz. 2000. The ubiquitin-mediated Download full-text
proteolytic pathway: mode of action and clinical implications. J. Cell. Biochem.
Suppl. 34: 40–51.
14. Paolini, R., and J. P. Kinet. 1993. Cell surface control of the multiubiquitination
and deubiquitination of high-affinity immunoglobulin E receptors. EMBO J. 12:
15. Wang, H. Y., Y. Altman, D. Fang, C. Elly, Y. Dai, Y. Shao, and Y. C. Liu. 2001.
Cbl promotes ubiquitination of the T cell receptor ? through an adaptor function
of Zap-70. J. Biol. Chem. 276: 26004–26011.
16. Paolini, R., R. Molfetta, L. O. Beitz, J. Zhang, A. M. Scharenberg, M. Piccoli,
L. Frati, R. Siraganian, and A. Santoni. 2002. Activation of Syk tyrosine kinase
is required for c-Cbl-mediated ubiquitination of Fc?RI and Syk in RBL cells.
J. Biol. Chem. 277: 36940–36947.
17. Molfetta, R., F. Belleudi, G. Peruzzi, S. Morrone, L. Leone, I. Dikic, M. Piccoli,
L. Frati, M. R. Torrisi, A. Santoni, and R. Paolini. 2005. CIN85 regulates the
ligand-dependent endocytosis of the IgE receptor: a new molecular mechanism to
dampen mast cell function. J. Immunol. 175: 4208–4216.
18. Take, H., S. Watanabe, K. Takeda, Z. X. Yu, N. Iwata, and S. Kajigaya. 2000.
Cloning and characterization of a novel adaptor protein, CIN85, that interacts
with c-Cbl. Biochem. Biophys. Res. Commun. 268: 321–328.
19. Borinstein, S. C., M. A. Hyatt, V. W. Sykes, R. E. Straub, S. Lipkowitz,
J. Boulter, and O. Bogler. 2000. SETA is a multifunctional adapter protein with
three SH3 domains that binds Grb2, Cbl, and the novel SB1 proteins. Cell. Signal.
20. Dikic, I. 2002. CIN85/CMS family of adaptor molecules. FEBS Lett. 529:
21. Dikic. I., and S. Giordano. 2003. Negative receptor. Curr. Opin. Cell Biol. 15:
22. Kowanetz, K., K. Husnjak, D. Holler, M. Kowanetz, P. Soubeyran, D. Hirsch,
M. H. Schmidt, K. Pavelic, P. De Camilli, P. A. Randazzo, and I. Dikic. 2004.
CIN85 associates with multiple effectors controlling intracellular trafficking of
epidermal growth factor receptors. Mol. Biol. Cell. 15: 3155–3166.
23. Taniguchi, T., T. Kobayashi, J. Kondo, K. Takahashi, H. Nakamura, J. Suzuki,
K. Nagai, T. Yamada, S. Nakamura, and H. Yamamura. 1991. Molecular cloning
of a porcine gene syk that encodes a 72-kDa protein-tyrosine kinase showing high
susceptibility to proteolysis. J. Biol. Chem. 24: 15790–15796.
24. Youssef, L. A., B. S. Wilson, and J. M. Oliver. 2002. Proteasome-dependent
regulation of Syk tyrosine kinase levels in human basophils. J. Allergy Clin.
Immunol. 110: 366–373.
25. MacGlashan, D., and K. Miura. 2004. Loss of syk kinase during IgE-mediated
stimulation of human basophils. J. Allergy Clin. Immunol. 114: 1317–1324.
26. Kao, J. P. Y., A. T. Harootunian, and R. Y. Tsien. 1989. Photochemically gen-
erated cytosolic calcium pulses and their detection by fluo-3. J. Biol. Chem. 264:
27. Paolini, R., R. Molfetta, M. Piccoli, L. Frati, and A. Santoni. 2001. Ubiquitination
and degradation of Syk and ZAP-70 protein tyrosine kinases in human NK cells
upon CD16 engagement. Proc. Natl. Acad. Sci. USA 98: 9611–9616.
28. Siraganian, R. P., J. Zhang, K. Suzuki, and K. Sada. 2002. Protein tyrosine kinase
Syk in mast cell signalling. Mol. Immunol. 38: 1229–1233.
29. Carpino, N., S. Turner, D. Mekala, Y. Takahashi, H. Zang, T. L. Geiger, and
P. Doherty. 2004. Regulation of ZAP-70 activation and TCR signalling by two
related proteins, Sts-1 and Sts-2. Immunity 20: 37–46.
30. Kowanetz, K., N. Corsetto, K. Haglund, M. H. Schmidt, C. H. Heldin, and
I. Dikic. 2004. Suppressor of T-cell receptor signalling Sts-1 and Sts-2 bind to
Cbl and inhibit endocytosis of receptor tyrosine kinase. J. Biol. Chem. 279:
31. Soubeyran, P., K. Kowanetz, I. Szymkiewicz, W. Y. Langdon, and I. Dikic. 2002.
Cbl-CIN85-endophilin complex mediates ligand-induced downregulation of EGF
receptors. Nature 416: 183–187.
32. Petrelli, A., G. F. Gilestro, S. Lanzardo, P. M. Comoglio, N. Migone, and
S. Giordano. 2002. The endophilin-CIN85-Cbl complex mediates ligand-depen-
dent downregulation of c-Met. Nature 416: 187–190.
