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INTRODUCTION |
Macrophages play a critical role in the host response to
inflammation and bacterial infection (1). During these processes, macrophages are primarily involved in the phagocytosis of bacteria and
host cell debris, antigen processing and presentation, and the
secretion of reactive nitrogen intermediates and inflammatory cytokines
(e.g. tumor necrosis factor 
, interleukin-1, and
interleukin-6) (1). In vitro exposure of macrophages to
lipopolysaccharide (LPS),1 a
major component of the outer wall of Gram-negative bacteria, mimics
many of the effects bacteria have on macrophages in vivo, namely inducing the secretion of reactive nitrites and inflammatory cytokines and enhancing their tumoricidal activity (1).
The activation of macrophages by LPS is mediated by the binding of LPS
to CD14, a glycosylphosphatidylinositol-anchored protein found on the
surface of monocytes/macrophages and neutrophils (2). Binding of LPS to
CD14 is enhanced by LBP, an LPS-binding protein found in serum (3).
Since CD14 lacks an intracellular domain, it has previously been
unclear how CD14 transduces signals across the plasma membrane in
response to the binding of LPS. Recent studies suggest that members of
the Toll-like receptor family, and in particular Toll-like receptor 4, may serve as cell surface co-receptors with CD14 to mediate
transmembrane signal transduction (4-11). Members of the Toll-like
receptor family are structurally characterized by an extracellular
domain containing leucine-rich repeats, a transmembrane domain, and an
intracellular domain with sequence homology to the intracellular domain
of the interleukin-1 receptor (12).
LPS stimulation of macrophages leads to the activation of a variety of
proteins involved in signal transduction, including protein kinase C
(13, 14), Raf (15), mitogen-activated protein kinase (15, 16), p38
stress-activated protein kinase (17), Jun kinase (18), and
ceramide-activated protein kinase (19). However, it is still unclear
how the activation of these various signal-transducing proteins
mediates the variety of biological responses of macrophages to LPS.
Significantly, both in vitro and in vivo studies
with tyrosine kinase inhibitors have revealed that the activation of
tyrosine kinases is necessary for a number of the biological responses
of macrophages to LPS (e.g. tumoricidal activation)
(20-22).
The Src family tyrosine kinases Hck, Lyn, and Fgr have all been
implicated in playing a role in the biological response of macrophages
to LPS. Stimulation of monocytes/macrophages with LPS induces a rapid
increase in the specific kinase activity of Hck, Lyn, and Fgr and
physical association of Lyn with CD14 (23, 24). Moreover, enforced
expression of an activated form of Hck in Bac1.2F5 macrophage cells
augments tumor necrosis factor production in response to LPS
stimulation (24). Chronic exposure of bone marrow-derived macrophages
to LPS induces an increase in the expression of both Hck and Lyn (25).
Analysis of the promoter region of the hck gene has
facilitated definition of an element that confers LPS responsiveness
(26). Although these observations suggest that Src family kinases play
a role in the response of macrophages to LPS, and hence in the response
of macrophages to bacterial infection, the critical substrates of Src
family kinases that mediate the various biological responses of
macrophage to LPS have yet to be identified.
In the present study we have sought to identify proteins that are
phosphorylated by Src family kinases following LPS stimulation of the
Bac1.2F5 macrophage cell line. We show that Cbl is a substrate of Hck
in LPS-stimulated Bac1.2F5 cells. Further, we show that the
phosphorylation of Cbl facilitates the physical association of the p85
subunit of PI 3-kinase with Cbl. Notably, LPS stimulation enhances the
adherence of Bac1.2F5 cells, an effect that is dependent on the
activity of Src family kinases and PI 3-kinase. On the basis of these
findings, we postulate that Hck, at least in part, enhances the
adherence of LPS-stimulated macrophages via Cbl and PI 3-kinase.
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EXPERIMENTAL PROCEDURES |
Reagents--
Cell culture medium and G418 were from Trace
Biosciences Ltd. (Melbourne, Australia) and Life Technologies, Inc.,
respectively. Fetal calf serum (low endotoxin) was from P. A. Biologicals (Hawera, New Zealand). A rabbit anti-Hck polyclonal
antibody was a generous gift from Dr. Clifford Lowell, while a rat
anti-murine Hck monoclonal antibody was developed in this laboratory.
The anti-phosphotyrosine monoclonal antibody (4G10) and anti-p85
polyclonal antibody were from Upstate Biotechnology, Inc. (Lake Placid,
NY). Polyclonal rabbit anti-Cbl antibodies were from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA) and Dr. Wallace Langdon. Protein
A-Sepharose and glutathione-Sepharose were from Amersham Pharmacia
Biotech. [
-32P]ATP (3000 Ci/mmol) was obtained from
Bresatec Ltd. (Adelaide, Australia). Lipopolysaccharide
(Escherichia coli 0111:B4) and mitomycin C were purchased
from Sigma. ECL reagents were from Amersham Pharmacia Biotech.
Pfu DNA polymerase was obtained from Stratagene. EZ-LinkTM
sulfo-N-hydroxysuccinimide-biotin was from Pierce. PP2, PP3,
and wortmannin were obtained from Calbiochem-Novabiochem. All other
reagents were of the highest grade available.
Plasmid Construction--
A cDNA encoding activated murine
Hck (i.e. Hck499F) was excised from the plasmid pCDM8
Hck499F (a gift from Dr. Margaret Hibbs) with XbaI and
subcloned into the XbaI sites of pEF-BOS (27) to create
pEF-Hck499F or "blunted" and subcloned into the HpaI site of the retroviral vector pRuf Neo (a generous gift from Dr. Tom
Gonda (28)) to create pRuf Hck499F. The mammalian expression vectors
pEF-Hck267M (in which lysine 267 is replaced with methionine) and
pEF-Hck91A/499F (in which tryptophan 91 and tyrosine 499 are replaced
with alanine and phenylalanine, respectively) were constructed by
polymerase chain reaction. The mammalian expression vectors pEF-Cbl
(encoding wild type human Cbl) or pEF-Cbl731F (in which tyrosine 731 is
replaced with phenylalanine) were created by excising cDNA inserts
from pGEM 4Z-Cbl or pAlter-Cbl731F (generous gifts from Dr. Wallace
Langdon), respectively, and subcloning the fragments into the
XbaI sites of pEF-BOS.
