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Lymphocyte Biology Section, Division of Rheumatology, Immunology, and Allergy, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115; and
Division of Hematology and Medical Oncology, Oregon Health Sciences University, Portland, OR 97201
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cbl lacks intrinsic enzymatic activity, but interacts with a variety of
Src homology 2 (SH2) and SH3 domain-containing proteins. For example,
the interactions between the proline-rich region of Cbl (residues
481–690) and SH3 domains mediate the constitutive association of Cbl
with Src family kinases, such as Fyn and Lck, and the adaptor proteins
Nck and Grb2 (1, 2, 3). Documented tyrosine phosphorylation
sites within the C-terminal portion of Cbl mediate its TCR
activation-dependent association with SH2 domain-containing proteins
such as Vav (Tyr700), the p85 subunit of
phosphatidylinositol-3 kinase (Tyr731), and the
Crk adaptor proteins (Tyr774) (7, 8). These features suggest that one role for Cbl may be to serve
as a scaffold to assemble signaling protein complexes (1, 2, 4, 5, 6). However, the C-terminal region that mediates these
protein-protein interactions is least conserved through evolution: both
Drosophila melanogaster (D-Cbl) and Caenorhabditis
elegans (SLI-1) Cbl homologues as well as the recently cloned
third mammalian Cbl-family gene product, Cbl-3, lack most of these
C-terminal motifs (1, 2, 3, 9). In contrast, the N-terminal
domains of Cbl are highly conserved through evolution, suggesting their
critical functional role(s): these domains include a RING finger domain
and the N-terminal region (Cbl-N) corresponding to sequences retained
in the v-cbl oncogene. Recent results have demonstrated that
Cbl-N functions as a tyrosine kinase binding (TKB) domain via a direct
interaction with a number of autophosphorylated tyrosine kinases,
including the lymphocyte tyrosine kinases ZAP-70 and Syk, and the
receptor tyrosine kinases PDGFR 
/ß and EGFR (10, 11, 12, 13, 14, 15, 16).
The crystal structure of the TKB domain of Cbl in complex with a
phosphopeptide, representing its binding site in ZAP-70, revealed that
this domain is composed of a four-helical domain, an EF hand, and a
variant SH2 domain, with all three of these modules required for
phosphopeptide binding (17).
Recent genetic studies support a novel role of Cbl as an evolutionarily conserved negative regulator of tyrosine kinases (1, 2, 5, 6, 18). The C. elegans Cbl homologue, SLI-1, was identified during a screen for negative regulators of LET-23, a homologue of mammalian EGFR. The loss of function mutations in SLI-1, including a single substitution G315E, mapped to the conserved TKB domain (19). The D. melanogaster Cbl homologue D-Cbl was shown to negatively regulate R7 photoreceptor development, a process mediated by an EGFR homologue (20, 21). Interestingly, ablation of c-cbl in mice leads to hyperproliferation in several lymphoid organs, such as the thymus and spleen, and increased ductal branching in the mammary fat pads (22). Furthermore, Cbl deficiency promoted the positive selection of CD4+ T cells when examined in the context of an anti-HY-specific transgene (23).
Expression of wild-type or oncogenic mutant Cbl proteins in mammalian
cells has further demonstrated the role of Cbl as a negative regulator
of EGFR and PDGFR tyrosine kinases (10, 24, 25, 26). In
addition, analyses of Cbl-deficient macrophages have demonstrated that
Cbl regulates ubiquitination and subsequent endocytosis of the CSF-1
receptor, another receptor tyrosine kinase (27). Notably,
overexpression of Cbl in a rat basophilic leukemia cell line was shown
to reduce the Fc
R1-mediated degranulation as well as Syk
autophosphorylation and kinase activity (28). A
Cbl-induced reduction of the kinase-active pool of Syk was also
demonstrated in a reconstituted COS cell system (14).
Importantly, the Cbl-TKB domain binding sites on ZAP-70
(13) and Syk (14, 15) correspond to a
negative regulatory phosphorylation site within the linker region
between the SH2 and kinase domains (ZAP-70-Y292 and Syk-Y323)
(29, 30, 31, 32). Notably, mutation of these residues leads to
enhanced signaling through ZAP-70 and Syk in vivo, yet ZAP-70-Y292F did
not exhibit any increase in kinase activity (31, 33).
These results suggested that the negative regulatory phosphorylation
site in the linker region recruits a negative regulator to ZAP-70 and
Syk. Given the specific interaction of Cbl with these sites, Cbl
represents a candidate negative regulator for these tyrosine kinases.
