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Department of Microbiology and Immunology (M.R.S.) Department
of Physiology (J.H., C.C.-S.) University of Michigan Medical
School Ann Arbor, Michigan 48109
Hagedorn Research
Institute (N.B.) DK-2820 Gentofte, Denmark
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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(7), FAK, paxillin, tensin (9),
p130cas, CrkII, c-Src, c-Fyn, c-Cbl, and Nck
(10). Although JAK2 is required to tyrosyl phosphorylate and/or
activate many of these signaling molecules, the factor(s) that
dephosphorylate these signaling molecules and terminate GH signaling
are poorly understood.
One possible mechanism for the termination of GH signaling is the
activation and/or recruitment of a protein tyrosine phosphatase to
GHR/JAK2 signaling complexes. This phosphatase would dephosphorylate
GHR and/or JAK2 and result in down-regulation of GH signaling.
Dephosphorylation of the critical activating tyrosine within the kinase
domain of JAK2 would be expected to deactivate JAK2, whereas
dephosphorylation of GHR and other tyrosines within JAK2 would be
expected to remove binding sites for various signaling proteins. Two
phosphatases that could potentially negatively regulate GH signaling
are the SH2 domain-containing phosphatases SHP-1 and SHP-2 (11). These
phosphatases are thought to be activated as a consequence of binding to
phosphorylated tyrosines (12). SHP-1 has been demonstrated to regulate
negatively JAK/STAT signaling in hematopoietic cells mediated by the
receptors for erythropoietin (EPO), interleukin-4 (IL-4), interferon
(IFN), or interleukin 3 (IL-3), members of the cytokine receptor
superfamily that bind Janus tyrosine kinases (JAKs) (13, 14, 15, 16). Recently,
it has been suggested that SHP-1 plays a role in the dephosphorylation
of JAK2 in liver in response to GH (17). However, SHP-1 is not
expressed in all GH-responsive tissues and cell lines (18), including
the highly responsive 3T3-F442A cells (M. Stofega and C. Carter-Su,
unpublished observation). Furthermore, a fusion protein encoding the N-
and C-terminal SH2 domains of SHP-1 does not associate with GHR from
GH-treated 3T3-F442A cells (8). These characteristics suggest that
SHP-1 may not be a general negative regulator of GHR signaling in all
GH-responsive tissues and cell lines.
We therefore investigated the role of the ubiquitously expressed
protein tyrosine phosphatase SHP-2 (11, 19) in GH signaling. Both GHR
and JAK2 have potential binding sites for the SH2 domains of SHP-2, and
SHP-2 is a signaling molecule for GH, based on the tyrosyl
phosphorylation of SHP-2 in response to GH (7, 8). Although SHP-2 has
been generally regarded as a positive regulator of growth factor
signaling and has been implicated as a positive regulator of signaling
by the cytokine receptor ligands IFN and PRL (14, 20), as well as
for GH (8), recent studies indicate that SHP-2 is a negative regulator
of signaling by leptin, ciliary neurotropic factor (CNTF), IFN,
IFN
, and insulin (21, 22, 23, 24, 25). Here, we demonstrate the direct
association of the SH2 domains of SHP-2 with tyrosyl-phosphorylated
GHR, but not JAK2, from GH-treated cells. Tyrosine 595 of rat GHR and
to a lesser extent tyrosine 487, mediates the association of the SH2
domains of SHP-2 with GHR. To assess the effects of SHP-2 on GH
signaling, stable cell lines were created that express
tyrosine-to-phenylalanine mutations of tyrosine 595 or 487.
