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CUTTING EDGE |
Departments of
*
Pharmacology and
Immunology, Mayo Graduate and Medical Schools, Mayo Clinic, Rochester, MN 55905
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Recently, a proline-rich, 503-amino acid protein named WAS-interacting
protein (WIP) was isolated (6). WIP contains a number of
putative src homology 3 (SH3)-binding domains and several domains
implicated in binding directly or indirectly to actin (6).
The association of WIP with WASp occurs through an interaction within
amino acids 377–503 of WIP with the first 100 amino acids of WASp
(6). Interestingly, the majority of mutations associated
with WAS occur in the first 100 amino acids of WASp, yet this region
only represents 
18% of WASp (7). Moreover, it has
recently been demonstrated that three specific point mutations within
the first 100 amino acids of WASp that are associated with the
development of WAS abrogate the interaction of WIP with WASp
(8). These data strongly suggest that a WIP-WASp
interaction is critical for normal immune cell function. However, the
exact role that the WIP-WASp interaction plays in the regulation of
hematopoietic cells is still unclear.
The observation that T cells from WAS patients fail to undergo TCR-induced proliferation suggests a potential defect in the integration of signals leading to the production of the T cell growth factor IL-2 (3). Moreover, using T cells from mice lacking WASp, it was recently demonstrated that they also fail to enter the cell cycle upon TCR cross-linking (9). Indeed, the generation of signals downstream of the TCR requires actin polymerization and cytoskeletal changes (10). The inability of WASp-deficient T cells to undergo actin polymerization, capping, or cellular proliferation following TCR cross-linking is similar to the defect observed in T cells from Vav-deficient mice (11, 12). It has been previously shown that Vav is a potent regulator of the IL-2 promoter, in particular NF-AT/AP-1-mediated gene transcription (13, 14). Whether or not WASp and Vav function in distinct or overlapping pathways in the regulation of IL-2 production following TCR cross-linking is unclear.
We have performed experiments to determine whether Vav and the WIP-WASP complex cooperate in the regulation of the IL-2 gene following TCR stimulation. To this end, we have identified that overexpression of WIP and Vav result in enhanced NF-AT/AP-1-mediated gene transcription following TCR stimulation. Interestingly, the ability of WIP to enhance NF-AT/AP-1 activity in the presence of Vav requires the interaction of WIP with WASp, as a WIP deletion mutant, lacking its WASp-interacting domain can no longer cooperate with Vav in the regulation of NF-AT/AP-1-mediated gene transcription. Furthermore, deletion analysis of WIP has identified a region in WIP that is responsible for the ability of WIP and Vav to activate NF-AT/AP-1-mediated gene transcription. These data identify a role for WIP in the regulation of IL-2 gene transcription. In addition, our data provide new insight into how mutations within WASp that affect the binding of WIP could adversely influence T cell activation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Unless otherwise stated, all chemicals were obtained from Sigma (St. Louis, MO) The Jurkat T cell line and anti-CD3-producing hybridoma OKT3 (murine IgG2a) were obtained from the American Type Culture Collection (Manassas, VA). Abs used in this study include the anti-FLAG murine mAb FLAG-M2 from Sigma and anti-CD3 mAb was purified by affinity chromatography over protein A-agarose. Keyhole limpet hemocyanin-conjugated peptides derived from WIP amino acids 154–179 (SPGHRSGPPEPQRNRMPPPRPDVGSK, anti-WIP.1), amino acids 468–492 (YVQTTKSYPSKLARNESRSGSNRRER, anti-WIP.2), and WASp amino acids 477–502 (RSRAIHSSDEGEDQAGDEDEDDEWDD, anti-WASp) were used to generate polyclonal rabbit anti-sera (Cocalico Biologicals, Reamstown, PA).