Consistent with this notion, the negative regulatory effect of Cbl on
Syk in a COS cell reconstitution system required an intact Cbl TKB
domain as well as the Y323 residue in Syk (14). More
recently, analyses of Cbl-deficient thymocytes demonstrated that more
ZAP-70 is associated with 
-chain and that ZAP-70 is
hyperphosphorylated compared with wild-type thymocytes
(34). However, the role of Cbl’s interaction with the
negative regulatory phosphorylation sites in ZAP-70 or Syk in lymphoid
cells has not been demonstrated.
In this study, we have used overexpression in Jurkat T cells as well as reconstitution in COS cells and ZAP-70-deficient Jurkat cells to provide direct evidence that Cbl can negatively regulate the ZAP-70 tyrosine kinase. Importantly, we demonstrate that Cbl-dependent negative regulation of ZAP-70 in T cells is mediated through the Cbl TKB domain binding to Tyr292, the site of negative regulatory phosphorylation in ZAP-70. The present results provide direct support for the idea that Cbl is a negative regulator that is specifically recruited to activated autophosphorylated ZAP-70 via its negative regulatory phosphorylation site.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Jurkat-JMC (a ZAP-70 and Syk-expressing Jurkat T cell line) and its SV40 T Ag-expressing derivative, JMC-T, were maintained as described (12). The Jurkat-derived ZAP-70/Syk-deficient p116 cell line was kindly provided by Dr. R. T. Abraham (Department of Immunology, Mayo Clinic, Rochester, NY), and was maintained as described (35). The retrovirally transfected clonal derivatives of Jurkat JMC (JMC-HA-Cbl), overexpressing HA-tagged Cbl protein, were maintained as described (36). COS-7 cells (American Type Culture Collection, Manassas, VA) were maintained as previously described (14).
The Abs used in this work were: mouse mAb 4G10 (anti-Tyr(P); IgG2a)
(37), mAb SPV-T3b (anti-CD3; IgG2a)
(38), mAb 12CA5 (anti-influenza hemagglutinin (HA)
epitope tag; IgG2b) (39), mAb anti-Syk (IgG2a; Santa
Cruz Biotechnology, Santa Cruz, CA), mAb anti-ZAP-70 (IgG2a;
Transduction Laboratories, Lexington, KY), mAb anti--chain
(IgG1; Santa Cruz Biotechnology), mAb W6/32 (anti-class I HLA;
IgG2a) (40), rabbit polyclonal anti-phospho MAP kinase
(New England Biolabs, Beverly, MA), rabbit polyclonal
anti-MAP kinase (New England Biolabs), and PE-conjugated goat
anti-mouse Ig (Jackson ImmunoResearch, West Grove, PA).
cDNAs and site-directed mutagenesis
The Lck mammalian expression vectors pdKCR-Lck and
pdKCR-Lck-505F (41) were kindly provided by Dr. Y. Minami
(Institute for Molecular and Cellular Biology, Osaka University, Osaka,
Japan). The pSRNeo-CD8- plasmid encoding a CD8
(extracellular and transmembrane domains) and TCR -chain
(cytoplasmic region) chimera has been previously described
(13). The pAlterMAX-ZAP-70, pAlterMAX-ZAP-70-Y292F,
pAlterMAX-HA-Cbl, and pAlterMAX-HA-Cbl-G306E have also been described
(14). pAlterMAX-HA-Cbl-70Z/3 was derived by subcloning the
appropriate fragment of pSRNeo-HA-Cbl-70Z/3 in pAlterMAX using
KpnI and SalI restriction sites.
pAlterMAX-HA-Cbl-70Z/3-G306E was generated by site-directed mutagenesis
of pAlterMAX-HA-Cbl-70Z/3 using the Altered Sites-II Mammalian
Mutagenesis System (Promega, Madison, WI), according to the
manufacturer’s protocol. The mutagenic oligonucleotide used was 5'-AGT
AAC ATA CTC AAT AGC CCA-3'. All constructs were verified by automated
DNA sequencing. All Cbl constructs encode N-terminally HA-tagged Cbl
proteins. The 70Z/3 Cbl encodes a protein with deletion of residues
366–382 and corresponds to an oncogenic Cbl mutant derived from the
murine B cell lymphoma cell line 70Z/3 (42). The NF-AT
luciferase reporter plasmid, designated NF-AT-Luc, was a generous gift
from Dr. A. Rao (Center for Blood Research, Harvard Medical School,
Boston, MA) and contains three repeats of the NF-AT binding sites from
the distal promoter of the murine IL-2 gene linked to the firefly
luciferase gene (43).