Tyrosine-to-phenylalanine mutation of tyrosine 595, and to a lesser
extent tyrosine 487, prolongs the tyrosyl phosphorylation of STAT5B in
response to GH, consistent with the effects of these mutations on the
level of SHP-2 binding to GHR. The increased duration of STAT5B tyrosyl
phosphorylation in cells expressing Y595F GHR correlates with prolonged
tyrosyl phosphorylation of GHR and JAK2. These results suggest that
SHP-2 binding to specific tyrosines in GHR negatively regulates GH
signaling.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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(7, 8). Using
glutathione-S-transferase (GST) fusion proteins, we have
also shown that SIRP from GH-treated cells binds to the SH2 domains
of SHP-2 (M. Stofega and C. Carter-Su, unpublished data). Kim et
al. (8) have shown a similar GH-dependent association of GHR/JAK2
complexes with the SH2 domains of SHP-2. To confirm that the SH2
domains of SHP-2 mediate its association with GHR and that GH
stimulates the association of the SH2 domains of SHP-2 with GHR, cell
lysates from untreated or GH-treated 3T3-F442A cells were incubated
with immobilized GST fusion protein fused to the N- and C-terminal SH2
domains of SHP-2. Bound proteins were eluted and immunoblotted with
antibody to GHR. As shown in Fig. 1
, lanes 3 and 4, GH stimulates the association of GHR with the SH2
domains of SHP-2. No GHR was detectable in cell lysates precipitated
with GST alone (Fig. 1, lanes 5 and 6). Interestingly, the GHR
coprecipitating with the SH2 domains of SHP-2 comigrates with the upper
portion of the GHR coimmunoprecipitated by GH (Fig. 1, lane 2),
suggesting that the SH2 domains of SHP-2 associate with a slower
migrating subset of GHR. This subset is presumably the
tyrosyl-phosphorylated form of GHR, because phosphorylation of proteins
often decreases the mobility of proteins in SDS-PAGE gels, and the SH2
domains of SHP-2 would be expected to bind only to the
tyrosyl-phosphorylated form of GHR. The results of Fig. 1 therefore
demonstrate that SHP-2 associates with GH-activated GHR via the SH2
domains of SHP-2.
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GHR and JAK2 immunoprecipitates were separated
by SDS-PAGE, transferred to nitrocellulose, and incubated first with
GST fused to the N- and C-terminal SH2 domains of SHP-2 followed by
SHP-2 (far Western blot). The blot was stripped and reprobed with
antiphosphotyrosine antibody (PY) to show the migrations and
relative amounts of tyrosyl-phosphorylated JAK2 and GHR in the JAK2
and GHR immunoprecipitates. In GHR immunoprecipitates, GHR
migrates as a broad tyrosyl-phosphorylated band, with a small amount of
tyrosyl-phosphorylated JAK2 migrating slightly slower than GHR (Fig. 2, lane 2). In JAK2
immunoprecipitates, the ratio between tyrosyl-phosphorylated GHR and
JAK2 is reversed. There is a smaller amount of tyrosyl-phosphorylated
GHR migrating slightly faster than a larger amount of
tyrosyl-phosphorylated JAK2 (Fig. 2, lane 4). Far Western blotting with
the SH2 domains of SHP-2 and SHP-2 reveals that the SH2 domains of
SHP-2 bind to proteins that comigrate with GHR and not JAK2 in both the
GHR and JAK2 immunoprecipitates (Fig. 2, lanes 6 and 8). Further,
the relative amounts of SH2 domain binding to proteins in the JAK2
and GHR immunoprecipitates correspond to the relative amounts of
tyrosyl-phosphorylated GHR in those immunoprecipitates. These results
therefore indicate that the SH2 domains of SHP-2 bind directly to
tyrosyl-phosphorylated GHR in response to GH.
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PY. As expected, tyrosyl
phosphorylated, wild-type GHR associates with the SH2 domains of SHP-2
(Fig. 3, lane 4). The association of
Y487F GHR with the SH2 domains of SHP-2 is reduced compared with that
of wild-type GHR, suggesting that Y487 may be a binding site for the
SH2 domains of SHP-2. Notably, the association of the SH2 domains of
SHP-2 with tyrosyl-phosphorylated Y595F GHR is undetectable (Fig. 3, compare lanes 4 and 8), suggesting that Y595 is a major site of
interaction of GHR with the SH2 domains of SHP-2. To ensure that the
reduced association of SHP-2 with Y487F or Y595F GHR was due to loss of
a specific SHP-2 interaction site, and not simply due to
tyrosine-to-phenylalanine mutation of GHR, the interaction of the SH2
domains of SHP-2 with Y627F GHR, which retains the SHP-2 interaction
motifs, was examined. As shown in Fig. 3, lane 10, mutation of Y627 to
phenylalanine does not impair association of SHP-2 with
tyrosyl-phosphorylated GHR. The differing abilities of GHR to bind to
the SH2 domains of SHP-2 were not due to different levels of GHR
expression, since the levels of wild-type GHR, Y595F GHR, Y487F GHR,
and Y627F GHR were similar, as judged by immunoprecipitation with
GHR and immunoblotting with GHR (data not shown). The SH2 domains
of SHP-2 do not associate with tyrosyl-phosphorylated JAK2 expressed in
the absence of GHR (Fig. 3, lane 2), in agreement with previous results
(27). The results of Fig. 3 suggest that tyrosine 595 of GHR and, to a
lesser extent, tyrosine 487 of GHR mediate the association of the SH2
domains of SHP-2 with tyrosyl-phosphorylated GHR.