Cell Culture, Retroviral Infection, and Transient
Transfection--
Bac1.2F5 cells were grown in Hepes-buffered
Dulbecco's modified Eagle's medium supplemented with 10%
heat-inactivated fetal calf serum and 25% L-cell conditioned medium
(as a source of colony-stimulating factor-1). For LPS stimulation
experiments cells were maintained in the above medium, but lacking
L-cell conditioned medium, for 16 h prior to stimulation.
2 cells were grown in Dulbecco's modified Eagle's
medium supplemented with 10% fetal calf serum. The retroviral constructs pRuf Neo and pRuf Hck499F were introduced into
2 cells by calcium phosphate-mediated transfection and
subjected to selection with 500 µg/ml G418 for 2 weeks. For infection
of Bac1.2F5 cells, 2 × 106 retrovirus-producing
2 cells were seeded in 10-cm tissue culture dishes. The
following day, the cells were treated with 10 µg/ml mitomycin C for
4 h and then washed extensively with Hepes-buffered Dulbecco's
modified Eagle's medium supplemented with 10% fetal calf serum.
Bac1.2F5 cells (2.5 × 106) were co-cultivated with
the mitotically inactivated 2 cells in the presence of 4 µg/ml polybrene for 2 days and then subjected to selection with 500 µg/ml G418 for 3 weeks. Individual clones were isolated by single
cell cloning. Human 293T cells were grown in RPMI medium supplemented
with 10% fetal calf serum and transfected with plasmid DNA using
polyethylenimine (29).
Cell Lysis, Western Blotting, and
Immunoprecipitation--
Bac1.2F5 or 293T cells were lysed directly in
tissue culture dishes with 1% Nonidet P-40 lysis buffer (20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 1% Nonidet P-40,
10% glycerol, 1 mM sodium orthovanadate, 0.1 mM sodium molybdate, 1 mM Pefabloc, 10 µg/ml
leupeptin, 100 units/ml aprotinin) for 30 min on ice. Lysates were
clarified by centrifugation at 13,000 × g for 10 min
at 4 °C, and protein concentrations were measured with a Bio-Rad
protein assay kit. To isolate the cytoskeletal fraction, Bac1.2F5 cells
were incubated on ice for 5 min and then washed three times with
ice-cold phosphate-buffered saline (PBS). Cells were lysed for 2 min
with 0.5 ml of cytoskeleton-stabilizing buffer (10 mM
Pipes, pH 6.8, 0.3 M sucrose, 0.5% Triton X-100, 100 mM KCl, 1 mM CaCl2, 2.5 mM MgCl2, 1 mM sodium
orthovanadate, 0.1 mM sodium molybdate, 1 mM
Pefabloc, 10 µg/ml leupeptin, 100 units/ml aprotinin). Plates were
rinsed briefly (<10 s) with 0.5 ml of cytoskeleton-stabilizing buffer
and pooled with the initial lysate. Cytoskeleton-stabilizing buffer
lysates (1.0 ml) were centrifuged at 13,000 × g for 10 min at 4 °C to remove cellular debris. This fraction is referred to
as the Triton X-100-soluble fraction. To extract the cytoskeletal
fraction, 1.0 ml of 1% Nonidet P-40 lysis buffer was added to the
residue remaining on the dishes and incubated for 30 min on ice. The
cytoskeletal fraction was clarified by centrifugation at 13,000 × g for 10 min at 4 °C. Western blotting and
immunoprecipitation of cell lysates was performed by standard techniques.
In Vitro Phosphorylation Assays--
The catalytic activity of
Hck was assessed by first immunoprecipitating the kinase from aliquots
of cell lysate containing 200 µg of protein. The immunoprecipitates
were washed three times with 1% Nonidet P-40 lysis buffer and then
once with kinase wash buffer (20 mM Hepes, pH 7.4, 0.1%
Nonidet P-40, and 0.1 mM sodium orthovanadate). The
immunoprecipitates were incubated at room temperature for 5 min in 30 µl of kinase buffer (20 mM Hepes, pH 7.4, 10 mM MnCl2, 0.1% Nonidet P-40, and 0.1 mM sodium orthovanadate) containing 10 µCi of
[-32P]ATP. Reactions were terminated by the addition
of an equal volume of 2× SDS-polyacrylamide gel electrophoresis sample
buffer and heating for 5 min at 95 °C. Phosphorylated proteins were
analyzed by SDS-polyacrylamide gel electrophoresis. Prior to exposure
to a PhosphorImager screen, gels were treated with 1 M KOH
at 55 °C for 2 h in order to reduce 32P label
incorporated into serine and threonine residues. To phosphorylate Cbl
in vitro, anti-Cbl immunoprecipitates were incubated for 30 min at 30 °C in kinase buffer containing 50 µM ATP and
100 ng of purified recombinant Hck.
GST-Hck Fusion Proteins: Generation, Biotinylation, Binding
Assays, and Far-Western Blotting--
The following glutathione
S-transferase (GST) fusion proteins of murine Hck were
generated: GST-Hck unique domain (amino acids 1-80 of the p59 isoform
of Hck), GST-Hck SH3 domain (amino acids 72-140), GST-Hck SH3 mutant
(tryptophan 91 mutated to alanine), GST-Hck SH2 domain (amino acids
142-250), and GST-Hck unique/SH3/SH2 domains (amino acids 1-250)
(referred to as GST-HckU32). The various domains were generated by
polymerase chain reaction using Pfu DNA polymerase and then
cloned into pGEX-2TH. The pGEX expression vectors were introduced into
competent DH5 E. coli bacteria, and the expressed GST
fusion proteins were purified by standard procedures with
glutathione-Sepharose beads (30). To biotinylate GST and GST-HckU32,
the affinity-purified proteins were dialyzed against 100 mM
sodium bicarbonate buffer (pH 8.5) and then incubated for 4 h on
ice with EZ-LinkTM sulfo-N-hydroxysuccinimide-biotin according to the manufacturer's instructions. Following biotinylation, the GST proteins were extensively dialyzed against 50 mM
Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, and
then stored at 
70 °C in the presence of 15% glycerol. Binding
assays were conducted by incubating 20 µl of glutathione-Sepharose
beads containing 2 µg of bound GST fusion protein with aliquots of
cell lysate containing 1 mg of protein for 2 h at 4 °C with
mixing. Beads were then washed four times with 1% Nonidet P-40 buffer
and subjected to Western blotting by standard techniques. Far-Western
blotting was performed by probing filters with biotinylated GST or
GST-HckU32 (100 ng/ml) for 5 h at 4 °C. Filters were washed
with TBST (Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20)
and then incubated for 1 h at 4 °C with
streptavidin-horseradish peroxidase in TBST. Following extensive washing, filters were developed with ECL reagents (Amersham Pharmacia Biotech).