NF-AT luciferase assay
JMC-T or p116 cells were transfected with the indicated amounts of NF-AT luciferase construct and Cbl or ZAP-70 plasmids by electroporation, as previously described (31). The cells were cultured for 12–24 h and then seeded in replicates of five for each stimulation condition (2 x 105 cells/well). The cells were stimulated for 6–8 h at 37°C with media alone, anti-CD3 (CD3; 1/2000 dilution of SpVT3b ascites; determined to be optimal in titration experiments), or 50 ng/ml PMA plus 1 µg/ml ionomycin (Sigma, St. Louis, MO). Cells were lysed using Cell Culture Lysis Reagent (Promega), and cleared lysates were normalized by the Bradford assay. Equal amount of each lysate was analyzed for luciferase activity using a Monolight 3010C luminometer (Analytical Bioluminescence Laboratory, San Diego, CA) and Luciferin Reagent (Promega), according to the manufacturer’s protocol. The mean (of five replicates) of luciferase units for unstimulated (medium alone) or anti-CD3-stimulated (CD3) cells was expressed as a percentage of the mean luciferase units for PMA plus ionomycin stimulation with the combined SE of both means used in the calculation. For biochemical analysis, an aliquot of the same cells that were used for the NF-AT luciferase assay was cultured for a total of 48 h before cell lysates were prepared.
Transient expression in COS cells
COS-7 cells were plated overnight and transfected for 6 h using Lipofectamine (Life Technologies, Gaithersburg, MD) in OPTI-MEM medium, according to the manufacturer’s protocol. The amounts of each plasmid used for transfection are indicated in the appropriate figure legend. The total amount of DNA for each transfection was held constant with pAlterMAX vector. Cells were lysed 48 h posttransfection in lysis buffer containing 0.5% Triton X-100, 50 mM Tris (pH 7.5), 150 mM sodium chloride, 1 mM PMSF, 1 mM sodium orthovanadate, 10 mM sodium fluoride, and 1 µg/ml each of leupeptin, pepstatin A, antipain, and chymostatin.
Surface TCR analysis of Jurkat T cell transfectants
For surface TCR staining, cells were washed twice with ice-cold PBS containing 2% FCS and incubated with the isotype-matched negative control mAb (anti-Syk), an anti-class I HLA mAb W6/32 (positive control), and anti-CD3 mAb (SPV-T3b). Cells were then washed and stained with a PE-conjugated goat anti-mouse secondary Ab. Flow cytometry, data collection, and analysis were performed on a FACSort machine using CellQuest software (Becton Dickinson, Franklin Lakes, NJ).
Cell lysis, immunoprecipitations, gel electrophoresis, and immunoblotting
Jurkat T cell transfectants were either left unstimulated or stimulated with anti-CD3 mAb (SPV-T3b) at 37°C and then lysed in the buffer described above (12). Immunoprecipitations from aliquots of COS-7 or Jurkat cell lysates were performed as described (12). Immunoprecipitated proteins or whole cell lysates were resolved by SDS-PAGE, transferred to polyvinylidine difluoride (PVDF) membranes (Immobilon-P; Millipore, Bedford, MA), and immunoblotted with the indicated Abs, essentially as described (13). Protein A-HRP (Cappel-Organon Technika, Durham, NC) was used as a secondary reagent for blotting. Blots were visualized using enhanced chemiluminescence (ECL) and exposed to XB1 film (New England Nuclear Life Sciences, Boston, MA). Blots were stripped and reprobed as described (14). Images of blots were generated by direct scanning of films using a Hewlett-Packard (Palo Alto, CA) ScanJet 4c scanner and Corel Draw version 6 software. Densitometry was conducted on directly scanned images using ScionImage for Windows software.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Overexpression of Cbl in Jurkat T cells reduces the level of TCR-induced tyrosine phosphorylation and MAP kinase activation
To assess the effect of Cbl on proximal events triggered by TCR
stimulation, we utilized Jurkat T cells overexpressing HA-tagged Cbl as
a result of retroviral transfection (36). Three
HA-Cbl-overexpressing clones were chosen for these analyses. Each of
these clones, C12, D3, and E10, expressed 
10-fold higher levels of
Cbl compared with the parental Jurkat JMC cell line (Fig. 1
A). Surface staining of the
CD3 complex revealed that all three clones expressed cell surface TCR
levels comparable with those on the parental Jurkat JMC cell line (Fig. 1B). Staining with a Vß8-specific Ab also revealed
comparable levels of surface TCR on Cbl-transfected clones vs parental
cells (data not shown).
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A). Essentially
all of the major substrates of tyrosine phosphorylation, such as Syk,
ZAP-70, LAT, and -chain, showed reduced levels of phosphorylation in
the Cbl-overexpressing clones, with the exception of the 120-kDa
polypeptide, which corresponds to overexpressed Cbl.
