From the Division of Allergy and Immunology,
NIAID, National Institutes of Health Asthma and Allergic Diseases
Research Center, Department of Internal Medicine, and the
§ Department of Microbiology and Immunology, University of
Texas Medical Branch, Galveston, Texas 77555
Received for publication, August 13, 2002, and in revised form, December 5, 2002
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ABSTRACT |
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Lyn, an Src-type tyrosine kinase, is associated
with the interleukin (IL)-5 receptor in eosinophils. The mechanism of
its activation is unknown. Through yeast two-hybrid screening we have cloned and characterized a new signaling molecule, Unc119, that associates with IL-5R One of the fundamental goals of cell biology is to understand the
mechanism of signal generation by receptors. Many receptors rely upon
kinases, especially tyrosine kinases, for receptor phosphorylation and
activation of signaling cascades. The Src family tyrosine kinases
(SrcTKs)1 frequently serve as
the trigger mechanism for cytosolic signals (1). Receptor-associated
SrcTKs exist in a non-active conformation and become transiently
activated following ligand binding and receptor oligomerization (2).
How receptor oligomerization leads to the activation of SrcTKs is unknown.
Two intramolecular interactions tightly regulate structural
conformation and enzymatic activity of SrcTKs. One is through Src
homology 2 (SH2) and the other is through the SH3 domain. All cellular
SrcTKs have a C-terminal regulatory tyrosine residue (Tyr527 for Src). Phosphorylated Tyr527
forms an intramolecular interaction with the SH2 domain of the kinase
(3). This interaction decreases the kinase activity. Dephosphorylation
of this tyrosine residue by phosphatases such as CD45 is required but
is not sufficient for kinase activation (4). Receptor-bound SrcTKs are
frequently found to be dephosphorylated under basal conditions, yet
lack appreciable catalytic activity (5). CD45-negative cells have
variable effects on SrcTKs. CD45 negatively regulates Lyn by
dephosphorylating both its negative and positive regulatory tyrosine
residues (6). The activity of Lyn and Hck is increased in macrophages
(7) and B cells (8) lacking CD45.
Regulation of SrcTK activity through other intramolecular interactions
was realized after the crystal structures of Src (9) and Hck (10) were
solved. The linker region that is located between the SH2 and the
kinase domains binds to the SH3 domain of the kinase rendering a
non-active conformation. Furthermore, a short amino acid stretch
connecting the SH2 and SH3 domains has recently been identified as an
additional negative regulator of Hck and Src activation (11). The SH3
domain of SrcTKs prefers to bind to the RXXPXXP
motif (a motif is defined as a short stretch of amino acid residues
that binds to a signaling domain) (12, 13). Although the linkers of
SrcTK bind to the SH3 domain, they do not have an optimal SH3-binding
motif. Src and Fyn do not even have the second proline residue in the
motif (13). Although Lyn has the canonical PXXP motif (amino
acids 230-233) like all other SrcTKs, it lacks the flanking arginine
residue. This "imperfect" SH3 motif with low affinity binding can
be easily displaced by a high affinity SH3 ligand. Accordingly, the
exogenous SH3 ligand Nef (a human immunodeficiency virus protein)
activates Hck, a member of SrcTKs, in an SH3
motif-dependent manner (14). However, the identity of
endogenous SH3 ligands that activate SrcTKs is largely unknown.
Our laboratory has been interested in the signal transduction mechanism
of IL-5R, which is composed of a unique Yeast Two-hybrid Screening--
A human fetal cDNA library
(Clontech) was screened using the LexA Matchmaker
Yeast Two-hybrid System (Clontech). The bait was
constructed by fusing the cytosolic and transmembrane portion of
IL-5R Primers, RT-PCR, and Southern Hybridization--
Primers were
designed using the Primer Designer software (1990-1991 Scientific and
Educational software) and synthesized at the University of Texas
Medical Branch Molecular Biology Core Lab. The PCRs were carried out in
a thermocycler (Idaho Technology) in capillary tubes. For RT-PCR, total
RNA was purified from eosinophils using TRIzol (Invitrogen). Twenty
nanograms of RNA was reverse-transcribed into cDNA using
Superscript II preamplification kit (Invitrogen) following the protocol
provided. PCR amplification was performed using CCGCAAGGCCATGAAGGTGA
and CAGTGCCTGAGTCCATGTCC as 5' and 3' primers, respectively. The PCR
mixture was separated on 3% NuSieve 3:1 agarose gel (FMC Bioproducts)
and then either transferred to nylon membrane (Schleicher & Schuell)
and hybridized with 32P-labeled (Amersham Biosciences)
Unc119 (labeled with DECAprime II kit, Ambion) or the
corresponding ~200-bp band was excised, purified, sequenced, and
aligned with that of Unc119 with the BLAST2 program
(www.ncbi.nlm.nih.gov/blast/bl2seq/bl2.html).