Adhesion Assays--
Bac1.2F5 cells were seeded in six-well
tissue culture plates and grown until approximately 90% confluent. The
cells were then treated with either 20 µM PP3 (an
inactive Src family kinase inhibitor), 20 µM PP2 (an
active Src family kinase inhibitor), or 100 nM wortmannin (a PI 3-kinase inhibitor) for 60 min, followed by stimulation with 1 µg/ml LPS for 30 min. Differences in the adherence of the cells were
then assessed by incubating the cells in 2 ml of PBS containing 10 mM EDTA (PBS/EDTA) for 30 min on a rotating platform. Following this treatment, the nonadherent cells (i.e. the
"nonadherent" fraction) were collected and counted with the aid of
a hemocytometer. Cells that remained attached to the dishes
(i.e. the "adherent" fraction) were collected by
vigorous pipetting in PBS/EDTA and counted.
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RESULTS |
LPS Induces the Src Family Kinase-dependent
Phosphorylation of Cbl in Bac1.2F5 Cells--
Bac1.2F5 cells, which
were derived from SV40-transformed mature splenic macrophages (31),
exhibit many of the immunological properties of mature primary
macrophages, including the ability to secrete reactive nitrogen
intermediates and tumor necrosis factor when stimulated with LPS as
well as the ability to kill bacterial and tumor cells (32). Thus,
Bac1.2F5 cells represent a good model system to study LPS-induced
signal transduction. Stimulation of Bac1.2F5 cells with LPS induced a
time-dependent increase in tyrosine phosphorylation of a
number of cellular proteins (p190, p150, p120, p110, p75, and p66) (see
Fig. 1A). Since tyrosine phosphorylation of Cbl has previously been reported to occur in response to a variety of stimuli (33-45), we were interested in determining if the p120 protein that became tyrosine-phosphorylated in
response to LPS was Cbl. Accordingly, Cbl was immunoprecipitated from
lysates of Bac1.2F5 cells that had been stimulated with LPS for various
periods of time, and the immunoprecipitates were Western blotted with a
monoclonal anti-phosphotyrosine antibody. As shown in Fig.
1B, Cbl became tyrosine-phosphorylated in response to LPS
stimulation; maximal tyrosine phosphorylation was observed within
15-30 min of LPS stimulation and remained elevated for at least 2 h. It should be noted that two closely migrating proteins were detected
when the Cbl immunoprecipitates were Western blotted with either
anti-phosphotyrosine or anti-Cbl antibodies (Fig. 1B). These
observations suggest that (i) Bac1.2F5 cells may express alternatively
spliced forms of Cbl, (ii) Bac1.2F5 cells may, in addition to Cbl,
express a Cbl-related protein of similar size, or (iii) Cbl may undergo
limited proteolytic cleavage upon cell lysis. To ascertain if the
LPS-induced phosphorylation of Cbl is mediated by Src family kinases we
employed the specific Src family kinase inhibitor PP2 and the inactive
form of the inhibitor PP3 as a control. Significantly, pretreatment of
Bac1.2F5 cells with PP2, but not PP3, completely abolished the ability
of LPS to induce tyrosine phosphorylation of Cbl (Fig. 1C).
Additionally, pretreatment with PP2 was also found to abolish the basal
tyrosine phosphorylation of Cbl in unstimulated Bac1.2F5 cells (Fig.
1C).
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Fig. 1.
Src family kinase-dependent
phosphorylation of Cbl in LPS-stimulated Bac1.2F5 cells. Bac1.2F5
cells were stimulated with 1 µg/ml LPS for the indicated periods of
time then lysed. A, aliquots of the whole cell lysates
(WCL) were Western blotted (WB) with an
anti-phosphotyrosine (-pY) monoclonal antibody. Positions
of molecular mass markers (in kDa) are indicated on the
left, while tyrosine-phosphorylated proteins are indicated
on the right. B, Cbl was immunoprecipitated from
the whole cell lysates in A and subjected to
anti-phosphotyrosine Western blotting. The filter was stripped and
reprobed to establish that equal amounts of Cbl had been
immunoprecipitated at each time point. C, Bac1.2F5 cells
were treated with either 20 µM PP3 or PP2 for 60 min and
then stimulated with 1 µg/ml LPS for 30 min. Cbl was
immunoprecipitated from lysates of the cells and sequentially Western
blotted with anti-phosphotyrosine and anti-Cbl antibodies.
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Phosphorylation of Cbl by Hck in Bac1.2F5 Cells--
Given that
Hck has previously been implicated in the response of macrophages to
LPS (23, 24), we wanted to establish if Cbl physically associates with
Hck in Bac1.2F5 cells. To this end, Hck was immunoprecipitated from
lysates of Bac1.2F5 cells that had been stimulated with LPS for various
periods of time, and then the immunoprecipitates were Western blotted
with a polyclonal anti-Cbl antibody. This experiment revealed that Hck
is physically associated with Cbl prior to LPS stimulation of Bac1.2F5
cells and that no change in the amount of Cbl physically associated with Hck occurs following LPS stimulation (Fig.
2A). Cbl was not detected in
control immunoprecipitation reactions in which a control (irrelevant)
antibody was employed.2 The
presence of Hck in anti-Cbl immunoprecipitates could not be
demonstrated, since the heavy chain of the rabbit anti-Cbl immunoglobulin used in the immunoprecipitation reaction
potentially obscured Hck protein in the immunoblot.2
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Fig. 2.