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B, upper
panel). In cells overexpressing Cbl, reduced levels of
autophosphorylated ZAP-70 were detected at all time points (Fig. 2B). Very low phosphotyrosine signal was detected in lysates
of Cbl transfectants at 30 min poststimulation (Fig. 2B,
lanes 8, 12, and 16) when
compared with the parental cell line in which signal was still
detectable (lane 4). Immunoblotting of
whole cell lysates with an anti-ZAP-70 Ab showed relatively
equivalent levels of ZAP-70 protein at various time points
poststimulation (Fig. 2B, lower panel). Thus, Cbl
overexpression in Jurkat T cells appears to lead to selective reduction
in the phosphorylated pool of ZAP-70.
In view of the reduced levels of TCR-induced autophosphorylated ZAP-70
and other T cell phosphoproteins in Cbl-overexpressing Jurkat T cells,
we wished to examine signaling events further downstream of TCR
activation. For this purpose, we chose to analyze the levels of
phospho-MAP kinases p42 and p44, which reflect the activation of the
Ras/Raf/MAP kinase signaling cascade, a key tyrosine kinase-dependent
downstream signaling pathway in T cells (44). Parental
Jurkat cells and a Cbl-overexpressing clone (C12) were stimulated
through the TCR, and the cell lysates were immunoblotted with a
phospho-MAP kinase-specific Ab (Fig. 2C). A substantial
reduction in the duration of MAP kinase activation was observed in
Cbl-overexpressing cells compared with the parental Jurkat JMC cell
line. An aliquot of the same cell lysates was blotted with a MAP kinase
Ab to demonstrate equivalent levels of MAP kinase. Thus, two
independent assay systems provided evidence that Cbl overexpression
reduces the level of activation events triggered by stimulation of the
TCR in a T cell system.
Reconstitution in COS cells reveals Cbl-mediated negative regulation of ZAP-70 and an essential role for the Cbl TKB domain and ZAP-70 Tyr292
Based on the above findings and the fact that Cbl interacts
directly with ZAP-70 in an activation-dependent manner, we wished to
determine whether Cbl-dependent negative regulation was due to a direct
effect on ZAP-70, and whether the interaction between the Cbl TKB
domain and ZAP-70 Tyr292 was required for this
negative regulation. For this purpose, we first utilized a COS cell
reconstitution system in which cotransfection of ZAP-70 with a Src
family kinase (Lck or Lck 505F in our analyses) and an immunoreceptor
tyrosine-based activation motif-bearing docking chain (a CD8-
chimera in our experiments) leads to ZAP-70 activation, as assessed by
ZAP-70 autophosphorylation and phosphorylation of relevant substrates
(e.g., CD8-).
COS cells were transfected with the indicated DNA constructs for
48 h, and cell lysates were prepared after a 2-h incubation with
100 µm orthovanadate. Anti-HA immunoprecipitations of cell lysates
were blotted with anti-ZAP-70 Ab to detect the Cbl/ZAP-70 protein
association (Fig. 3). As expected
(12), ZAP-70 coimmunoprecipitated with wild-type Cbl (Fig. 3, second panel, lane 3), but not with the
Cbl-G306E mutant (second panel, lane 5).
Conversely, the ZAP-70-Y292F mutant failed to coimmunoprecipitate with
Cbl (second panel, lane 4). Notably, the
Cbl-70Z/3 mutant also associated with ZAP-70, in fact at a higher level
compared with wild-type Cbl (second panel, compare
lanes 1 and 3), and this interaction
was also abrogated by the Y292F mutation in ZAP-70 (second
panel, lane 2). In each case, immunoprecipitation of
HA-tagged Cbl proteins was confirmed by anti-HA immunoblotting
(Fig. 3, first panel), and equivalent levels of ZAP-70
expression were confirmed by immunoblotting of whole cell lysate with
anti-ZAP-70 Ab (Fig. 3, third panel). Taken together,
these results indicated that Cbl associates with ZAP-70 in a
TKB-dependent manner, and that the association of both wild-type Cbl as
well as the Cbl-70Z/3 mutant protein requires an intact
Tyr292 on ZAP-70.
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), a potential negative regulator, and the hyperresponsive
phenotype of ZAP-70-Y292F or ZAP-70 mutants with a deletion of the
entire SH2-kinase linker when expressed in lymphocytes (33, 45), we hypothesized that Cbl’s association with phosphorylated
Tyr292 would lead to a negative regulatory effect
on ZAP-70. To directly test this idea, we examined the effect of Cbl
coexpression on the autophosphorylation of ZAP-70. COS cells were
cotransfected with cDNA constructs encoding ZAP-70 or the ZAP-70-Y292F
mutant together with CD8- and the Src family kinase Lck. In
addition, graded amounts of Cbl or its G306E mutant were coexpressed,
and whole cell lysates were analyzed by anti-phosphotyrosine,
anti-HA, anti-ZAP-70, and anti- immunoblotting (Fig. 4). As previously observed, coexpression
of Lck in this system was essential for tyrosine phosphorylation of
CD8- and ZAP-70 (Fig. 4A, top panel,
lane 15).