Expression and Purification of Recombinant Unc119--
The
encoding cDNA sequence of Unc119 was PCR-amplified using
GGCGAATTC-CCATGAAGGTGAAGAAGG and AATGTCGA-CGTGGGATCAGGGTGT
as 5' and 3' end primers, respectively. The PCR fragment was fused in-frame into pGEX-4T2 (Amersham Biosciences) after EcoRI
and SalI restriction (Invitrogen) reactions. After the
correct reading frame was confirmed with sequencing, it was
electroporated into the E. coli BL21 strain. The expression
and purification of the recombinant Unc119 was performed with the
modification of the protocol provided. Briefly, the transformed cells
were grown in 2× YTA medium overnight with 200 rpm shaking at
37 °C. The overnight culture was diluted 1:100 and incubated at
25 °C with shaking until the A600
reached ~1.50. At this time the
isopropyl-1-thio- Peptide Synthesis and Antibody Generation--
Experiments were
performed according to the protocols of The Animal Resources Center and
the Institutional Animal Care and Use Committee at the University of
Texas Medical Branch. All peptides were synthesized at the Protein
Biochemistry Core Lab of the University of Texas Medical Branch. A
sequence of an immunogenic peptide from Unc119 was determined by
hydropathy plot using Protscale at the Expasy site
(www.expasy.ch/cgi-bin/protscale.pl). The peptide was synthesized and
conjugated to keyhole limpet hemocyanin (Pierce) according to the
protocol provided. The peptide-keyhole limpet hemocyanin conjugate (300 µg) or whole recombinant Unc119 (200 µg) was mixed with either
Imject Alum (Pierce) or Titermax Gold (Sigma) as adjuvant and injected
into a New Zealand White rabbit subcutaneously after obtaining a sample
of pre-immune serum. Three booster injections were performed at the
4th, 6th, and 8th weeks after first immunization. A blood sample was
drawn before each booster and 2 weeks after the third booster (10th
week). The serum was obtained according to Ref. 24. Briefly the blood
was kept at room temperature for 4 h and then at 4 °C
overnight. The serum was centrifuged at 12,000 × g at
4 °C to precipitate any particulate and aliquoted and stored at
Eosinophil and Mononuclear Cell Purification and
Stimulation--
All experiments were conducted in compliance with the
Institutional Review Board protocol. Eosinophils were purified as
described (17). In short, peripheral blood from volunteers was left at room temperature in 1.2% Hetastarch, 25 mM EDTA for 30 min. The buffy coat was transferred into new tubes and washed 2 times
with Hanks' balanced saline solution before Percoll gradient
(Amersham Biosciences) centrifugation. The polymorphonuclear fraction
was taken, and the red blood cells were lysed with hypotonic shock. Eosinophils were negatively selected after incubation with anti-CD16 antibody-coated microbeads and passage through a magnetic column (Miltenyi Biotec). In some experiments the mononuclear cell fractions were used as the source of lysates. When needed the cells were cultured
in RPMI and stimulated with 10 ng/ml IL-5.
Immunoprecipitation, Gel Electrophoresis, and Western
Blotting--
The immunoprecipitation was performed as explained (17).
Shortly, the stimulated and non-stimulated cells were lysed in the
lysis buffer containing 50 mM Tris-HCl, pH 7.4, 75 mM NaCl, 1 mM EDTA, 1 mM NaF, 1 mM Na3VO4, 0.5% Nonidet P-40, 1 µg/ml each of the protease inhibitors aprotinin, leupeptin, and
pepstatin, and 1 mM phenylmethylsulfonyl fluoride. After
incubation on ice for 30 min, detergent-insoluble materials were
removed by centrifugation at 4 °C at 12,000 × g.
After preclearing, 1-2 µg of an appropriate antibody was added to
the lysate and incubated at 4 °C rotating for 1 h followed by
addition of 20 µl of protein A/G-agarose. The incubation was
continued for 2 h or in some cases overnight. The mixture was
centrifuged at 12,000 × g for 5 min, and the pellet was washed with 1 ml of lysis buffer 5 times or 3 times with lysis buffer and 3 times with kinase buffer in the case of kinase reactions. The samples were separated with SDS-PAGE and blotted onto
nitrocellulose or PVDF membranes for immunoblotting. The membranes were
incubated in 5% non-fat dried milk or 5% bovine serum albumin in
TBS-T buffer followed by incubation in the primary antibody solutions
at a concentration of 0.01-0.03 µg/ml in 2% bovine serum
albumin/TBS-T. The membranes were then washed 3 times in the TBS-T
buffer for 10 min each and incubated with horseradish
peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology)
solution (0.01 µg/ml). After washing again the membranes were
developed with enhanced chemiluminescence (ECL or ECL Plus) substrate
(Amersham Biosciences). To strip and reprobe, the membranes were
incubated in the stripping buffer (100 mM
2-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl, pH 6.7) at
55 °C for 30 min, washed, blocked, and immunoblotted with a proper
antibody as explained above. Densitometric analyses of select
Western-blotted protein bands were performed with the software "Metamorph" version 6.4r8 (Universal Imaging Corporation,
Downingtown, PA).
GST Pull-down--
GST or GST fusion proteins (Unc119 or Lyn)
were incubated with the cell lysates or recombinant Unc119 at 4 °C
rotating for 2 h. Twenty µl of glutathione-agarose was then
added, and incubation was continued for 2-4 h or overnight. The
samples were centrifuged at 12,000 × g for 5 min, and
the pellets were washed with lysis buffer, separated with SDS-PAGE,
transferred to PVDF membrane, and Western-blotted.
In Vitro Kinase Assay--
To assess the effect of Unc119 on the
activation of individual kinases, Lyn, Hck, Itk, or p38 was
immunoprecipitated with respective antibodies (Santa Cruz
Biotechnology) in bulk from the mononuclear cell lysates. The
respective kinases were immunoprecipitated from the lysates
corresponding to 50-60 million cells, aliquoted in 20-30 samples, and
kept at Chariot Protein Transduction--
Purified eosinophils (>98%)
were used for Unc119 or anti-Unc119 antibody (affinity-purified)
transduction experiments. HSA and purified rabbit IgG were used as
controls for Unc119 and anti-Unc119 antibody, respectively. TF-1 cells
were used for transduction with FITC-labeled ovalbumin (Molecular
Probes, Eugene, OR). For each reaction 6 µl of Chariot
(Active Motif, Carlsbad, CA) suspension was diluted to 100 µl with
H2O, combined with Unc119 or HSA (0.5 and 5 µg/106 cells), or anti-Unc119 antibody or rabbit IgG (2, 4, and 6 µg per 106 cells), resuspended in 100 µl of
PBS, and incubated at room temperature for 30 min. After 2 washes in
PBS, 1 × 106 cells were resuspended in 200 µl of
Chariot-protein complex. After the addition of 400 µl of RPMI, the
cells were incubated for 1 h at 37 °C. Fetal calf serum and
RPMI were then added to bring final transfection volume to 1 ml at 5%
fetal calf serum. The cells were incubated for an additional 2 h.