33. Szymkiewicz, I., K. Kowanetz, P. Soubeyran, A. Dinarina, S. Lipkowitz, and
I. Dikic. 2002. CIN85 participates in Cbl-b-mediated down-regulation of receptor
tyrosine kinases. J. Biol. Chem. 277: 39666–39672.
34. Gout, I., G. Middleton, J. Adu, N. N. Ninkina, L. B. Drobot, V. Filonenko,
G. Matsuka, A. M. Davies, M. Waterfield, and V. L. Buchman. 2000. Negative
regulation of PI 3-kinase by Ruk, a novel adaptor protein. EMBO J. 19:
35. Borthwick, E. B., I. V. Korobko, C. Luke, V. R. Drel, Y. Y. Fedyshyn,
N. Ninkina, L. B. Drobot, and V. L. Buchman. 2004. Multiple domains of Ruk/
CIN85/SETA/CD2BP3 are involved in interaction with p85? regulatory subunit
of PI3-kinase. J. Mol. Biol. 343: 1135–1146.
36. Schmidt, M. H., I. Dikic, and O. Bogler. 2005. Src phosphorylation of Alix/AIP1
modulates its interaction with binding partners and antagonizes its activities.
J. Biol. Chem. 280: 3414–3425.
37. Aissouni, Y., G. Zapart, J. L. Iovanna, I. Dikic, and P. Soubeyran. 2005. CIN85
regulates the ability of MEKK4 to activate the p38 MAP kinase pathway. Bio-
chem. Biophys. Res. Commun. 338: 808–814.
38. Oliver, J. M., D. L. Burg, B. S. Wilson, J. L. McLaughlin, and R. L. Geahlen.
1994. Inhibition of mast cell Fc?R1-mediated signalling and effector function by
the Syk-selective inhibitor, piceatannol. J. Biol. Chem. 269: 29697–29703.
39. Costello, P. S., M. Turner, A. E. Walters, C. N. Cunningham, P. H. Bauer,
J. Downward, and V. L. Tybulewicz. 1996. Critical role for the tyrosine kinase
Syk in through the high affinity IgE receptor of mast cells. Oncogene 13:
40. Zhang, J., E. H. Berenstein, R. L. Evans, and R. P. Siraganian. 1996. Transfection
of Syk protein tyrosine kinase reconstitutes high affinity IgE receptor-mediated
degranulation in a Syk-negative variant of rat basophilic leucemia RBL-2H3
cells. J. Exp. Med. 184: 71–79.
41. Moriya, K., J. Rivera, S. Odom, Y. Sakuma, K. Muramato, T. Yoshiuchi,
M. Miyamoto, and K. Yamada. 1997. ER-27319, an acridone-related compound,
inhibits release of antigen-induced allergic mediators from mast cells by selective
inhibition of Fc? receptor I-mediated activation of Syk. Proc. Natl. Acad. Sci.
USA 94: 12539–12544.
42. Kepley, C. L., L. Youssef, R. P. Andrews, B. S. Wilson, and J. M. Oliver. 1999.
Syk deficiency in nonreleaser basophils. J. Allergy Clin. Immunol. 104: 279–284.
43. Lavens-Phillips, S. E., and D. W. MacGlashan, Jr. 2000. The tyrosine kinases
p53/56lynand p72sykare differentially expressed at the protein level but not at the
messenger RNA level in nonreleasing human basophils. Am. J. Respir. Cell Mol.
Biol. 266: 566–571.
44. Ota, Y., and L. E. Samelson. 1997. The product of the proto-oncogene c-cbl: a
negative regulator of the Syk tyrosine kinase. Science 276: 418–420.
45. Ryan, P. E., G. C. Davies, M. M. Nau, and S. Lipkowitz. 2006. Regulating the
regulator: negative regulation of Cbl ubiquitin ligases. Trends Biochem. Sci. 31:
46. Siegel, R., U. Kim, A. Patke, X. Yu, X. Ren, A. Tarakhovsky, and R. G. Roeder.
2006. Nontranscriptional regulation of SYK by the coactivator OCA-B is re-
quired at multiple stages of B cell development. Cell 125: 761–774.
47. Qu, X., K. Sada, S. Kyo, K. Maeno, S. M. Miah, and H. Yamamura. 2004.
Negative regulation of Fc?RI-mediated mast cell activation by a ubiquitin-protein
ligase Cbl-b. Blood 103: 1779–1786.
48. Zhang, J., Y. J. Chiang, R. J. Hodes, and R. P. Siraganian. 2004. Inactivation of
c-Cbl or Cbl-b differentially affects signalling from the high affinity IgE receptor.
Immunology 173: 1811–1818.
49. Chu, D. H., N. S. C. van Oers, M. Malissen, J. Harris, M. Elder, and A. Weiss.
1999. Pre-T cell receptor signals are responsible for the down-regulation of Syk
protein tyrosine kinase expression. J. Immunol. 163: 2610–2620.
50. Lankester, A. C., G. M. van Schijndel, C. E. van der Schoot, M. H. van Oers,
C. J. van Noesel, and R. A. van Lier. 1995. Antigen receptor nonresponsiveness
in chronic lymphocytic leukemia B cells. Blood 86: 1090–1097.
2096 DOWN-REGULATION OF Syk EXPRESSION BY CIN85
by guest on June 13, 2013