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A, first panel, compare lanes 1–4
with lane 14). In contrast, the level of ZAP-70
phosphorylation remained unchanged upon expression of the Cbl TKB
domain mutant Cbl-G306E (first panel, compare
lanes 5–8 with lane 14). Notably, the
ZAP-70-Y292F mutant was completely resistant to the effects of Cbl, as
shown by its unchanged level of phosphorylation upon wild-type Cbl
coexpression (first panel, compare lanes
9–12 with lane 14). These findings clearly
demonstrated that Cbl expression leads to a loss of the phosphorylated
pool of ZAP-70. Under these experimental conditions, Cbl expression led
to a small decrease in CD8- phosphorylation; however, this was
seen both in ZAP-70- and ZAP-70-Y292F-transfected cells
(first panel, compare lanes 1–4 with
lanes 9–12), consistent with the observation that
CD8- phosphorylation was Lck dependent.
Anti-ZAP-70 blots of whole cell lysates revealed that the decrease in
ZAP-70 tyrosine phosphorylation was paralleled by a decrease in ZAP-70
protein levels (Fig. 4A, third panel, compare
lanes 1–4 with lane 14). The decrease in ZAP-70
protein levels was not seen upon coexpression of the Cbl-G306E mutant,
nor were protein levels of the ZAP-70-Y292F mutant affected by its
coexpression with wild-type Cbl (third panel, compare
lanes 5–12 with lane 14). The levels of Cbl
protein were lower compared with those of Cbl-G306E protein at
equivalent input DNA amounts, while levels of Cbl protein were
comparable in cells coexpressing ZAP-70 or the ZAP-70-Y292F mutant
(second panel). No major differences in CD8- protein
levels were observed when either ZAP-70 or its Y292F mutant was
expressed with Cbl or Cbl-G306E (fourth panel).
Analysis of cell lysates prepared in SDS-containing lysis buffer
revealed that the Cbl-dependent decrease in ZAP-70 protein levels was
apparently not due to sequestration in a Triton X-100-insoluble
fraction (data not shown).
To quantify the decrease in ZAP-70 phosphorylation and protein levels,
densitometric analysis was conducted on the data shown in Fig. 4A. The intensity of ZAP-70 bands (lanes
1–12) was expressed as a ratio to that of the ZAP-70 band in the
absence of coexpressed Cbl (lane 14). This analysis
further confirmed that coexpression of wild-type Cbl and ZAP-70 results
in a Cbl dose-dependent loss of phosphorylated ZAP-70 protein, whereas
this effect is not observed when ZAP-70 is coexpressed with Cbl-G306E,
nor when the ZAP-70-Y292F mutant is coexpressed with Cbl (Fig. 4, B and C).
Cbl-70Z/3 mutant fails to exert a negative regulatory effect on ZAP-70
The oncogenic 70Z/3 Cbl mutant has been shown to enhance the basal
as well as Ca2+-ionophore-induced NF-AT
luciferase activity in transfected Jurkat cells (46, 47).
It was therefore of interest to ascertain whether or not this Cbl
mutant, which prominently associated with ZAP-70 (Fig. 3), retained the
ability to negatively regulate ZAP-70. This was tested using the COS
cell transfection system described above. In contrast to the effect of
overexpressing increasing amounts of wild-type Cbl (Fig. 5A, first panel,
compare lanes 1–4 with lane 9), expression of
the Cbl-70Z/3 mutant did not result in a decrease in the levels of
tyrosine-phosphorylated ZAP-70 (first panel, compare
lanes 5–8 with lane 9), even though higher
levels of Cbl-70Z/3 protein were expressed compared with wild-type Cbl
protein (second panel). In addition, expression of the
Cbl-70Z/3 mutant did not result in a decrease in ZAP-70 protein levels
(third panel, compare lanes 1–4 with
lanes 5–8). Densitometric analysis confirmed the lack of an
effect of Cbl-70Z/3 on ZAP-70 phosphorylation and protein levels
compared with the effects of wild-type Cbl (Fig. 5, B and
C).
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Although the analyses in the COS cell system clearly demonstrated
a Cbl TKB domain-dependent and ZAP-70-Y292-dependent inhibitory effect
of Cbl on ZAP-70 (Fig. 4), we wished to establish this further in the
context of the TCR stimulation in a lymphoid cell system. For this
purpose, we examined the effects of various Cbl proteins on basal and
TCR stimulation-induced activity of the NF-AT luciferase reporter, a
ZAP-70-mediated distal readout of TCR signaling (35).