DNA constructs and recombinant vaccinia generation
The WIP-coding sequence was amplified by PCR using the
5'-oligonucleotide containing a HindIII site
(5'-CGCATAAGCTTCCACCATGGGTCCTGTCCCTCCCCC-3')
and a 3'-oligonucleotide containing a NotI site distal to
the stop codon for subcloning
(5'-AGAGCGGCCGCCAAAGATCACCTCGGGATGGG-3')
from cDNA prepared from NK cell total RNA using a previously published
method (15). The WIP sequence was subcloned as an
HindIII/NotI fragment into similarly digested
pCDNA3. In contrast to the originally published WIP sequence
(6), the sequence we obtained is in agreement with a
recent report that identified a difference in the nucleotides that
encode amino acids 303–309 (SSQAPPP instead of PHRPHLR)
(8). WIP mutants were generated using the site-directed
mutagenesis kit (Clontech, Palo Alto, CA) as described previously
(16). The selection oligonucleotide containing a mutant
XbaI site is (5'-CGAGCATGCATCCAGAGGGCCCTATT-3'). The mutant
oligonucleotides are as follows: WIP3'
(5'-GACCCGCCAGGCCGATGAGGCCCCCTCCCACCACCTCCTCCAG-3');
WIP112
(5'-CAGGCTGGAATGCCGAAGCTTAGATCCACGGCCAAC-3');
WIP237
(5'-TTGGGAGGAGGCTCAAAGCTTCAGTCCCCCTTGAGC-3');
WIP293
(5'-CCTCCTCAGAACAACAAGCTTCCAGTGCCTTCCACTCCG-3');
and WIP362
(5'-CTTCCTCCCCCGCCCATGGAGAGACCCCCACCT-3'). The
bolded nucleotides represent the introduced restriction site or stop
codon for generation of the mutant WIP proteins.
Electroporation and luciferase assays
The indicated amount of DNA was electroporated into Jurkat T cells. Cell stimulations and luciferase assays were done as described previously (17). The percentage of maximal reporter activity was determined by dividing the luciferase activity obtained in the absence or presence of stimulation by that obtained by stimulation with PMA + ionomycin. The maximum reporter responses obtained with the combination of PMA and ionomycin did not differ significantly between transfection conditions.
Immunoblot analysis
Jurkat T cells (107) were electroporated with the indicated constructs and lysed on ice for 10 min in 0.5 ml of passive lysis buffer (Promega, Madison, WI). Recombinant proteins were analyzed as previously reported (16). Anti-FLAG proteins were detected using anti-FLAG mAb followed by sheep anti-mouse IgG coupled to HRP (Amersham, Buckinghamshire, U.K.) and the enhanced chemiluminescence detection system from Amersham. In some instances, WIP and WASP proteins were detected using specific anti-WIP and anti-WASP rabbit polyclonal antisera followed by protein A-linked to HRP (Amersham) and the enhanced chemiluminescence detection system.
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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The Vav protooncogene is a known regulator of IL-2 gene
transcription following anti-CD3 stimulation. In particular, it was
shown that when overexpressed, Vav regulates the activity of the
NF-AT/AP-1 transcription factor complex of the IL-2 promoter (13, 14). To determine whether WIP or WIP and Vav can influence
NF-AT/AP-1-mediated gene transcription, we electroporated a WIP
expression construct, Vav expression construct, or a combination of
both into Jurkat T cells along with an NF-AT/AP-1 luciferase reporter
construct, and assayed their ability to regulate NF-AT/AP-1-mediated
gene transcription following various stimulation conditions. In
repeated experiments, WIP by itself had little to no effect on
NF-AT/AP-1 activity under any of the stimulation conditions tested
(Fig. 1
A). To evaluate whether
WIP could enhance Vav-dependent activation of NF-AT/AP-1, suboptimal
amounts of Vav-encoding DNA were electroporated into the Jurkat T cell
line. For example, 1 µg of Vav by itself had only modest effects on
NF-AT/AP-1 activity (Fig. 1A, compare Vav with pCDNA3).
However, when WIP and the suboptimal amount of Vav are expressed
simultaneously, there is enhanced NF-AT/AP-1-mediated gene
transcription following anti-CD3 + PMA stimulation, and a smaller
but reproducible increase following anti-CD3 stimulation alone
(Fig. 1A, compare Vav + WIP to either WIP or Vav alone).