Hck is physically associated with Cbl in
Bac1.2F5 cells and can directly phosphorylate Cbl. A,
Hck was immunoprecipitated from lysates of Bac1.2F5 cells that had been
stimulated with 1 µg/ml LPS for the indicated periods of time. The
anti-Hck immunoprecipitates were then Western blotted with an anti-Cbl
antibody. B, Hck was immunoprecipitated from lysates of
Bac1-Neo4 (Neo-4) and Bac1-Hck499F (Hck499F) cells. The
immunoprecipitates were then assessed for Hck kinase activity by
subjecting the immunoprecipitates to an autophosphorylation reaction.
C, whole cell lysates (WCL) of Bac1-Neo4 and
Bac1-Hck499F cells were subjected to anti-phosphotyrosine Western
blotting. Positions of molecular mass markers (in kDa) are indicated on
the left, while tyrosine-phosphorylated proteins are
indicated on the right. D, Cbl was
immunoprecipitated from lysates of Bac1-Neo4 and Bac1-Hck499F cells and
then sequentially Western blotted with anti-pY and anti-Cbl antibodies.
E, Cbl was immunoprecipitated from transiently transfected
293T cells and incubated in the absence () or presence (+) of
purified recombinant Hck and ATP. Cbl was then reimmunoprecipitated and
subjected to Western blotting with anti-phosphotyrosine and anti-Cbl
antibodies.
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In order to determine if Hck is capable of phosphorylating Cbl in
Bac1.2F5 cells, an activated form of Hck (i.e. Hck499F) was
introduced into Bac1.2F5 cells using the retroviral vector pRuf Neo
(28). The ectopically expressed Hck499F is enzymatically active, as
demonstrated by an autophosphorylation assay (Fig. 2B), and
induces tyrosine phosphorylation of primarily three proteins (p120,
p110, and p66) in the absence of LPS stimulation (Fig. 2C).
In order to determine if the p120 protein was Cbl, anti-Cbl antibodies
were employed in immunoprecipitation reactions using lysates of
Bac-Hck499F cells and a corresponding lysate of cells containing the
parental pRuf Neo vector (i.e. Bac-Neo4 cells). Western
blotting of the immunoprecipitates with an anti-phosphotyrosine antibody revealed that Cbl is indeed highly tyrosine-phosphorylated in
Bac1.2F5 cells expressing an activated form of Hck (Fig.
2D). Although this finding indicates that Hck can induce
tyrosine phosphorylation of Cbl in Bac1.2F5 cells, it does not allow us
to conclude that Hck directly mediates its phosphorylation. Thus, an
in vitro phosphorylation assay was performed to determine if
Hck can directly phosphorylate Cbl. Specifically, Cbl was
immunoprecipitated from transiently transfected 293T cells and then
incubated in the absence or presence of ATP and purified recombinant
Hck. The data shown in Fig. 2E clearly indicate that Hck can
phosphorylate Cbl. In an attempt to demonstrate that Hck is directly
involved in the LPS-induced tyrosine phosphorylation of Cbl in Bac1.2F5
cells, we generated clonal cell lines expressing a dominant-negative
form of Hck (i.e. Hck267M) following infection of the
parental Bac1.2F5 cells with the retroviral vector pRuf-Hck267M.
However, LPS-induced tyrosine phosphorylation of Cbl was not
significantly perturbed in the Bac-Hck267M cell lines
tested.2 Notably though, Western blotting of lysates
derived from various Bac-Hck267M cell lines with an anti-Hck antibody
revealed that the kinase-inactive mutant of Hck was only expressed at
levels that were approximately 2-3-fold above that of endogenous
Hck.2 It seems unlikely that this level of overexpression
of kinase-inactive Hck would be sufficient to exert a dominant-negative
effect over endogenous Hck.
The SH3 Domain of Hck Directly Mediates the Physical Association of
Hck with Cbl--
We next sought to identify which domain of Hck was
responsible for mediating the interaction of Hck with Cbl in Bac1.2F5
cells and, further, whether this is a direct physical interaction
between the two proteins. To address the first of these two questions, the ability of Cbl in lysates of unstimulated or LPS-stimulated Bac1.2F5 cells to associate with GST fusion proteins of Hck domains was
examined. The results shown in Fig.
3A indicate that the isolated SH3 domain of Hck can mediate the association of Cbl with Hck. It was
estimated that at least 20% of the total Cbl present in the cell
lysates was capable of associating with GST fusion proteins containing
the SH3 domain of Hck under the conditions employed.2 In
agreement with our co-immunoprecipitation experiments, Cbl from both
unstimulated and LPS-stimulated Bac1.2F5 cells associated equally well
with GST fusion proteins containing the SH3 domain of Hck. No
association of Cbl with the GST-Hck SH2 domain fusion protein was
observed, although Cbl had become tyrosine-phosphorylated in response
to LPS stimulation (Fig. 3A). Since this apparent lack of
binding of tyrosine-phosphorylated Cbl to the SH2 domain of Hck may
have simply been due to limited sensitivity of our assay, Cbl and
Hck499F were transiently co-expressed in 293T cells. Cell lysates
derived from 293T cells transfected with a plasmid encoding Cbl alone
or co-transfected with plasmids encoding Cbl and Hck499F were then
incubated with GST fusion proteins of Hck domains. As shown in Fig.
3B (upper panel), even under these
conditions only a very small fraction of the total Cbl present in the
cell lysates associated with the GST-Hck SH2 domain fusion protein. It
was estimated that greater than 50% of the total Cbl present in the
lysates associated with the GST-Hck SH3 domain fusion
protein.2 The filter was reprobed with anti-phosphotyrosine
antibodies in order to determine if tyrosine-phosphorylated Cbl binds
the SH3 domain of Hck in preference to the SH2. The results shown in
the lower panel of Fig. 3B revealed that approximately
2-3-fold more tyrosine-phosphorylated Cbl associated with the GST-Hck
SH3 domain fusion protein when compared with that which associated with
the GST-Hck SH2 domain fusion protein. To determine if the association
of Cbl with the SH3 domain of Hck is direct, Cbl was immunoprecipitated
from lysates of unstimulated Bac1.2F5 cells. The immunoprecipitates
were then subjected to far-Western blotting in which a biotinylated GST
fusion protein containing the unique, SH3, and SH2 domains of Hck
(referred to as GST-HckU32) was used as a probe. This experiment
revealed that Cbl could directly bind to Hck (Fig. 3C).