Unstimulated JMC-T cells transiently transfected with increasing
amounts of Cbl or Cbl-G306E showed no significant change in the level
of NF-AT luciferase reporter activity compared with mock-transfected
cells (Fig. 6A). In contrast,
a Cbl dose-dependent decrease in anti-CD3 Ab-induced NF-AT
luciferase activity was observed when wild-type Cbl was expressed
in these cells. Notably, the effect of Cbl overexpression was
essentially abrogated by the TKB domain-inactivating G306E mutation
(Fig. 6A), similar to the effect of this mutation on the
negative regulation of ZAP-70 in COS cells (Fig. 4). The lack of effect
of the Cbl-G306E mutant was not due to a difference in protein
expression (Fig. 6A, second panel).
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B). Neither
Cbl-N nor Cbl-N-G306E had an effect on basal NF-AT luciferase activity.
These observed differences in the effects of Cbl-N and Cbl-N-G306E were
not due to differences in protein levels (Fig. 6B,
second panel). A simultaneous comparison of Cbl, Cbl-N, and
their G306E mutants further substantiated these results (Fig. 6C). Cbl-70Z/3 mutant-induced enhancement of basal and anti-CD3-induced NF-AT activity is TKB domain dependent
Previous studies have shown that the oncogenic Cbl mutant 70Z/3
enhances the basal and Ca2+-ionophore-induced
NF-AT luciferase activity when overexpressed in Jurkat cells
(46); however, its effects on anti-CD3-induced NF-AT
luciferase activity and the role of the TKB domain in this process have
not been clarified. Because Cbl-70Z/3 clearly and prominently
associated with ZAP-70, but not with ZAP-70-Y292F (Fig. 3), it appeared
likely that a TKB domain-mediated interaction contributed to the effect
of Cbl-70Z/3 on NF-AT activity. As expected (46) (Fig. 5),
overexpression of Cbl-70Z/3 in Jurkat cells led to a marked increase in
NF-AT luciferase activity in the absence of anti-CD3 triggering
(Fig. 7). Anti-CD3 stimulation led to a
further increase in the NF-AT luciferase activity; however, the fold
increase was markedly lower compared with the vector-transfected
control cells, apparently due to high basal activity. Importantly, the
G306E mutation in Cbl-70Z/3 led to a complete lack of enhancement of
the basal as well as anti-CD3-induced NF-AT luciferase activity
when compared with cells transfected with Cbl-70Z/3 (Fig. 7A). Experiments using ZAP-70-deficient p116 T cells
reconstituted with ZAP-70 yielded similar results to those obtained
using JMC-T cells (Fig. 7B). Therefore, the Cbl-70Z/3 effect
on T cell activation, as assessed by NF-AT luciferase activity,
appeared to be dependent on the TKB domain. The requirement of the TKB
domain for the Cbl-70Z/3-induced increase in NF-AT luciferase activity
in the absence of TCR stimulation has been previously observed
(47, 48).
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Because the JMC-T cell line used in the experiments described
above expresses ZAP-70 as well as Syk (36), both of which
can be targeted by Cbl, the effects of Cbl on NF-AT luciferase could
not be conclusively ascribed to ZAP-70. To directly demonstrate that
the effects of Cbl-mediated negative regulation reflected its influence
on ZAP-70, we utilized the Jurkat E6-1 (Syk negative, ZAP-70
positive)-derived mutant cell line p116, which is ZAP-70 deficient
(35). As expected, the lack of ZAP-70 in these cells
results in a block of anti-CD3-stimulated increase in NF-AT
luciferase activity (Figs. 7B and
8), as well as early activation events
such as tyrosine phosphorylation of cellular proteins (data not shown).
However, reconstitution of ZAP-70 expression by transient transfection
restored the anti-CD3-induced stimulation of NF-AT activity (Figs. 7B and 8). Overexpression of Cbl in the absence of ZAP-70
had no effect on NF-AT luciferase induction. Cotransfection of Cbl and
ZAP-70, however, resulted in a decrease in NF-AT luciferase activity
compared with that seen with ZAP-70 transfection alone (Fig. 8),
confirming results obtained using the JMC-T cell line (Fig. 6).
Importantly, coexpression of the Cbl-G306E mutant with ZAP-70 failed to
reduce the anti-CD3-stimulated NF-AT luciferase activity, thus
confirming the critical role of the Cbl TKB domain.
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). Importantly, coexpression of Cbl failed to decrease
ZAP-70-Y292F-dependent NF-AT luciferase activity, indicating that this
mutant is insensitive to the negative regulatory effects of Cbl.