IL-5 was added to appropriate cells at 1 × 10 Measurement of Apoptotic Eosinophils by Flow
Cytometry--
Cells were washed twice in PBS and then resuspended in
Binding Buffer (10 mM HEPES, pH 7.4, 140 mM
NaCl, 2.5 mM CaCl2) at 1 × 106 cells/ml according to the manufacturer's protocol for
annexin V staining (BD Biosciences). Five µl of annexin V FITC and 6 µl of 50 µg/ml propidium iodide (Sigma) were added to 100 µl of
cell suspension. Cells were gently mixed and incubated 15 min at room temperature in the dark. After adding 400 µl of Binding Buffer to
each tube, the samples were analyzed by flow cytometry.
Construction of Bait and Cloning Unc119--
In order to search
for IL-5R Unc119 Is Expressed in Eosinophils and Mononuclear
Cells--
Unc119/HRG4 was shown previously (25) to be preferentially
expressed in retina. However, its expression in hematopoietic cells was
not studied. Because we cloned it from fetal liver, an early
hematopoietic organ, we critically examined its expression in myeloid
cells. We demonstrated the presence of its message in eosinophils by
RT-PCR followed by Southern hybridization (Fig. 1A). We subcloned the
RT-PCR-amplified Unc119 cDNA and confirmed its identity by
sequencing (data not shown). We raised a rabbit polyclonal antibody and
used it to detect the presence of Unc119 in eosinophils, mononuclear
cells, and polymorphonuclear granulocytes by Western blotting. Immune
serum but not the preimmune serum detected two bands of proteins of
approximate molecular mass of 37 and 39 kDa (Fig.
1B, left panel). In order to identify the exact
Unc119 band, we depleted the anti-Unc119 antibody from the immune serum
by incubating the latter with excess amounts of GST-Unc119. The
anti-Unc119-depleted serum failed to stain the lower band (37 kDa) but
not the upper band (Fig. 1B, right panel). The
depleted serum also failed to detect recombinant Unc119 that was loaded in the 1st lane. The results suggest that our antibody
detects a 37-kDa Unc119 protein, which is expressed in myeloid and
lymphoid cells. By using this antibody we Western-blotted tissue lysate from mouse retina, heart, liver, lung, and spleen. As reported, Unc119
was predominantly expressed in the retina (Fig. 1C).
However, small detectable amounts of Unc119 were also present in other organs.
Unc119 Is Associated with IL-5R Unc119 Has SH3- and SH2-binding Motifs--
SH3-binding motifs
contain the canonical PXXP sequence (12, 13) that is
frequently flanked by a conserved arginine (Table I). Unc119 has one complete and one
incomplete SH3-binding motif at the N terminus. It also has
phosphorylation sites for SrcTKs and one SH2-binding motif (Table I).
The SH2-binding motif (26), which also conforms to the consensus
tyrosine phosphorylation site for SrcTKs, is located at the C
terminus.
Unc119 Associates with Lyn and Hck--
In the next step we
investigated whether Unc119 was associated with SH2 and SH3
domain-containing signaling molecules. For this purpose we examined the
association of Unc119 with Lyn, Hck, Itk, and phosphatidylinositol
3-kinase (PI3K) p110 Unc119 Associates with Lyn through SH2 and SH3 Domains--
In
order to examine the physical association of the native proteins in the
cell, we immunoprecipitated Lyn and Western-blotted with the
anti-Unc119 antibody (Fig.
3A). As a control for rabbit immunoprecipitating anti-Lyn antibody, we used the anti-
Like other SrcTKs, Lyn has a unique domain, followed by a SH3 domain, a
SH2 domain, a linker region, and a kinase domain. In order to map the
Unc119-binding site of Lyn, we used GST fusion proteins of Lyn unique,
SH2, and SH3 domains in a direct binding assay. As controls we used the
SH3 domain of Grb2 and the SH2 domain of Shc. Both SH2 and SH3 domains
of Lyn were able to associate with rUnc119 (Fig. 3B). The
SH2 and SH3 domains of Fyn, another member of SrcTKs, were also able to
associate with Unc119 (Fig. 3B). In contrast, the SH3 domain
of Grb2 and the SH2 domain of Shc had no or negligible association with
Unc119 (Fig. 3B). Direct coprecipitation of GST-Lyn with
rUnc119 indicates that the association is not mediated by IL-5R or
adapter proteins that might be present in a cell lysate. It is
important to note that the binding of the SH2 domain of Lyn to Unc119
occurs in a non-phosphotyrosinedependent manner, because we
have used bacterially expressed recombinant Unc119.
In a next set of experiments, we examined whether the SH3 or the SH2
motif peptides derived from Unc119 bind to Lyn kinase. To this goal we
synthesized a peptide encompassing the conserved RXXPXXP (residues 55-64) of the SH3 motif of
Unc119 (PSH3) and a second peptide that had the conserved
arginine and proline residues substituted with alanine (proline to
alanine mutant, PP Unc119 Activates Lyn and Hck--
The mechanism of activation of
SrcTKs following receptor stimulation is unclear. Because of its
interaction with SH3 and SH2 domains, we studied the effect of Unc119
on Lyn and Hck activation. Initially we examined the effect of Unc119
on Lyn autophosphorylation in an in vitro kinase assay.
Unc119 stimulated autophosphorylation of Lyn in a
dose-dependent manner with the optimum autophosphorylation occurring at a concentration between 7 and 70 nM Unc119
(Fig. 4A). To assess whether
the increase in autophosphorylation was associated with enhanced
catalytic activity, enolase was used as a Lyn substrate in a similar
kinase assay. Unc119 induced enolase phosphorylation by Lyn in a
dose-dependent manner (Fig. 4B, left panel). In order to confirm the kinase activity with another
substrate, we used Sam68, a known substrate for Src (30). Unc119 was
also able to induce the phosphorylation of recombinant Sam68 by Lyn (Fig. 4B, right panel).
Next, we investigated the effect of Unc119-derived motif peptides
(PSH3, PP
Like the PSH3 peptide, the PSH2 and
pPSH2 peptides induced Lyn autophosphorylation at
relatively low concentrations (Fig. 4E, left
panel). Both SH2 peptides were also able to induce substrate enolase phosphorylation by Lyn, although the PSH2 peptide
seems to be more effective than the pPSH2 peptide (Fig.