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Fig. 3.
The SH3 domain of Hck directly mediates the
physical association of Hck with Cbl. A, Bac1.2F5 cells
were either untreated () or stimulated (+) with 1 µg/ml LPS for 60 min and then lysed. Aliquots of cell lysate were incubated with
glutathione-Sepharose beads containing immobilized GST
(GST); GST-Hck unique domain (GST-unique);
GST-Hck SH3 domain (GST-SH3); GST-Hck SH2 domain
(GST-SH2); or GST-Hck unique, SH3, and SH2 domains
(GST-U32). The ability of Cbl to bind to the various fusion
proteins was then determined by Western blotting with anti-Cbl
antibodies. B, Cbl was expressed in the absence () or
presence (+) of Hck499F in 293T cells. Lysates of the transfected cells
were then incubated with glutathione-Sepharose beads containing
immobilized GST, GST-Hck SH3 domain, or GST-Hck SH2 domain. The ability
of Cbl to bind to the fusion proteins was determined by Western
blotting with anti-Cbl antibodies. C, lysates of
unstimulated Bac1.2F5 cells were subjected to immunoprecipitation
reactions in the absence () or presence (+) of anti-Cbl antibodies,
and the immunoprecipitates were subjected to Far-Western blotting
(FWB) with a biotinylated GST-HckU32 fusion protein. The
filter was then stripped and reprobed with anti-Cbl antibodies.
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The SH3 Domain of Hck Is Important for Phosphorylation of
Cbl--
Given the above findings, we wanted to establish if Hck
requires a functional SH3 domain to phosphorylate Cbl in
vivo. Accordingly, an SH3 domain mutant of Hck499F was created by
mutating tryptophan 91 to alanine. GST binding experiments confirmed
that the mutation abolished the ability of the Hck SH3 domain to bind
Cbl (Fig. 4A). The relative
ability of Hck499F and the SH3 domain mutant (i.e.
Hck91A/499F) to phosphorylate Cbl was tested by transiently co-expressing the proteins in 293T cells. Significantly, Hck91A/499F was found to be approximately 4-5-fold less efficient than Hck499F in
phosphorylating Cbl under these conditions (Fig. 4B).
Western blotting of the cell lysates with an anti-Hck antibody revealed that Hck499F and Hck91A/499F were expressed to similar levels (Fig.
4B). Intriguingly, Hck91A/499F exhibited a slightly reduced electrophoretic mobility in comparison with Hck499F (Fig.
4B). To exclude the possibility that the mutation may
detrimentally affect its specific activity, the ability of Hck91A/499F
to phosphorylate other proteins was investigated. Expression of
Hck91A/499F in 293T cells induced equivalent levels of tyrosine
phosphorylation of endogenous cellular proteins (Fig. 4C) or
co-transfected paxillin (Fig. 4D) as did Hck499F.
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Fig. 4.
The SH3 domain of Hck is important for the
in vivo phosphorylation of Cbl. A,
lysates of 293T cells transiently expressing Cbl were incubated with
glutathione-Sepharose beads containing immobilized GST, GST-Hck SH3
domain (GST-SH3), or GST-Hck SH3 domain mutant
(GST-SH3 mutant). The ability of Cbl to bind to the fusion
proteins was determined by Western blotting with anti-Cbl antibodies.
B, Cbl was immunoprecipitated from lysates of 293T cells
transiently expressing Cbl alone or together with Hck499F or
Hck91A/499F. The immunoprecipitates were then sequentially Western
blotted with anti-phosphotyrosine (-pY) and anti-Cbl
antibodies. Whole cell lysates (WCL) of the transfected
cells were then subjected to Western blotting with an anti-Hck
antibody. C, whole cell lysates of 293T cells transiently
expressing Hck499F or Hck91A/499F were subjected to Western blotting
with anti-phosphotyrosine and anti-Hck antibodies, respectively.
D, paxillin alone or together with Hck499F or Hck91A/499F
was transiently expressed in 293T cells. Paxillin was
immunoprecipitated from lysates of the 293T cells and sequentially
Western blotted with anti-phosphotyrosine and anti-paxillin antibodies.
Whole cell lysates of the transfected cells were then subjected to
Western blotting with an anti-Hck antibody.
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LPS Induces the Association of Cbl with p85--
It has previously
been reported that the p85 regulatory subunit of PI 3-kinase can
physically associate with tyrosine-phosphorylated Cbl (35, 37, 39, 40,
42, 46). Moreover, it has been reported that phosphorylated tyrosine
731 serves as a binding site on Cbl for p85 (37, 46). We were therefore
interested in determining if Cbl bound p85 following LPS stimulation of
Bac1.2F5 cells. It can be seen from Fig.
5A that p85 is physically
associated with Cbl prior to LPS stimulation of Bac1.2F5 cells but that
association of p85 with Cbl is quantitatively enhanced following LPS
stimulation of the cells. Notably, physical association of p85 with Cbl
(both basal and LPS-stimulated) was completely abolished by
pretreatment of the cells with the Src family kinase inhibitor PP2
(Fig. 5A).
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Fig. 5.
LPS stimulation enhances the association of
Cbl with the p85 subunit of PI 3-kinase. A, the
anti-Cbl immunoprecipitates shown in Fig. 1C were
sequentially Western blotted with antibodies that recognize p85 and
Cbl, respectively. B, Cbl was immunoprecipitated from
lysates of 293T cells transiently expressing either Cbl or Cbl731F
alone or together with Hck499F or Hck267M. The immunoprecipitates were
then sequentially Western blotted with anti-phosphotyrosine
(-pY), anti-p85, and anti-Cbl antibodies. Whole cell
lysates (WCL) of the transfected cells were Western blotted
with anti-p85 and anti-Hck antibodies.