Together, these results show that Cbl can negatively regulate the
ZAP-70-dependent activation of T cells, and that this effect is
contingent upon an interaction mediated by the Cbl TKB domain binding
to the negative regulatory phosphotyrosine 292 in ZAP-70. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The negative regulatory effect of Cbl on ZAP-70 was demonstrated in two
different cellular systems using distinct readouts. First, we used
HA-Cbl-overexpressing Jurkat T cell clones to study the effect of Cbl
on early events of T cell activation. Each of the three
Cbl-overexpressing clones showed a reduction in the level of
anti-CD3-induced tyrosine phosphorylation of cellular substrates
compared with the parental T cell line (Fig. 2). Specifically, we
observed a reduction in the level of ZAP-70 tyrosine phosphorylation in
all three Cbl-overexpressing clones. The inhibitory effect of Cbl
overexpression was also observed using a distal readout of T cell
activation, the phosphorylation of MAP kinases.
These results in Jurkat Cbl transfectants, and previous findings that
the Cbl TKB domain interacts with the negative regulatory linker
phosphorylation site Y292 in ZAP-70 (13) were consistent
with a role for Cbl as a negative regulator of ZAP-70. Direct evidence
for this idea was provided by analyses in the COS cell reconstitution
system, in which coexpression with Cbl led to a marked reduction in the
autophosphorylated pool of ZAP-70 (Fig. 4). Another measure of
Cbl-dependent negative regulation in COS cells was provided by a
decrease in ZAP-70 protein levels, analogous to Cbl-dependent decrease
in Syk protein levels in the COS cell system (14).
Although the mechanisms of reduction in Syk/ZAP-70 protein levels in
the COS cell system are presently unclear, it provides a useful index
of the effect of Cbl on these tyrosine kinases in COS cells.
The Cbl-dependent negative regulation of ZAP-70 in the COS cell system
required the conserved TKB domain of Cbl and phosphorylated
Tyr292 in ZAP-70, both of which are required for
the physical association of these two proteins (Fig. 4). However, the
TKB domain-mediated physical association of Cbl with ZAP-70 is not by
itself sufficient for negative regulation, as demonstrated by analyses
of the oncogenic Cbl-70Z/3 mutant. Cbl-70Z/3 carries a 17-aa deletion
near the N-terminal boundary of the highly conserved RING finger
domain. This mutant prominently associated with ZAP-70 (Fig. 3), but
did not induce a decrease in ZAP-70 phosphorylation or protein levels
(Fig. 5). Similarly, Cbl-N interacts with ZAP-70 and Syk, but does not
lead to a reduction in the autophosphorylated pool of these tyrosine
kinases (S. Ota, N. Rao, and H. Band, unpublished results). Thus,
domains C terminal to the TKB domain, such as the RING finger and/or
other regions, appear to be important for the negative regulatory
effect of Cbl on ZAP-70. Recent analyses have revealed that the TKB and
RING finger domains are sufficient for Cbl-dependent negative
regulation of Syk in a COS cell system (S. Ota, N. Rao, and H. Band,
unpublished results).
To directly assess the negative regulatory effect of Cbl on ZAP-70
function in T cells, further analyses were conducted in a Jurkat T cell
line and its ZAP-70-deficient mutant. Using the TCR-induced NF-AT
luciferase reporter activity as a measure of ZAP-70-dependent T cell
activation, we demonstrated that wild-type Cbl expression led to a
dose-dependent inhibition of T cell activation in a TKB
domain-dependent manner (Fig. 6, A and C). The
importance of the TKB domain-mediated interaction of Cbl with ZAP-70
was also revealed by the inability of the 70Z/3 Cbl mutant with an
inactive TKB domain (G306E mutation) to enhance basal and TCR-induced
NF-AT luciferase activity (Fig. 7). Other groups have also recently
shown that the G306E mutant of Cbl-70Z/3 was unable to enhance the
basal NF-AT luciferase activity in Jurkat cells (47, 48);
however, the effect of Cbl-70Z/3 on TCR stimulation-induced NF-AT
activation was not investigated in these studies (46, 47).
Finally, the expression of the Cbl TKB domain (Cbl-N) by itself led to
a substantial increase in TCR-induced NF-AT luciferase activity, and
this effect was abrogated by the G306E mutation (Fig. 5, B
and C). Apparently, the mutant Cbl proteins, such as
Cbl-70Z/3 and Cbl-N, bind to ZAP-70 and prevent the endogenous
wild-type Cbl from exerting a negative regulatory effect. This
phenotype of oncogenic Cbl mutants is strongly indicative of a negative
regulatory role of the endogenous Cbl for ZAP-70.
Given the presence of endogenous ZAP-70 in Jurkat JMC-T cells, and the
expression of Syk in the particular cell line used in our analyses (
(36) and data not shown), we extended our analyses to a
ZAP-70 and Syk-deficient Jurkat T cell line, p116 (35). As
previously reported (35), stimulation of this cell line
through the TCR did not induce NF-AT luciferase activity, and Cbl
expression by itself had no effect (Fig. 8). The expression of
wild-type ZAP-70 reconstituted the TCR-induced NF-AT luciferase
activity; Cbl coexpression, but not Cbl-G306E coexpression, reduced the
NF-AT activity. As expected from studies in wild-type Jurkat cells
(31), the ZAP-70 Y292F mutant demonstrated a
hyperresponsive phenotype when expressed in p116 cells (Fig. 8).