4E, right panel). An irrelevant peptide
(PC) derived from Unc119 did not stimulate enolase
phosphorylation by Lyn (Fig. 4F).
To assess whether Unc119 is a specific activator of SrcTKs, we examined
the effect of Unc119 and its motif peptides on the kinase activity of
Hck, Itk, and p38 mitogen-activated protein kinase (Fig.
5). We chose Itk as a non-Src tyrosine
kinase and p38 as a serine/threonine kinase. Unc119, the
PSH3 and PSH2 but not the PP IL-5 Stimulates Unc119 Association with Lyn and Its Kinase
Activity--
In order to assess the biological relevance of Unc119 in
IL-5 signaling, we stimulated eosinophils with IL-5 and examined the
association of Unc119 with Lyn and Unc119-bound Lyn activity. IL-5
enhanced the association of Unc119 with Lyn and its catalytic activity
(Fig. 6A). The amount of
kinase activity that coprecipitated with Unc119 was similar to that
associated with the anti-Lyn immunoprecipitate.
Transduced Unc119 Activates Lyn and Promotes Cell Survival in the
Absence of Growth Factors--
Next, we examined whether Unc119
modulated SrcTK-dependent cellular functions. IL-5
regulates eosinophil survival by activating SrcTKs (19-21) among
others. The eosinophil is a terminally differentiated non-proliferating
cell and cannot be easily transfected with expression vectors or
propagated in selection medium. To overcome this problem we used a new
protein transduction reagent called Chariot. This reagent combines the
nuclear localization sequences with the retroviral gp41 fusion domain
(32). In order to test its capacity to transduce protein into cells, we
incubated cells with FITC-labeled ovalbumin in the presence or absence
of Chariot. A significant quantity of FITC-labeled ovalbumin was
internalized in the presence of Chariot (Fig. 6B). Next, we
examined the effect of Unc119 transduction on Lyn kinase activation in
eosinophils. Unc119-transduced cells show increased Lyn activation
(Fig. 6C), which was comparable with or better than that
stimulated by IL-5 (10
Next we studied the effect of the anti-Unc119 antibody on
IL-5-stimulated eosinophil survival. Eosinophils were transduced with
the affinity-purified anti-Unc119 antibody in the presence of Chariot
and then examined for IL-5-stimulated eosinophil survival. The
anti-Unc119 antibody (39 versus 84% with IL-5) but not a
control IgG (73 versus 84% with IL-5) partially inhibited
IL-5-induced eosinophil survival suggesting that Unc119 is
physiologically relevant to IL-5 signaling (Fig.
7).
Lyn, a member of SrcTKs, plays an important role in many
hematopoietic cells, especially in B cells (33), mast cells (34), and
eosinophils (17). The activation of eosinophils through the IL-5
receptor is critically dependent upon Lyn kinase (19-21). The
mechanism of activation of Lyn following receptor stimulation is
unknown. We have cloned Unc119 using the IL-5R Immunoprecipitation experiments suggest that only a small fraction of
Unc119 is associated with Lyn in vivo (Fig. 3A).
The result may suggest that this association is physiologically not important. However, because the binding of Unc119 leads to the activation of Lyn, it is likely that Unc119 associates with Lyn only
transiently. Indeed, the association of Unc119 with Lyn increases after
IL-5 stimulation (Fig. 5A). The Unc119-associated Lyn shows increased kinase activity. More importantly, the entire activable and
detergent-soluble Lyn fraction is associated with Unc119 following IL-5 stimulation.
Unc119 (also known as HRG4 for human retinal gene protein 4) was
originally cloned as a retina-specific gene (25). It had been localized
in ribbon synapses of retina (35). It has 57% homology to the
unc-119 gene from Caenorhabditis elegans, which is expressed in the neural tissue and is involved in feeding, locomotion, and chemosensation of the nematode (36). Recently, a single
patient with cone-rod dystrophy who expressed a truncated form of HRG4
has been identified (37). This patient is heterozygous, and it is not
clear from the report whether this patient displays any other
abnormalities. Rhodopsin promoter-driven overexpression of this
truncated HRG4 in the retina causes its degeneration in mice (37). The
foregoing observation is exciting and clearly indicates an important
role for HRG4/Unc119 in retina. However, the exact function of
HRG4/Unc119 in retina and other organs remains unknown.
Crystallographic studies predict that the binding of the SH3 domain to
an external SH3 ligand would release the linker region leading to a
conformational change in the kinase domain and its activation. Indeed,
studies using human immunodeficiency virus Nef, an exogenous SH3
ligand, have demonstrated robust activation of SrcTKs in
vitro (14). Nef is not the only SH3 ligand that activates SrcTKs.
Two cell surface receptors, CD28 and CD2, have proline-rich sequences
that conform to the SH3-binding motif. In support of our findings both
CD28 and CD2 activate Lck and Fyn, respectively, in an SH3
motif-dependent manner (31). Heterotrimeric guanine
nucleotide-binding regulatory protein (G protein) can directly activate
Src and Hck (38). Unc119 is not the first molecule that binds an
Src-type kinase at two sites and regulates its function. It has been
shown that p130Cas (Csk-associated substrate) binds Src
kinase through its SH2 and SH3 domains (39). The binding through the
SH3 domain activates Src. A mutation in the SH3-binding motif of
p130Cas causes significant reduction in Src
kinase activity (39). We have seen activation of Lyn by both SH2 and
SH3 peptides. However, higher concentrations of the SH2 peptide are
needed to activate Lyn. We speculate that the SH3-binding motif may be
physiologically more relevant than the SH2-binding motif for Lyn activation.
Unc119 and Unc119-derived motif peptides induce autophosphorylation of
Lyn as well as kinase activity for the substrate. However, we have
observed that autophosphorylation occurs at a concentration of Unc119
that is frequently lower than required for the induction of kinase
activity. The discrepancy between autophosphorylation and substrate
phosphorylation has been noted previously by other investigators (40).