This functional synergy between WIP and Vav does not appear to require
a direct physical interaction since coimmunoprecipitation studies have
not identified a functional complex between these two molecules (data
not shown). The ability of WIP to cooperate with Vav in the regulation
of NF-AT/AP-1 gene transcription is specific since coexpression of WIP,
along with suboptimal concentrations of another protein known to
regulate NF-AT/AP-1 activation, SLP76 (18), does not
result in enhanced activation of NF-AT/AP-1-mediated gene transcription
under any of the stimulation conditions tested (Fig. 1B).
Taken together, these data suggest that WIP and Vav can cooperate in
the regulation of NF-AT/AP-1 gene transcription.
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The observation that 50% of the mutations in WAS
(7) occur in the region of WASp where WIP interacts
suggests that the formation of a WIP-WASp complex is critical for the
generation of functional intracellular signaling. To determine whether
the ability of WIP to cooperate with Vav in the regulation of
NF-AT/AP-1 required a functional interaction with WASp, we generated a
WIP mutant (WIP3') lacking amino acids 377–502 which should no
longer interact with WASp. To confirm that this mutant does not
associate with WASp, we electorporated Jurkat T cells with pCDNA3
control vector or FLAG-tagged expression constructs containing Vav,
WIP, or WIP3'. Following an overnight incubation, the recombinant
FLAG-tagged proteins were immunoprecipitated using anti-FLAG mAb,
resolved by SDS-PAGE, and transferred to a nylon membrane. As shown in
Fig. 2A, equivalent amounts of
WIP and WIP3' were expressed as demonstrated by probing the blot
with rabbit polyclonal anti-WIP.1 antisera (upper panel,
compare lanes 3 and 4). Significantly, reprobing
the blot for WASp demonstrates that WASp interacts with WIP, but does
not associate with the WIP3' mutant (Fig. 2A, lower
panel, compare lanes 3 and 4). The
association of WIP with WASp is specific since no WASp is observed in
the FLAG immunoprecipitates of pCDNA3 transfected or Vav-expressing
Jurkat T cells (Fig. 2A, lanes 1 and
2).
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does not associate with WASp, we tested this mutant
for its ability to cooperate with Vav in the regulation of the
NF-AT/AP-1 reporter. As shown in Fig. 2
B, Jurkat T cells
coexpressing Vav and WIP lead to enhanced activation of
NF-AT/AP-1-mediated gene transcription in the presence of anti-CD3
and PMA stimulation. In contrast, coexpression of Vav and WIP3' does
not result in enhanced activation of the NF-AT/AP-1 reporter under any
stimulation conditions tested (Fig. 2B, compare Vav +
WIP3' to Vav + WIP). The ability of WIP, but not WIP3', to
cooperate with Vav is not due to the levels of overexpressed protein as
both WIP and WIP3' are expressed at equivalent levels (Fig. 2B, right inset). Our data suggest that the
ability of WIP to regulate NF-AT/AP-1 activity requires an interaction
with WASp, since the WIP3' mutant cannot regulate NF-AT/AP-1
activity. It is therefore conceivable that T cells from WAS patients
harboring a mutation that would diminish or abolish the interaction
with WIP would not signal properly because WIP would not be brought
into the signaling complex. Other mutations in WAS that do not affect
the WIP interaction may affect other molecular interactions that are
also required for normal WASp activation. Indeed, several mutations
have been identified that would potentially affect the ability of WASp
to interact with the low m.w. GTP-binding proteins (7).