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To ascertain if Hck can create a binding site on Cbl for p85, wild type
Cbl or Cbl731F (in which tyrosine 731 is replaced with phenylalanine)
was expressed either alone, or together with a kinase-active
(i.e. Hck499F) or kinase-inactive (i.e. Hck267M) form of Hck in 293T cells. The Cbl proteins were then
immunoprecipitated from lysates of the transiently transfected cells
using anti-Cbl-specific antibodies. Both wild type Cbl and Cbl731F
became tyrosine-phosphorylated when co-expressed with kinase-active
Hck, although wild type Cbl exhibited a somewhat higher degree of
tyrosine phosphorylation when compared with Cbl731F (Fig.
5B). This finding suggests that in addition to being capable
of phosphorylating Cbl on tyrosine 731, Hck is also capable of
phosphorylating Cbl at another site(s). The ability of endogenous p85
to physically associate with transfected wild type Cbl or Cbl731F was
tested by determining if p85 was present within the anti-Cbl
immunoprecipitates. As shown in Fig. 5B, co-expression of
wild type Cbl with an active, but not with an inactive, form of Hck
resulted in co-immunoprecipitation of p85 with Cbl. In contrast, no
co-immunoprecipitation of p85 with the Cbl731F mutant was detected,
although the mutant form of Cbl had become tyrosine-phosphorylated when
co-expressed with an active form of Hck (Fig. 5B). We have
found that co-expression of wild type Cbl with an activated form of Lyn
in 293T cells also facilitates the co-immunoprecipitation of endogenous
p85 with Cbl.2
LPS Induces Partial Translocation of Hck to the Cytoskeleton of
Bac1.2F5 Cells--
LPS stimulation of Bac1.2F5 cells induces
significant cell spreading, which presumably involves reorganization of
the cell's cytoskeleton. We were therefore curious to determine if
Hck, Cbl, or p85 became associated with the cytoskeleton of Bac1.2F5
cells in response to LPS stimulation. Partial translocation of both the
p59 and p56 isoforms of Hck from the soluble fraction to the cytoskeletal fraction of Bac1.2F5 cells was observed within 30 min of
LPS stimulation and increased further by 60 min (see Fig. 6A). Although it is not yet
clear why the p56 isoform of Hck found in the cytoskeletal fraction
exhibits a reduced electrophoretic mobility on SDS-polyacrylamide gel
electrophoresis gels in comparison with the p56 isoform in the soluble
fraction, it is most likely to be due to differences in their
phosphorylation status. Although there is a suggestion from Fig.
6A that Cbl and p85 may also partially translocate to the
cytoskeleton, we have been unable to conclusively demonstrate
translocation of these two proteins in response to LPS stimulation of
Bac1.2F5 cells.
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Fig. 6.
LPS induces partial translocation of Hck to
the cytoskeleton of Bac1.2F5 cells. Bac1.2F5 cells were stimulated
with 1 µg/ml LPS for the indicated periods of time then the Triton
X-100-soluble and cytoskeletal fractions were isolated as described
under "Experimental Procedures." Equal volumes of the Triton X-100
soluble (S) and cytoskeletal (C) fractions were
Western blotted with antibodies that recognize Hck, Cbl, and p85,
respectively (A) or far-Western blotted (FWB)
with a biotinylated GST-HckU32 fusion protein (B). The
asterisks in B indicate proteins that bound the
biotinylated GST-HckU32 fusion protein in an LPS-dependent
manner. Positions of molecular mass markers (in kDa) are indicated on
the left.
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It is possible that the partial translocation of Hck may be due, at
least in part, to the formation of Hck-binding sites in the
cytoskeleton of LPS-stimulated Bac1.2F5 cells. The presence of
Hck-binding proteins in the cytoskeleton of Bac1.2F5 cells was
investigated by subjecting the soluble and cytoskeletal fractions of
unstimulated and LPS-stimulated cells to far-Western blotting with a
GST-Hck fusion protein. Whereas GST only bound two major proteins (p125
and p75), the GST-Hck fusion protein (i.e. GST-HckU32) bound
a number of additional proteins (p120, p68, p62, p60, and p44) (Fig.
6B). Binding of GST-HckU32 to p68 was more pronounced in the
cytoskeletal fraction, and notably, binding of the fusion protein to
p68 increased in response to LPS stimulation of the cells. Likewise,
binding of GST-HckU32 to p44 also increased in response to LPS
stimulation; however, p44 binding occurred exclusively in the
cytoskeletal fraction (see Fig. 6B). The identity of p68 and
p44 remains to be determined.
LPS Enhances the Adherence of Bac1.2F5 Cells in a Src Family Kinase
and PI 3-Kinase-dependent Manner--
Phosphorylation of Cbl by Src
family kinases and the binding of PI 3-kinase to
tyrosine-phosphorylated Cbl have previously been shown to be important
for 
1-integrin-mediated macrophage adhesion (47). To
determine if LPS alters the adherence of Bac1.2F5 cells, an adhesion
assay that relied upon the ability of EDTA coupled with mechanical
shaking to promote detachment of cells from tissue culture dishes was
used to assess the relative adherence of unstimulated and
LPS-stimulated Bac1.2F5 cells. Using this assay, LPS stimulation
induces an approximately 2.5-fold increase in the adherence of Bac1.2F5
cells (Fig. 7A).
Significantly, the ability of LPS to enhance the adherence of Bac1.2F 5 cells could be completely abolished by pretreating the cells with the
Src family kinase inhibitor PP2 prior to LPS stimulation (Fig.
7A). Pretreating the cells with PP2 was also found to
perturb the basal adherence of unstimulated Bac1.2F5 cells, suggesting
that both basal and LPS-enhanced Bac1.2F5 cell adhesion are dependent
on Src family kinase activity (Fig. 7A). To determine if PI
3-kinase activity is required for the observed enhancement in the
adherence of LPS-stimulated Bac1.2F5 cells, adhesion assays were
performed on cells that had been treated with the PI 3-kinase inhibitor wortmannin prior to stimulation with LPS. As was the case for PP2,
pretreatment with wortmannin suppressed both the basal and LPS-enhanced
adherence of Bac1.2F5 cells (Fig. 7B).