The interplay between autophosphorylation and kinase activity of SrcTKs
is very complex and is dependent upon multiple factors including
divalent cation and ATP concentrations and affinity for substrates and
activators. This complex process was recently investigated by Sun
et al. (41), who demonstrated that autophosphorylation of
Src is inhibited during the peak of substrate phosphorylation.
One of the important functions of IL-5 and other growth
factors is to delay eosinophil apoptosis and prolong eosinophil
survival. Lyn kinase has been shown previously (19, 20) to play a
non-redundant role in this process. Inhibition of Lyn blocks eosinophil
survival. When transduced into eosinophils Unc119 activated Lyn kinase
and inhibited eosinophil apoptosis in the absence of growth
factors. The anti-Unc119 antibody blocks eosinophil survival suggesting that Unc119 is important for Lyn activation in vivo. In
other work2 we have demonstrated that overexpression of
Unc119 induces activation of Lck and Fyn in T cells. Unc119-deficient
cells are unable to activate Lck and Fyn following T cell
stimulation. As a consequence these cells are unable to produce IL-2
and proliferate poorly. Thus, Unc119 appears to be a
receptor-associated activator of SrcTKs. We believe that in eosinophils
Unc119 and Lyn form an inactive complex with IL-5R and Src family tyrosine kinases. Unc119 induces the catalytic activity of these kinases through interaction with Src homology 2 and 3 domains. IL-5 stimulation of eosinophils increases Unc119 association with Lyn and induces its catalytic activity. Lyn is important for eosinophil survival. Eosinophils that
are transduced with Unc119 have increased Lyn activity and demonstrate
prolonged survival in the absence of IL-5. Inhibition of Unc119
down-regulates eosinophil survival. To our knowledge Unc119 is the
first receptor-associated activator of Src family tyrosine kinases.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and a common
subunit
(15). The earliest event of IL-5R signaling in eosinophils is the
activation of SrcTKs and Janus kinases (16-18). One of the SrcTKs that
plays a dominant role in eosinophils is Lyn kinase. We and others
(19-21) have shown that Lyn is important for eosinophil survival and
differentiation. The molecular mechanism of activation of Lyn kinase by
IL-5 receptor is unknown.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
in-frame with LexA DNA binding domain in pLexA plasmid. The
cytosolic portion of IL-5R
(98 amino acids) was PCR-amplified from
IL-5R
cDNA (a kind gift from Dr. J. Tavernier) using
AGCGAATTC-AGTGAGTGGAGCCAACCTA and AGAGGATCC-GCATGTGTGAGTTCATCAG
as 5' and 3' end primers, respectively, and ligated into pLexA after
EcoRI and BamHI restriction enzyme (Invitrogen)
reactions. The correct reading frame was confirmed by sequencing. The
bait construct and the fetal human cDNA library in the pB42AD
(Clontech) were cotransformed into EGY48 yeast
strain carrying the reporter plasmid p8opLacZ. A set of negative and positive controls was carried out according to the protocol provided. The transformed colonies were grown on SD/-His-Trp-Ura selection plates
for 5 days. The colonies were then replica-plated on induction plates
SD/Gal-Raf-His-Leu-Trp-Ura plates containing
5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside (X-gal)
(Invitrogen). The grown blue colonies on induction plates were streaked
on selection plates and then again on induction plates to allow
possible plasmid segregation and eliminate transition interactions. A
stock for each of the double positive colonies (Leu+, LacZ+;
grown blue colonies) was prepared and stored at
80 °C. These
colonies then were individually cultured in SD/
Trp media overnight
for plasmid segregation and selection for pB42AD-cDNA constructs.
The plasmid DNA was isolated (22) and electroporated (23) into
Escherichia coli KC8 cells. The transformants carrying pB42AD-cDNA were selected on M9/
Trp/Amp plates for two rounds followed by plasmid purification. The plasmids were purified from individual colonies using QIAprep Spin Miniprep kit (Qiagen) and were
cut with EcoRI and XhoI restriction enzymes
(Invitrogen) for confirmation of cDNA inserts which was then
partially sequenced using pB42AD sequencing primers at the University
of Texas Medical Branch Molecular Biology Core Lab. The partial
sequence of each colony was searched for homology to known sequences
with the BLAST program (www.ncbi.nlm.nih.gov/BLAST) with non-redundant
data base.
-D-galactopyranoside (Invitrogen) was
added to a final concentration of 0.1 mM and incubated for
4 h. The cells were then pelleted, resuspended in ice-cold PBS,
and sonicated. Triton X-100 (Sigma) was added to a final concentration
of 1% and was mixed and centrifuged at 12,000 × g,
4 °C, for 10 min. The recombinant protein was batch-purified with
glutathione-agarose (Santa Cruz Biotechnology). One ml of glutathione-agarose was added to 25 ml of sonicate and incubated at
room temperature for 1 h with rotation. The mixture was
centrifuged at 5,000 × g for 5 min. The pellet was
washed 5 times with 20 ml of PBS and was eluted with elution buffer (10 mM reduced glutathione in 50 mM Tris-HCl, pH 8;
1 ml of elution buffer per 1 ml of glutathione-agarose used). In some
cases it was subjected to thrombin cleavage before elution. 100 cleavage units of thrombin (Amersham Biosciences) was added to 5 mg of
GST-Unc119 and incubated at 22 °C with rotation for 20 h. The
GST then was separated by glutathione-agarose pull-down. The
supernatant containing cleaved Unc119 was aliquoted and stored at
20 °C. The purity of GST-Unc119 and Unc119 was assessed with Coomassie Blue staining. The amount of recombinant or lysate protein was determined with Coomassie Plus Protein Assay Kit (Pierce) following
the protocol provided.
20 °C until use.
