COMMUNICATION
Activation of a CrkL-Stat5 Signaling Complex by Type I
Interferons*
Eleanor N.
Fish
,
Shahab
Uddin§,
Mete
Korkmaz§,
Beata
Majchrzak
,
Brian J.
Druker¶, and
Leonidas C.
Platanias§
From the
Department of Medical Genetics and
Microbiology, University of Toronto, Toronto, Ontario M5S 3E2, Canada,
the § Section of Hematology-Oncology, University of Illinois
and West Side Veterans Affairs Hospital, Chicago, Illinois 60607, and
the ¶ Division of Hematology and Medical Oncology, Oregon Health
Sciences University, Portland, Oregon 97201
 |
ABSTRACT |
Type I interferons (IFN
and IFN
) transduce
signals by inducing tyrosine phosphorylation of Jaks and Stats, as well
as the CrkL adapter, an SH2/SH3-containing protein which provides a
link to downstream pathways that mediate growth inhibition. We report that Stat5 interacts constitutively with the IFN receptor-associated Tyk-2 kinase, and during IFN
stimulation its tyrosine-phosphorylated form acts as a docking site for the SH2 domain of CrkL. CrkL and Stat5
then form a complex that translocates to the nucleus. This IFN-inducible CrkL-Stat5 complex binds in vitro to the
TTCTAGGAA palindromic element found in the promoters of a subset of
IFN-stimulated genes. Thus, during activation of the Type I IFN
receptor, CrkL functions as a nuclear adapter protein and, in
association with Stat5, regulates gene transcription through DNA binding.
 |
INTRODUCTION |
Type I interferons
(IFN
,1 IFN
, and IFN
)
are pleiotropic cytokines that exhibit multiple biological effects
including antiviral and growth-inhibitory activities (1, 2). Following
engagement of the Type I IFN receptor by IFN
or IFN
, two kinases
of the Janus family, Tyk-2 and Jak-1, are activated and phosphorylate the Stat proteins: Stat1, Stat2, Stat3, Stat4, and Stat5 (3, 4).
Activated Stat proteins form distinct signaling complexes to regulate
gene transcription. Stat1 and Stat2 form a heterodimer that associates
with a member of the IFN regulatory factor family, p48, resulting in
the formation of the mature ISGF3 complex that translocates to the
nucleus to initiate gene transcription by binding to
interferon-stimulated response elements (3, 4). Stat1 and Stat3 homo-
and heterodimers and homodimers of Stat4, Stat5a, and Stat5b bind a
palindromic sequence found in the promoters of IFN-stimulated genes (4,
5).
In addition to the Stat pathway, other signaling cascades are activated
downstream of Jaks in IFN
signaling. These include the insulin
receptor substrate (IRS) pathway that regulates activation of the
phosphatidylinositol 3'-kinase (6-8) and the CrkL pathway that links
the functional Type I IFN receptor complex to the growth-inhibitory C3G/Rap-1 cascade (9). In the present study we determined whether the
CrkL pathway functions in coordination with the Stat pathway. Our data
demonstrate that Stat5 is constitutively associated with the Tyk-2
kinase, and its IFN-phosphorylated form provides a docking site for the
SH2 domain of CrkL. The resulting CrkL-Stat5 complex translocates to
the nucleus to regulate gene transcription via GAS elements. Viewed
together, these findings provide evidence for a novel function of CrkL
as a nuclear adapter protein.
 |
EXPERIMENTAL PROCEDURES |
Cells and Reagents--
The Daudi and KG1 human cell lines were
grown in RPMI 1640 (Life Technologies, Inc.) supplemented with 10%
(v/v) fetal bovine serum (Life Technologies, Inc.) and antibiotics.
Human recombinant IFN
2 was provided by Hoffmann-La Roche. Human
recombinant IFN
-consensus (IFNCon1) was provided by Amgen Inc. Human
recombinant IFN
was provided by Biogen Inc. (Cambridge, MA). The
anti-CrkL and anti-Stat5b polyclonal antibodies were obtained from
Santa Cruz Biotechnology (Santa Cruz, CA). The production of the
pGEX-CrkLSH2 construct has been described previously (10).