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Fig. 7.
LPS enhances the adherence of Bac1.2F5
cells. Bac1.2F5 cells were treated with 20 µM PP3 or
PP2 (A) or with Me2SO (DMSO) or 100 nM wortmannin (B) for 60 min and then left
unstimulated () or stimulated (+) for 30 min with 1 µg/ml LPS. The
cells were then treated with 10 mM EDTA in PBS for 30 min
on a rotating platform, and the number of nonadherent cells (as a
percentage of the total number of cells) was determined. The data
presented are the averages of three independent experiments in which
each treatment was performed in duplicate.
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DISCUSSION |
In the present study, we sought to identify proteins that are
phosphorylated by Src family kinases following LPS stimulation of
Bac1.2F5 cells, since these proteins may play a critical role in the
biological response of macrophages to LPS. We have shown that one of
the proteins that became tyrosine-phosphorylated in an Src family
kinase-dependent manner following LPS stimulation of
Bac1.2F5 cells was Cbl. Tyrosine phosphorylation of Cbl was found to be
both gradual and persistent. Maximal tyrosine phosphorylation of Cbl
did not occur until 15-30 min after LPS stimulation and remained
elevated for at least 2 h. Such kinetics of tyrosine phosphorylation of Cbl contrast with the rapid and transient tyrosine phosphorylation of Cbl following colony-stimulating factor-1
stimulation of Bac1.2F5 cells (34) but are somewhat similar to those
observed upon plating macrophages onto fibronectin-coated tissue
culture dishes (43). Significantly, we found that Cbl is physically associated with Hck in Bac1.2F5 cells and that enforced expression of a
constitutively activated form of Hck in Bac1.2F5 cells induces tyrosine
phosphorylation of Cbl in the absence of LPS stimulation. Additionally,
Hck was shown to be capable of directly phosphorylating Cbl in
vitro. Taken together, these findings are consistent with the
notion that Hck directly mediates, at least in part, the
phosphorylation of Cbl in LPS-stimulated Bac1.2F5 cells.
By employing GST fusion proteins of Hck, we have been able to
demonstrate that the association of Cbl with Hck is mediated by a
direct interaction of Cbl with the SH3 domain of Hck. The interaction
of Cbl with the SH3 domain of Hck is likely to be important for its
subsequent phosphorylation, since an SH3 domain mutant of Hck499F was
found to be 4-5-fold less efficient than Hck499F in phosphorylating
Cbl in transiently transfected 293T cells. The reduced ability of the
SH3 domain mutant to phosphorylate Cbl does not appear to be a
consequence of the mutation negatively impacting on its specific
activity, since its ability to phosphorylate other proteins
(e.g. endogenous cellular proteins or co-transfected paxillin) was comparable with that of Hck499F. Since these experiments were performed in an overexpression system (i.e. 293T
cells), the 4-5-fold lower phosphorylation of Cbl by the Hck499F SH3
domain mutant may actually underestimate the contribution of the SH3 domain of Hck to the phosphorylation of Cbl when the proteins are
expressed at physiologically relevant levels (e.g. in
Bac1.2F5 cells).
Our finding that the SH3 domain of Hck is sufficient to bind Cbl
contrasts with two previous reports describing interactions between Hck
and Cbl (35, 48). The GST-SH3 domain fusion protein utilized in this
study encompassed amino acids 72-140 of murine Hck, whereas the
GST-SH3 domain fusion proteins employed in the previous studies
encompassed amino acids 87-137 (35, 48). X-ray crystallographic and
NMR studies, however, have revealed that the SH3 domain of Hck is
formed by amino acids 80-135 (49, 50). Thus, the fact that the GST
fusion proteins employed in the prior studies lacked amino acids 80-86
may potentially explain the inability of those fusion proteins to bind
Cbl. Our observation that the SH3 domain of Hck is capable of binding
Cbl is consistent with previous reports that GST fusion proteins
encompassing just the SH3 domain of other Src family kinases
(e.g. Fyn, Lck, and Lyn) are capable of binding Cbl (38,
51).
Even when tyrosine-phosphorylated, 2-3-fold more Cbl bound the SH3
domain of Hck when compared with that which bound the SH2 domain of
Hck. Preferential binding of tyrosine-phosphorylated Cbl to the SH3
domain of Hck would have at least two important consequences. First, it
may allow Hck to simultaneously bind another protein via its SH2
domain; second, the tyrosine residue(s) on Cbl that is phosphorylated
by Hck may remain accessible to bind SH2 domain-containing proteins,
thus potentially mediating the formation of a multiprotein-signaling
complex. Tyrosine phosphorylation of Cbl has previously been reported
to occur in response to a variety of stimuli, including cytokine
stimulation (33-37), activation of the T-cell and B-cell receptors
(38-42), cell adhesion (43), and oncogenic transformation (44, 45).
Cbl is able to bind a number of SH3 domain-containing proteins involved
in signal transduction (e.g. Grb2), and when
tyrosine-phosphorylated bind SH2 domain-containing proteins
(e.g. p85 subunit of PI 3-kinase) (33, 35-37, 39-43, 45).
These observations have led to the suggestion that Cbl may serve as a
docking protein to facilitate assembly of multiprotein signaling
complexes. Our finding that Hck phosphorylates Cbl in response to LPS
stimulation of Bac1.2F5 cells suggests that Hck may mediate the
formation of such a multiprotein-signaling complex in LPS-stimulated
macrophages. Indeed, we have found that association of the p85 subunit
of PI 3-kinase with Cbl is enhanced following LPS stimulation of
Bac1.2F5 cells and that this association of p85 with Cbl is dependent
on Src family kinase activity. Transient expression experiments in 293T
cells have allowed us to demonstrate that phosphorylation of Cbl by Hck
can facilitate the physical association of Cbl with p85. Moreover, we
have been able to demonstrate that phosphorylation of Cbl on tyrosine
731 is necessary for its physical association with p85. Tyrosine 731 is
found within the sequence CTYEAMYN, which conforms to the
minimal consensus binding sequence of YXXM for the SH2
domains of p85 (52).