80 °C for future kinase assays. The kinase assays were
performed in the presence or absence of Unc119 or its SH3 motif
(PSH3, QRKQPIGPED), proline to alanine mutant
(PP-A, QAKQAIGAED), SH2 motif (PSH2,
TCEHIYDFPPLS), phosphorylated SH2 motif (pPSH2,
TCEHIpYDFPPLS), an immunogenic peptide (PC, SERLPINRRDLDPNAGRC), or an SH3 peptide from CD2 (CD2 PSH3,
QKGPPLPRPRVQPKPPCG) (28) in a kinase buffer containing 20 mM Tris, pH 7.4, 2 mM MgCl2, 0.5 µM cold ATP, and 2 µCi of [
-32P]ATP
for 5 min. In the reactions in which the effect of Unc119 on the
activation of Lyn was evaluated, 2 µM Enolase (Sigma) or ATF-2 (Santa Cruz Biotechnology) was added to the reactions as the
substrate. This buffer was modified from Moarefi et al. (14) to decrease the high base-line phosphorylation of Lyn. The reactions were carried out at 30 °C for different times and were stopped by
addition 6× Laemmli's buffer. The reactions then were separated by
SDS-PAGE, transferred to PVDF membrane, and autoradiographed.
10
M, and cells were stimulated for 5 min or cultured
overnight. The cells were then lysed for kinase assays or used for
survival/apoptosis assay.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-interactive signaling molecules, we have used the LexA
Matchmaker Yeast Two-hybrid System. We constructed the bait pLexA-5R
by fusing the cytoplasmic tail of IL-5R
to the DNA binding domain of
LexA. By using this bait, we have screened the human fetal liver
cDNA library for IL-5R
interacting proteins. One of the
cDNAs was HRG4/human Unc119 (98% homology) (GenBankTM
accession number U40998) with a 1.4-kbp cDNA and a 240-amino acid
open reading frame (25).
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Fig. 1.
Unc119 is expressed in hematopoietic
cells and interacts with the IL-5 receptor. A, Unc119
mRNA expression in eosinophils. Total RNA from eosinophils was
reverse-transcribed into cDNA (RT-PCR). In the control
reaction (NO-RT) reverse transcriptase was omitted from the
reaction. For a positive control, we PCR-amplified (PCR)
Unc119 from pB42AD-Unc119. The reaction mixtures were separated,
transferred to a nylon membrane, and probed with
32P-labeled Unc119 cDNA followed by autoradiography.
B, Western blot showing the expression of Unc119 in
hematopoietic cells. Cell lysates from eosinophils (Eos),
mononuclear cells (MNC), and polymorphonuclear leukocytes
(PMN) were separated by SDS-PAGE and Western-blotted
(WB) with preimmune and rabbit anti-Unc119 antibody
(left panel). Another sample of the cell lysates were
Western-blotted (right panel) with the anti-Unc119
antibodies ( -Unc119) or the anti-Unc119 antibody-depleted
serum (
-Unc119 depleted) (n = 3).
C, comparison of Unc119 expression in various mouse organs.
Tissue lysates (60 µg of protein) from retina, heart, liver, lung,
and spleen was directly Western-blotted. The last lane shows
the position of rUnc119. D, Unc119 interacts with IL-5R
in the yeast. pLexA-5R
and pB42AD-Unc119 constructs were transfected
into the yeast EGY48 alone or together and plated first on selection
and then on induction plates and were checked for the activation of the
reporter gene lacZ. 53-T (pLexA-53 + pB42AD-T) and
Pos (pLexA-53 fused to AD-T) were positive and
Lam (pLexA-53 + pB42AD-Lam) was a negative control for the
experiment (n = 2). E, Unc119 is associated
with IL-5R
but not with common
c chain in eosinophils. Lysates
from non-stimulated or IL-5-stimulated eosinophils were
immunoprecipitated (IP) with either anti-IL-5R
or
anti-
c antibodies and Western-blotted with anti-Unc119 antibody
(above). In the 1st lane rUnc119 (50 ng) was loaded alone in
order to identify the position. The same membrane was stripped and
reprobed with either anti-IL-5R
(lower right) or with
anti-
c (lower left) antibodies (n = 3).
--
We reconfirmed the
interaction of IL-5R
and Unc119 in the yeast. Cotransfection of the
bait construct pLexA-5R
and pB42AD-Unc119 into the yeast activated
the reporter lacZ gene, indicating the interaction of
IL-5R
and Unc119. Neither the bait nor the Unc119 alone activated
the reporter gene (Fig. 1D). Next we assessed this
interaction in vivo in eosinophils in coimmunoprecipitation experiments. Unc119 was physically associated with IL-5R
in lysates from both IL-5-stimulated and non-stimulated eosinophils (Fig. 1E), which is anticipated from the result of the yeast
two-hybrid experiment. Under the same conditions, Unc119 did not
associate with the common
c chain. The anti-
c antibody is a
rabbit antibody and served as a control for the anti-Unc119 antibody.
SH2 and SH3 motifs of Unc119
. Lyn and Hck are members of SrcTKs and contain
SH2 and SH3 domains. Itk is a non-Src tyrosine kinase and has SH2, SH3,
and pleckstrin homology domains. The lipid kinase (PI3K) p110
contains no SH2 or SH3 domains. The physical association of Unc119 with
the foregoing molecules was studied in GST pull-down experiments (Fig.
2). The results show that Lyn and Hck but
not Itk or PI3K
associate with Unc119. Lyn exists in two molecular
weight isoforms (53 and 56 kDa), and both are coprecipitated with
GST-Unc119. In additional studies GST-Unc119 did not associate with
FAK, Abl, p85 subunit of PI-3 kinase, Raf-1, ERK and p38
mitogen-activated protein
kinase.2
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Fig. 2.
Unc119 associates with Lyn and Hck but not
with Itk or PI3K. A, GST (G) or GST-Unc119
(G-Unc) were incubated with the whole cell lysate
(WCL). The bound proteins were pulled down with
glutathione-agarose, separated by SDS-PAGE, and Western-blotted
(WB) with the respective antibodies (n = 4).
B, the membranes that were Western-blotted with anti-Hck and
anti-PI3K antibodies were stripped and reprobed with anti-GST
antibodies to confirm the presence and assess the quantity of GST or
GST-Unc119 in the binding reactions (n = 2).
c antibody. Unc119 coprecipitated with Lyn but not with the control antibody (see
also Fig. 1B). The bottom panel shows the amount
of Unc119 that did not bind to Lyn and was detectable in the
supernatant. The densitometric concentrations of rUnc119 (50 ng),
Lyn-bound Unc119, and non-bound Unc119 are 1.4, 0.8, and 1.8 units,
respectively. After correcting for the dilution factor of the
supernatant (one-fifth of the supernatant was loaded onto the gel), the
approximate densitometric concentration of Unc119 in eosinophils
(2 × 106) is 9.8 units (Lyn-bound and non-bound).