Notably, coexpression of Cbl failed to down-regulate the ZAP-70-Y292F
mutant-mediated NF-AT luciferase activation in p116 cells. The
reconstitution studies in p116 cells demonstrate that Cbl indeed
impinges on ZAP-70 function via a mechanism that requires an intact Cbl
TKB domain and an intact negative regulatory phosphorylation site,
Tyr292, in ZAP-70.
Our conclusion that Cbl is a negative regulator of ZAP-70 is consistent with the phenotype of c-cbl-deficient mice, which display hypercellularity in thymus and spleen (22). The thymocytes of these mice displayed higher levels of tyrosine-phosphorylated proteins, including ZAP-70, upon TCR stimulation. Furthermore, when the c-cbl deficiency was bred into TCR transgenic mice, an enhancement of positive selection of CD4+ thymocytes was observed (23). Our studies, however, provide direct evidence that the negative regulatory effect of Cbl on ZAP-70 is indeed exerted via its linker phosphorylation site.
Demonstration of a role for Cbl as a negative regulator of ZAP-70 is of considerable interest in relation to the shared and divergent regulation of Syk and ZAP-70 (49). Compared with ZAP-70, Syk has a substantially higher intrinsic kinase activity and is less dependent on Src family kinases for its activation (49, 50, 51, 52). However, Cbl does function as a negative regulator of Syk, as demonstrated by experiments in a transfected RBL 2H3 mast cell line, COS cells, and more recently in lymphoid cells (14, 28) (S. Ota and H. Band, unpublished results). Notably, Cbl association with Syk also requires an intact Cbl TKB domain and Syk Tyr323; the latter corresponds to ZAP-70 Tyr292 (14, 15, 32, 49). Thus, Cbl represents a negative regulator that is recruited to activated ZAP-70/Syk kinases via the regulatory phosphorylation sites outside their kinase domains.
Coexpression of Cbl and ZAP-70 in COS cells resulted in a decrease in
the amount of ZAP-70 (Fig. 4), similar to our earlier observation of
Cbl-dependent decreases in the levels of coexpressed Syk
(14). In both cases, the Cbl TKB domain and the cognate
binding sites on Syk/ZAP-70 are required for a decrease in the protein
levels of these tyrosine kinases. However, we did not observe a
detectable decrease in ZAP-70 protein levels in Cbl-overexpressing
Jurkat T cells, even though a clear reduction in ZAP-70 phosphorylation
was observed. This discrepancy might reflect the activation of a
smaller pool of ZAP-70 in T cells, as opposed to COS cells, in which
the coexpression of Lck probably leads to activation of a larger pool
of ZAP-70. Consistent with this idea, a small decrease in Syk protein
levels was observed in anti-IgM-stimulated Ramos B cells, and this
decrease was accentuated when Cbl was overexpressed (S. Ota and H.
Band, unpublished results). Furthermore, a recent report showed a
decrease in the levels of ZAP-70 in Ag-activated T cells, although the
role of Cbl in these effects was not investigated (53). It
is, however, possible that the Cbl-induced loss of ZAP-70 protein that
we observed is not mechanistically relevant to T cells. Further
analyses will be required to address these possibilities.
In conclusion, our studies show that the negative regulatory phosphorylation site within ZAP-70 provides a means to recruit Cbl as a negative regulator in activated T lymphocytes, and that loss of this site renders ZAP-70 insensitive to Cbl-mediated negative regulation. Further studies of the Cbl-ZAP-70 interaction are therefore likely to elucidate a physiologically relevant pathway of regulating tyrosine kinase function in T cells.
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Acknowledgments |
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Footnotes |
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2 Current address: ICOS Corp., Bothell, WA 98021.
3 Current address: Laboratory for Physiological Chemistry, Utrecht University, Utrecht, The Netherlands.
4 Address correspondence and reprint requests to Dr. Hamid Band, Smith Building, Room 538C, 1 Jimmy Fund Way, Boston, MA 02115.
5 Abbreviations used in this paper: PDGFR, platelet-derived growth factor receptor; Cbl-N, N-terminal transforming region of Cbl; EGFR, epidermal growth factor receptor; HA, hemagglutinin; MAP, mitogen-activated protein; PVDF, polyvinylidene difluoride; SH, Src homology; TKB, tyrosine kinase binding.
Received for publication September 14, 1999. Accepted for publication February 24, 2000.
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