What then is the biological significance of the LPS-induced tyrosine
phosphorylation of Cbl by Hck? With regard to this question, it is
worth noting that macrophages derived from mice simultaneously carrying
null mutations at the hck, lyn, and
fgr loci exhibit no discernible defects in terms of nitrite
or inflammatory cytokine secretion when stimulated in vitro
with LPS (53). Similarly, activation of the MAP kinase and JNK kinase
signal transduction pathways by LPS appears normal in
hck
/lyn/fgr/
triple mutant macrophages (53). However, the ability of these macrophages to kill tumor cells in vitro is partially
impaired (53). Given that cell adhesion plays a role in tumor cell
killing, this partial defect in macrophage function may be due to a
cell adhesion defect. Indeed, Meng and Lowell (47) have subsequently reported that
hck/lyn/fgr/
triple mutant macrophages have a major defect in 1
integrin-mediated cell adhesion and spreading. In vivo this
defect manifests itself in the form of reduced migration of macrophages
into the peritoneum of mice injected with the inflammatory stimulus
thioglycolate (47). Taken together, the findings by Meng and Lowell
(47, 53) suggest that at the molecular level Hck, Lyn, and Fgr are not
components of the signal transduction pathways that control nitrite and
inflammatory cytokine secretion by macrophages, or if they are, another
tyrosine kinase(s) can fulfill this function in their absence. However,
these findings do suggest the three Src family kinases are
indispensable components of the signal transduction pathways
controlling macrophage adhesion/spreading and migration. Interestingly,
a recent report has proposed that the activation state of Hck might
serve as a "molecular switch" to regulate myelomonocytic motility
and adherence in response to urokinase (54).
Significantly, we have found that LPS stimulation increases the
adherence of Bac1.2F5 cells. Moreover, the effect of LPS on Bac1.2F5
cell adhesion was found to be dependent on the activity of Src family
kinases and PI 3-kinase and to correlate with the tyrosine
phosphorylation status of Cbl. Accordingly, we propose that LPS may
enhance the adherence of Bac1.2F5 cells via a Hck-Cbl-PI 3-kinase
signal transduction pathway. Specifically, we propose that Hck directly
phosphorylates Cbl on tyrosine 731 in response to LPS stimulation, thus
facilitating the physical association of Cbl with p85 and leading to
the coordinated activation of PI 3-kinase. However, since Lyn is also
capable of phosphorylating Cbl in 293T cells and facilitating the
physical association of p85 with Cbl, we cannot exclude the possibility
that Lyn (and possibly Fgr) might also contribute to the tyrosine
phosphorylation of Cbl and its association with p85 in LPS-stimulated
Bac1.2F5 cells. The fact that a similar mechanism has been proposed for 1-integrin-mediated macrophage cell adhesion (47)
suggests that Src family kinases, Cbl, and PI 3-kinase may regulate the adherence of macrophages in response to various stimuli.
Further investigation will be required to elucidate how activation of
this Hck-Cbl-PI 3-kinase signal transduction pathway enhances the
adherence of LPS-stimulated Bac1.2F5 cells. However, since the
phosphorylation products of PI 3-kinase (e.g. PI
3,4,5-trisphosphate) can interact with a subset of pleckstrin homology
domains (55), it seems likely that a pleckstrin homology
domain-containing protein might be involved in regulating the adherence
of macrophages in response to LPS. Notably, Hmama et al.
(56) have recently proposed a model in which LPS-induced monocyte
adherence is mediated by the pleckstrin homology domain-containing
protein cytohesin-1 (57, 58). Specifically, Hmama et al.
have postulated that the activation of PI 3-kinase following LPS
stimulation leads to the generation of PI 3,4,5-P3, which
binds to, and modifies the properties of, cytohesin-1 (56). Engagement
of the cytoplasmic tail of CD18 by cytohesin-1 would then lead to the
conversion of low avidity LFA-1 (CD11a/CD18) molecules into high
avidity molecules capable of increased binding to intercellular
adhesion molecule 1 (56). This model for LPS-induced
monocyte/macrophage cell adhesion can possibly now be extended further
to incorporate the signal transduction events we have described in this
report, namely that the activation of PI 3-kinase in response to LPS
stimulation might be mediated by the phosphorylation of Cbl by Hck.
We have been unable to maintain the phenotype of clonally derived
Bac1-Hck499F cells. Upon extended passages, the cells (i) became
morphology heterogeneous and (ii) exhibited levels of Hck activity and
tyrosine phosphorylation of cellular proteins indistinguishable from
that seen in Bac1.2F5 cells infected with the parental
retrovirus.2 Thus, it has not been possible to ascertain if
enforced expression of a constitutively activate form of Hck alone is
sufficient to enhance the adherence of Bac1.2F5 cells. Additionally, it
is unclear if tyrosine phosphorylation of Cbl (and its association with
p85) alone accounts for the enhanced adherent properties of
LPS-stimulated Bac1.2F5 cells. Intriguingly, we have detected partial
translocation of Hck to the cytoskeleton of LPS-stimulated Bac1.2F5
cells. While the precise nature of this process remains to be
established, it appears to be an F-actin-dependent process,
since the actin-depolymerizing drug cytochalasin D perturbs Hck
translocation.2 Additionally, far-Western blotting
experiments revealed the presence of two proteins (p68 and p44) in the
cytoskeletal fraction of Bac1.2F5 cells that bind Hck in an
LPS-inducible fashion. However, the role these two proteins play, if
any, in the translocation of Hck to the cytoskeleton is not known.
Given that the cell's cytoskeleton is intimately involved in cell
adhesion, it is tempting to speculate that in addition to the
phosphorylation of Cbl, phosphorylation of cytoskeletal or
cytoskeletally associated proteins by Hck may also contribute to
enhancing the adherence of LPS-stimulated macrophages. Identification
of additional substrates of Hck in LPS-stimulated Bac1.2F5 cells should
provide further insight into the molecular mechanisms governing
macrophage adhesion.
,
TOP
ABSTRACT
INTRODUCTION
To whom correspondence should be addressed. Tel.: 61-3-9341-3155;
Fax: 61-3-9341-3191; E-mail: Glen.Scholz@ludwig.edu.au.