Based upon the densitometric analysis of rUnc119, we calculate that the
concentration of Unc119 is ~175 ng/106 eosinophils. This
calculation also indicates that about 8% of cellular Unc119 is
associated with Lyn kinase. We understand that this is not an accurate
estimate of Unc119 concentration because we did not take into account
the recovery of cellular Unc119 during lysis and processing.
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Fig. 3.
Unc119 interacts with Lyn through SH2 and SH3
domains. A, Unc119 coprecipitates with Lyn. An aliquot of
IL-5-stimulated eosinophil lysate was incubated with rabbit polyclonal
anti-Lyn ( -Lyn) or with rabbit polyclonal anti-
c
(
-
c) antibodies and immunoprecipitated
(IP). Fifty nanograms of recombinant Unc119
(rUnc), the immunoprecipitated pellets (Pellet,
upper panel), or one-fifth of each supernatants
(Sup, lower panel) was separated with SDS-PAGE
and Western-blotted with anti-Unc119 antibodies (n = 3). B, Unc119 associates with the SH2 and SH3 domains of
Lyn. rUnc119 (2 µM) was incubated with 2 µM
GST-fused recombinant domains (U, unique N terminus; SH2 and
SH3, SH2 and SH3 domains) of Lyn, Grb2, Shc, or Fyn in the lysis
buffer, pulled down with glutathione-agarose, and Western-blotted with
anti-Unc119 antibody (upper panel). One-fifth of each
supernatant was separated on another gel and processed as above
(lower panel) (n = 3). C, SH2 and
SH3 motif peptides of Unc119 bind Lyn. An aliquot of leukocyte lysate
was incubated with no peptide (
) or various concentrations of
biotinylated SH3 (PSH3), mutated SH3 in which conserved
arginine and proline residues were substituted with alanine
(PP-A) (left panel), non-phosphorylated SH2
(PSH2) or phosphorylated SH2 (pPSH2)
(right panel) motif peptides, precipitated with
streptavidin-agarose, and Western-blotted with the anti-Lyn antibody
(Pellet). One-fifth of the supernatant from each sample was
also separated and processed as above to confirm the presence of Lyn in
the lysates (lower panels, Sup)
(n = 3).
A). Similarly we synthesized a third
peptide encompassing the SH2 motif of Unc119 (residues 189-200)
(PSH2) and a fourth peptide that had the tyrosine residue
phosphorylated (pPSH2). The association between the SH2
domains and their cognate SH2 peptide is typically mediated through
phosphorylation of the tyrosine residue within the motif peptide.
However, several groups (27-29) have reported that this association
also occurs independent of phosphorylation. For example, the binding of
the SH2 domain of SLAM-associated protein with non-phosphorylated
signaling lymphocyte activation molecule (SLAM) peptides has been
examined by crystallography, and the nature of the binding interaction
has been fully delineated (29). The SH3 motif peptide
(PSH3) but not PP
A was able to associate
with and pull down Lyn from the cell lysate (Fig. 3C,
left panel). On the other hand, both the phosphorylated and non-phosphorylated SH2 motif peptides bound to Lyn kinase in a dose-dependent manner (Fig. 3C, right
panel). It should be noted that the binding through the SH3 domain
occurs at much lower concentrations than that through the SH2 domain.
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Fig. 4.
Unc119 activates Lyn kinase. Lyn was
immunoprecipitated from the cell lysates and incubated with rUnc119
(Unc) ± enolase or Sam68 as the substrate in a kinase buffer
containing [ -32P]ATP for 5 min. The reaction mixtures
were separated by SDS-PAGE, transferred to PVDF membrane, and
autoradiographed. A, Unc119 induces autophosphorylation of
Lyn (n = 3). B, Unc119 induces kinase
activity of Lyn for enolase (left) or Sam68
(right) phosphorylation. In the absence of Lyn, Unc119 did
not induce the phosphorylation of the substrates (not shown).
C, Unc119-derived SH3 motif peptide induces Lyn
autophosphorylation and substrate phosphorylation. Immunoprecipitated
Lyn was incubated in the kinase buffer with the SH3 (PSH3)
or the mutant (PP-A) peptide for 5 min in the absence
(left panel) or presence (right panel) of enolase
as the substrate. The kinase reactions were separated by SDS-PAGE and
autoradiographed (upper panels). The membranes were then
Western-blotted with the anti-Lyn antibody (lower panels)
(n = 3). D, a CD2-derived SH3 peptide also
induced Lyn autophosphorylation (upper panel) and substrate
phosphorylation (lower panel). E, Unc119-derived
non-phosphorylated (PSH2) or phosphorylated
(pPSH2) SH2 motif peptides induce Lyn autophosphorylation
and substrate phosphorylation. F, an additional control
peptide of similar length from Unc119 (PC) did not activate
Lyn.
A, PSH2, and
pPSH2) on Lyn activation. The PSH3 peptide
induced autophosphorylation of Lyn (Fig. 4C, left panel) and kinase activity for enolase (Fig. 4C,
right panel) at low concentrations (0.02 to 0.2 µM).
In contrast, the PP
A peptide induced negligible
autophosphorylation and no substrate phosphorylation by Lyn.
Previously, a SH3 motif peptide derived from CD2 was shown to induce
Fyn activation at relatively high concentrations (1 mM)
(31). We tested this peptide for Lyn activation under our assay
conditions. This peptide induced autophosphorylation of Lyn (Fig.
4D, upper panel) and kinase activity for enolase (Fig. 4D, lower panel) at
20 µM
concentration. The Unc119-derived SH3 peptide compares favorably with
the CD2 derived SH3 peptide in regard to Lyn activation.