Immunoprecipitations, Immunoblotting, and Glutathione
S-Transferase Binding Studies--
Cells were stimulated with
104 units/ml of the indicated interferons as described
previously (9). After stimulation, the cells were lysed in
phosphorylation lysis buffer, and immunoprecipitations and
immunoblotting using the ECL method were performed as described previously (9). Production of glutathione S-transferase
fusion proteins and binding experiments using lysates from
IFN
-untreated or -treated cells were performed as described
previously (9, 11).
Genomic DNA Affinity Chromatography and Mobility Shift
Assays--
Preparation of nuclear extracts, genomic DNA affinity
chromatography, and mobility shift assays were performed essentially as
described previously (12). A double-stranded oligodeoxynucleotide specific for Stat5 binding (AGATTTCTAGGAATTCAAATC), derived from the
-casein promoter, was synthesized and used in gel shift assays.
 |
RESULTS AND DISCUSSION |
We sought to identify the tyrosine kinase that regulates
IFN
-induced activation of Stat5 and the mechanisms by which the protein is activated and binds DNA. When lysates from IFN
-stimulated KG1 myeloid cells were immunoprecipitated with an anti-Tyk-2 antibody and immunoblotted with antiphosphotyrosine, we noted that a tyrosyl phosphoprotein migrating as a doublet at 96/94 kDa was complexed with
Tyk-2 (Fig. 1A). This protein
corresponded to Stat5, as determined by immunoblotting with a specific
anti-Stat5 antibody (Fig. 1B). The interaction of Stat5 with
Tyk-2 was present prior to IFN
treatment and increased further after
IFN
stimulation, suggesting that Stat5 interacts constitutively with
Tyk-2 and thus may provide a link between this kinase and downstream
signaling elements. Similarly, a constitutive interaction of Stat5 with
Tyk-2 was seen in studies with the IFN
-sensitive Daudi
lymphoblastoid cell line (Fig. 1, C and D).

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Fig. 1.
Interaction of Stat5a and Stat5b with the
Type I IFN receptor-associated Tyk-2 tyrosine kinase.
A, KG-1 cells were incubated with IFN for the indicated
times at 37 °C. Cell lysates were immunoprecipitated with an
anti-Tyk-2 antiserum or nonimmune rabbit serum (NRS), and
proteins were analyzed by SDS-PAGE and immunoblotted with an
antiphosphotyrosine monoclonal antibody (4G-10, Upstate Biotechnology).
B, the blot shown in A was stripped and reprobed
with an antibody against Stat5b that recognizes both forms of Stat-5
(Santa Cruz Biotechnology). C, Daudi cells were incubated at
37 °C for the indicated times in the presence or absence of IFN .
Cell lysates were immunoprecipitated with anti-Tyk-2 antiserum or
nonimmune RIgG, and proteins were analyzed by SDS-PAGE and
immunoblotted with an anti-Stat5b antibody. D, the blot
shown in C was stripped and reprobed with a monoclonal
antibody against Tyk-2 (Transduction Laboratories).
|
|
In previous studies, we have demonstrated that the adapter protein CrkL
interacts in an IFN
-dependent manner with Tyk-2 and is
tyrosine-phosphorylated during IFN
stimulation, providing a link
between the Type I IFN receptor and the C3G-Rap-1 growth-inhibitory pathway (9). Accordingly, we examined whether CrkL associates with
Stat5 during IFN
or IFN
stimulation. In time course studies, Daudi cells were left untreated or treated for 10 or 20 min with IFN
or IFN
. Cell lysates were prepared, immunoprecipitated with an
anti-CrkL antibody, and then immunoblotted with an anti-Stat5 antibody.
Stat5 was detected in association with CrkL after IFN
stimulation,
establishing that this member of the Stat family of proteins associates
with CrkL in an IFN
-dependent manner (Fig. 2,
A and B). In
contrast, there was no Stat1 present in anti-CrkL immunoprecipitates
from IFN
-treated cells,2
indicating that the CrkL-Stat5 interaction is selective. As Stat5 was
found to be constitutively associated with the Tyk-2 kinase, while the
CrkL interaction was IFN
-dependent, we determined
whether Stat5 undergoes IFN
-induced tyrosine phosphorylation and
subsequently functions as a docking site for the SH2 domain of CrkL. In
experiments in which Daudi or KG-1 cells were treated with IFN
for
different times and the status of phosphorylation of Stat5 was
examined, we observed that Stat5 is phosphorylated on tyrosine,
suggesting that it acts as a substrate for the kinase activity of the
associated Tyk-2 protein (Fig. 3, A and
B, and data not shown). In
addition, the SH2 domain of CrkL bound to the phosphorylated/activated
form of both Stat5 isomers (a and b) in an IFN
-dependent
manner (Fig. 3, C and D), confirming that Stat5
acts as a docking site for the CrkL SH2 domain and strongly suggesting
that such an interaction mediates the formation of the CrkL-Stat5
complex.