These mutations would potentially impact on the activation of the
WIP/WASp-signaling complex by blocking its interaction with GTP-bound
Rac and Cdc42. Deletion analysis localizes a critical region in WIP required for NF-AT/AP-1 activation
As diagrammed in Fig. 3A,
WIP contains a number of putative SH3-binding domains, three potential
profilin-binding domains, and a WH2 domain which is also found in the
yeast protein verprolin (6, 8). Indeed, WIP has been shown
to interact with profilin (6) and to associate with the
adaptor molecule Nck through one of its SH3-binding domains
(19). In addition, WIP was recently found to compensate
for the yeast verprolin protein in the regulation of yeast cytoskeletal
asymmetry (20). Interestingly, the ability of WIP to
compensate for verprolin required an intact WH2 domain and the final
putative profilin-binding domain (20). In an attempt to
determine what part of WIP is required for its ability to cooperate
with Vav in the regulation of NF-AT/AP-1 gene transcription, we
generated a series of amino-terminal WIP deletion mutants (see Fig. 3A). Since the interaction of WIP with WASp is critical to
the ability of WIP to enhance NF-AT/AP-1 activity when overexpressed
with Vav, we initially wanted to determine whether these deletion
mutants could functionally interact with WASp. As shown in Fig. 3B, all of the WIP deletion mutants are expressed following
electroporation into Jurkat T cells (Fig. 3B, upper
panel) and they all retain their ability to interact with WASp
(Fig. 3B, lower panel). Although the WIP237 and
WIP293 mutant proteins appear as a broad band, analysis of the
expression plasmids confirms that they contain the correct sequence
(data not shown). Therefore, these truncations may alter the migration
of the mutant proteins due to changes on conformation or
posttranslational modification.
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C, compare WIP + Vav to
WIP112 + Vav). However, WIP237, which is lacking an additional 125
amino acids, or further deletions (WIP293 and WIP362) yield proteins
that can no longer cooperate with Vav in the regulation of NF-AT/AP-1
activity (Fig. 3C). The observed difference between the
ability of the various WIP mutant proteins to enhance NF-AT/AP-1
activity was not due to the level of expressed protein as similar
levels of protein were observed in each sample (data not shown).
Interestingly, in our reporter assay, the ability of WIP to regulate
NF-AT/AP-1 activity was not eliminated by removal of the WH2 domain
(WIP112), suggesting that regulation of gene transcription in T cells
is not dependent on the presence of this domain (see Fig. 3C, compare WIP to WIP112). In addition, the ability of WIP
to cooperate with Vav in the regulation of NF-AT/AP-1 activity was not
affected by WIP mutants harboring specific point mutations within the
WH2 domain or the carboxyl-terminal profilin-binding domain (our
unpublished observation). The region of WIP that is critical to its
ability to regulate NF-AT/AP-1 activity was localized to a region
encompassing amino acids 112 and 237 (see Fig. 3C). It will
be important to determine what structural features within this region
are responsible for WIP-regulated NF-AT/AP-1 activity. One functional outcome following the interaction of a TCR with peptide/MHC is the production of the T cell growth factor IL-2. This is a critical event required for the production of a functional immune response. T cells from patients with WAS fail to produce IL-2 following TCR cross-linking (3, 9), indicating a pivotal role for WASp in the regulation of signals leading to IL-2 gene transcription. It has been previously shown that Vav can positively regulate the activity of Rac and Cdc42 (21, 22, 23), two upstream regulators of WASp function. Furthermore, overexpression of a dominant-negative form of Cdc42 has been shown to negatively affect NF-AT/AP-1-mediated gene transcription regulated by the serine/threonine kinase PAK1 (24). Together, these data indicate a role for low m.w. GTP-binding proteins in the regulation of NF-AT/AP-1 activation through the regulation of their downstream effector molecules. Our data suggest that the one such downstream effector molecule, WASp, and its binding partner WIP are also involved in the regulation of NF-AT/AP-1 activity. More important, we have identified that the interaction of WIP with WASp is necessary for the observed enhancement and provide insight into the mechanism by which mutational changes in WASp that impact WIP interaction could potentially affect T cell activation. The identification of the structural domains of WIP required for the formation of a functional WIP/WASp-signaling module will be important in unraveling the many cellular defects associated with WAS.
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Acknowledgments |
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Footnotes |
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2 D.N.S. and D.D.B. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Paul J. Leibson, Department of Immunology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905. E-mail address:
4 Abbreviations used in this paper: WAS, Wiskott-Aldrich syndrome; WASp, WAS protein; WIP, WASp-interacting protein; SH3, src homology 3.
Received for publication December 6, 1999. Accepted for publication January 21, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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-Opioid receptors expressed by Jurkat T cells enhance IL-2 secretion by increasing AP-1 complexes and activity of the NF-AT/AP-1-binding promoter element. J. Immunol. 159:5431.[Abstract]
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