A or
pPSH2 peptide increased Hck activation (Fig. 5A). In contrast, neither Unc119 nor the peptides modified
the catalytic activity of Itk or p38 (Fig. 5, B and
C). Enolase and transcription factor ATF-2 were used as the
substrates for Itk and p38, respectively. Itk from pervanadate-treated
mononuclear cells was active and phosphorylated enolase (Fig.
5B). Similarly, p38 from granulocyte-macrophage
colony-stimulating factor (GM-CSF)-treated mononuclear cells was active
and able to phosphorylate its substrate, ATF-2 (Fig.
5C).
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Fig. 5.
Unc119 activates Hck but not Itk or p38.
Immunoprecipitated Hck (A), Itk (B), or p38
(C) was incubated in the kinase buffer ± Unc119 (700 nM) or Unc119-derived peptides (20 µM) in the
presence substrates (enolase for Hck and Itk, and ATF for p38,
n = 3). Itk from pervanadate-treated and p38 from
granulocyte-macrophage colony-stimulating factor
(GM-CSF)-treated cell lysates (1st lanes
in each panel) were used as positive controls.
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Fig. 6.
A, IL-5 stimulates Unc119-associated Lyn
kinase activity. Eosinophils were incubated with buffer ( ) or IL-5
(10
10 M) (+) for 5 min, immunoprecipitated
(IP) with the preimmune, anti-Unc119, or anti-Lyn antibodies
followed by kinase assay using enolase (Enol) as a
substrate. *, Lyn immunoprecipitate was loaded alone in the absence of
enolase in the 1st lane in order to identify the position of
Lyn kinase. One of two separate experiments is shown. B,
protein transduction by Chariot. Cells were incubated with
OVA-FITC ± chariot and then examined by flow cytometry.
C, activation of Lyn by Chariot-transduced Unc119.
Eosinophils were incubated with Chariot and Unc119 (5 µg/106 cells) ± IL-5 (10
10
M) and then lysed, immunoprecipitated with anti-Lyn
antibody, followed by kinase assay using enolase as the substrate. One
of two separate experiments is shown. D, effect of Unc119 on
eosinophil apoptois. Eosinophils were incubated with chariot ± Unc119 or human serum albumin (HSA) (both at 5 µg/106 cells) and then cultured ± IL-5
(10
10 M). Eosinophil apoptosis was assessed
by flow cytometry on day 3 following staining for annexin V and
propidium iodide. Results of one of two separate experiments are
shown.
10 M). The likely
explanation for this strong activation is that we have used a
relatively high concentration of Unc119 (5 µg/106 cells)
in order to maximize the outcome and to mimic protein overexpression
that is typically achieved with expression plasmids. By using this
experimental model we studied the effect of Unc119 on eosinophil
survival. Unc119 significantly prevented eosinophil apoptosis (Fig.
6D, annexin V
and annexin V+ propidium-positive cells) and
increased survival (annexin V and propidium negative cells). In the
absence of growth factors 9-16% eosinophils were alive on day 3. The
number of live eosinophils was dramatically improved to 70% in
Unc119-transduced cells. The effect of Unc119 (5 µg/106
cells) was comparable with that seen with IL-5 (10
10
M). Based upon the results of the kinase experiment (Fig.
6C), we speculate that the transduced Unc119 promoted
eosinophil survival by activating Lyn kinase. However, we understand
that the effect of interaction of Unc119 with other unidentified
signaling molecule(s) in promoting eosinophil survival cannot be ruled out.
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Fig. 7.
The effect of the anti-Unc119 antibody on
eosinophil survival. Affinity-purified rabbit anti-Unc119 antibody
(raised against full-length recombinant Unc119) or normal rabbit serum
IgG (6 µg per 106 eosinophils) were transduced into
eosinophils using Chariot as described in Fig. 6. The cells were then
cultured with and without IL-5 (10 10 M) for
24 h, and their survival was assessed by flow cytometry after
staining with propidium iodide and annexin V. Results of one of three
separate experiments are shown.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
subunit as the bait
in the yeast two-hybrid screening. Unc119 has SH2- and SH3-binding
motifs. It associates with IL-5R
and Lyn kinase in eosinophils. More
importantly, it activates Lyn kinase in an SH2- and
SH3-dependent manner. Unc119 also binds to Hck but not to Itk or PI3K p110
. Furthermore, it does not interact with the SH2 or
SH3 domains of Grb2 and Shc, respectively. The SH3 motif peptide of
Unc119 activates both Lyn and Hck suggesting that a common principle is
applicable to Unc119 interaction with SrcTKs. Interestingly Itk, which
has both SH2 and SH3 domains, does not bind to nor is activated by
Unc119. The results suggest the existence of microspecificity of SH3
interaction with their ligands.
under basal
conditions, and Unc119 is inaccessible to the Lyn SH3 domain. Receptor
activation changes the conformation allowing the association of Unc119
with the Lyn SH3 domain and leading to kinase activation. The
association through the SH2 domain may have an additive effect. By
activating SrcTKs Unc119 may provide a novel signal-generating
mechanism for receptors and, therefore, may have broad biological relevance.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. J. Tavernier for the IL-5R
cDNA and Dr. G. Inana for a sample of anti-HRG4 antibody.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants AI PO1 46004, ES06676, and AI50179, the John Sealy Memorial Fund, and the McLaughlin Fellowship Fund.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.
¶ Supported in part by Harran University, SanliUrfa, Turkey.
To whom correspondence should be addressed: National Jewish
Medical and Research Center, 1400 Jackson St., Denver, CO 80206. E-mail: alamr@njc.org.
Published, JBC Papers in Press, December 19, 2002, DOI 10.1074/jbc.M208261200
2 M. Gorska, S. J. Stafford, and R. Alam, submitted for publication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: SrcTKs, Src family tyrosine kinases; IL, interleukin; HRG4, human retinal gene protein 4; SH2, Src homology 2 domain; SH3, Src homology 3 domain; GST, glutathione S-transferase; PBS, phosphate-buffered saline; RT, reverse transcribed; PVDF, polyvinylidene difluoride; FITC, fluorescein isothiocyanate; HSA, human serum albumin; PI3K, phosphatidylinositol 3-kinase.
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