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Fig. 2.
IFN and IFN induce the association of
CrkL with Stat5 in intact cells. A, Daudi cells were
treated for the indicated times with IFN or IFN . Cell lysates
were first precleared with nonimmune RIgG and after immunoprecipitation
with the indicated antibodies, analyzed by SDS-PAGE and immunoblotted
with an anti-Stat5b antibody. B, the blot shown in
A was stripped and reprobed with the anti-CrkL antibody to
demonstrate equal loading.
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Fig. 3.
The IFN -induced tyrosine-phosphorylated
form of Stat5 functions as a docking site for the SH2 domain of
CrkL. A, Daudi cells were incubated in the presence or
absence of IFN for 20 min. Cell lysates were immunoprecipitated with
an antibody against Stat5b and immunoblotted with antiphosphotyrosine.
B, the blot shown in A was stripped and reprobed
with the anti-Stat5b antibody. C, Daudi cells were incubated
at 37 °C for 30 min in the presence or absence of IFN . Cell
lysates were bound to a glutathione S-transferase fusion
protein encoding the SH2 domain of CrkL (GST-CkLSH2) or GST alone used
as control. Bound proteins were analyzed by SDS-PAGE and immunoblotted
with an antibody against Stat5b. D, KG-1 cells were
incubated at 37 °C for 30 min in the presence or absence of IFN .
Cell lysates were bound to glutathione S-transferase fusion
protein encoding the SH2 domain of CrkL (GST-CkLSH2) or GST alone used
as control. Bound proteins were analyzed by SDS-PAGE and immunoblotted
with an antibody against Stat5b.
|
|
Previous studies have shown that CrkL functions as an adapter, linking
tyrosine kinases or their substrates to guanine exchange factors for
small G proteins. Our finding that the protein interacts in an
SH2-dependent manner with Stat5 suggested that it may also participate in the formation of DNA binding complexes that regulate transcription of interferon-stimulated genes. To test this hypothesis, we evaluated the ability of CrkL to bind DNA using genomic DNA affinity
chromatography (GDAC) (12). Specifically, nuclear extracts from Daudi
cells treated with IFN
or IFN
were subjected to GDAC, and then
the DNA-bound fraction was resolved by SDS-PAGE and immunoblotted for
CrkL (Fig. 4A). CrkL bound DNA
in an IFN
- or IFN
-dependent manner (Fig.
4A), and its DNA-bound form migrated at approximately 140 kDa, strongly suggesting that such DNA binding occurs in a complex with
Stat5. Furthermore, as CrkL was detectable in immunoblots of the
nuclear extracts only after Type I IFN treatment, these data suggested
that the protein translocates to the nucleus in a Type I
IFN-dependent manner. Similarly, when the IFN-induced DNA-bound fractions collected following GDAC were immunoblotted with
Stat5, we noticed that Stat5 was detectable in the same complex with
CrkL (Fig. 4B), strongly suggesting that it forms a
DNA-binding complex in association with CrkL.

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Fig. 4.
Identification of Type I IFN-inducible CrkL
by genomic DNA affinity chromatography. Nuclear extracts were
prepared from Daudi cells incubated with or without 104
IU/ml IFN or IFN for 10 min at 37 °C, then analyzed by GDAC.
GDAC eluates were resolved by 11% SDS-PAGE and after Western blotting
probed with anti-CrkL antibody (A) or anti-Stat5 antibody
(B).
|
|
Further analyses of these Type I IFN-induced nuclear extracts by gel
shift assays, employing an oligonucleotide specific for Stat5 binding
derived from the
-casein promoter, identified the presence of
IFN
- or IFN
-inducible DNA-binding complexes, whose mobilities
were affected by inclusion of anti-CrkL antibodies (Fig.
5A) but not control RIgG (Fig.
5A and data not shown). The presence of Stat5 in these
CrkL-containing complexes in the electrophoretic mobility shift assay
was confirmed by immunoblotting with antibodies to Stat5 (Fig.
5B). Thus, CrkL forms DNA-binding complexes in association
with Stat5, strongly suggesting that it is involved in the regulation
of Type I IFN-dependent gene expression.

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Fig. 5.
IFN and IFN activate Stat5-CrkL DNA
binding complexes in Daudi cells. A, Daudi cells were
either not treated or treated with 104 IU/ml IFN or
IFN for 10 min. Nuclear extracts were prepared, incubated with or
without antisera to CrkL or nonimmune rabbit immunoglobulin, and
reacted with a 32P-labeled oligonucleotide, specific for
Stat5 binding (AGATTTCTAGGAATTCAAATC), derived from the -casein
promoter. The resultant complexes were resolved using 4.5% native PAGE
and visualized by autoradiography. B, nuclear extracts (10 µg/ml, lanes 1 and 2, or 2 µg/ml, lanes
3 and 4) from IFN -treated or -untreated cells were
reacted with a 32P-labeled oligonucleotide specific for
Stat5 binding (as in A). The resultant complexes were
resolved using 4.5% native PAGE and visualized by autoradiography
(left panel). To confirm the presence of Stat5 in
the complexes seen in A and the left
panel, aliquots of nuclear extracts from the same experiment
shown in the left panel were reacted with the
unlabeled oligonucleotide, analyzed by native PAGE, and Stat5 was
detected by immunoblotting with an anti-Stat5 antibody (Santa Cruz
Biotechnology) (right panel).
|
|
Our data provide strong evidence that CrkL, in cooperation with Stat5,
binds DNA, and this complex likely functions as a transcription factor
in IFN
/
-induced signaling. Indeed, we have observed this IFN-induced CrkL-Stat5 complex in other IFN-sensitive cell lines, namely human glial T98G and human osteosarcoma U2OS cells (data not
shown). Such a role for CrkL was unexpected and raises the possibility
that other related proteins, e.g. CrkII, Grb-2, may function
as transcriptional activators in other signaling cascades. The members
of this family of proteins have been previously shown to function as
adapters, providing a link between receptor tyrosine kinases or their
substrates and guanine exchange factors. Until now, there has been no
evidence of their exhibiting DNA binding activity. CrkL has been shown
to interact primarily with C3G (9, 13-15), which acts as a guanine
exchange factor for Rap-1 (16), a small G protein that antagonizes Ras
and has tumor suppressor activity (17-19). Regulation of Rap-1
activation by the CrkL-C3G complex appears to be critical for
inhibition of T-cell proliferation and induction of anergy (20). In
addition, recent studies have demonstrated that CrkL interacts with a
newly cloned member of the IRS family of proteins, IRS-4, in an
IGF-1-dependent manner and that it has oncogenic potential
when overexpressed in cell lines (21).
The current report implicates Stat5 in the engagement of CrkL in IFN
signaling, as shown by the requirement of Stat5 as a docking site for
the SH2 domain of CrkL. Most importantly, for the first time these data
demonstrate that a Stat protein can act as a docking protein for the
SH2 domain of a non-Stat protein to form a DNA-binding complex with it.
Although, in the case of CrkL, this function appears to be specific for
Stat5, it is likely that other Stats will be found to function in a
similar manner in other systems. Recent reports have suggested that
Stat5 is involved in IFN
signaling in myeloid cell lines and HeLa
cells (5), and its activation has been observed in response to
differentiation and growth arrest signals (22, 23). Our results
strongly suggest that such functions for Stat5 require its interaction
with and formation of a signaling complex with CrkL.
 |
FOOTNOTES |
*
This work was supported by Grant CA77816 from the National
Institutes of Health (to L. C. P.), by the Hairy Cell Leukemia Foundation (to L. C. P.), and a Medical Research Council of Canada grant (to E. N. F.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Section of
Hematology-Oncology, University of Illinois at Chicago, MBRB, MC-734, Rm. 3150, 900 S. Ashland Ave, Chicago, IL 60607-7173. Tel.:
312-355-0155; Fax: 312-413-7963; E-mail: Lplatani{at}UIC.Edu.
The abbreviations used are:
IFN, interferon; GST, glutathione S-transferase; STAT, signal transducer and
activator of transcription; IRS, insulin receptor substrate; PAGE, polyacrylamide gel electrophoresis; GDAC, genomic DNA affinity chromatography.
2
S. Uddin and L. C. Platanias, unpublished observations.
 |
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