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, that the SH2
domains of SHP-2 directly associate with wild-type GHR from GH-treated
CHO-GHR cells. In preliminary experiments, mutating tyrosine 487 of GHR
and tyrosine 595 of GHR reduced the binding of the SH2 domains of SHP-2
to GHR to about 35% and 10%, respectively, of that of wild-type GHR
(data not shown), consistent with the COS cell experiment (Fig. 3). We
next examined the effect of disruption of SHP-2 binding to GHR on GH
signaling. The time course of tyrosyl phosphorylation of STAT5B in
response to GH in CHO cells expressing wild-type GHR, Y487F GHR, and
Y595F GHR was investigated. Tyrosyl phosphorylation of STAT5B is
required for it to bind DNA and transactivate target genes (29). CHO
cells were stimulated with GH for the indicated times, and STAT5B was
immunoprecipitated with STAT5B, immunoblotted with PY (Fig. 4A, upper panel) and
reprobed with STAT5B (Fig. 4A, lower panel). The amount
of tyrosyl-phosphorylated STAT5B over time was quantified for multiple
(10, 11, 12, 13) experiments and graphed as a percent of maximum tyrosyl
phosphorylation vs. time of incubation with GH in Fig. 4B.
Tyrosyl phosphorylation of STAT5B in wild-type GHR cells was reduced to
30% maximum after 1 h and 19% maximum after 4 h of GH
treatment (Fig. 4B). In contrast, tyrosyl phosphorylation of STAT5B in
Y487F GHR cells decreased to only 62% maximal after 1 h and 50%
maximal after 4 h of GH treatment. In cells expressing Y595F GHR,
the levels of tyrosyl phosphorylation of STAT5B only decreased to 95%
of maximum after 1 h of GH treatment and were 64% of maximum
after 4 h of GH treatment (Fig. 4B). Notably, basal levels of
tyrosyl-phosphorylated STAT5B in Y595F GHR or Y487F GHR cells were not
elevated, as compared with wild-type GHR cells nor did maximal levels
of tyrosyl-phosphorylated STAT5B differ significantly between cell
lines (Fig. 4A). Further, the experimental results in Fig. 4 were not
different in those instances in which the levels of
125I binding in CHO cells stably expressing
wild-type GHR, Y487F GHR, or Y595F GHR were similar. These data
indicate that mutation of Y487F or Y595F of GHR prolongs the duration
of tyrosyl phosphorylation of STAT5B in response to GH, with Y595F GHR
exhibiting the most dramatic prolongation.
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JAK2 and immunoblotted with PY (Fig. 5A). The level of tyrosyl phosphorylation
of JAK2 was quantified and was graphed as percent maximum
vs. time of GH stimulation in Fig. 5B. Tyrosyl
phosphorylation of JAK2 in wild-type GHR cells was maximal at 5 min and
reduced to an average 19% maximum after 15 min, 24% after 1 h,
and 11% after 4 h of GH treatment (Fig. 5B). The level of tyrosyl
phosphorylation of JAK2 was significantly prolonged in cells expressing
Y595F GHR, compared with cells expressing wild-type GHR.
Phosphorylation was maximal at 5 min in three of four experiments and
delayed until 15 min in the fourth experiment. It was reduced minimally
to an average 89% maximal by 15 min GH treatment. After 1 h and
4 h GH treatment, it was reduced to 80% and 24%, respectively
(Fig. 5B). Tyrosyl phosphorylation of JAK2 in Y487F GHR cells was not
prolonged, although there was a trend toward higher tyrosyl
phosphorylation of JAK2 at 15 min GH in Y487F GHR compared with
wild-type GHR cells. No tyrosyl phosphorylation of JAK2 was observed in
the absence of GH in any of the cell lines, and maximal levels of
tyrosyl phosphorylation of JAK2 detected at either 5 or 15 min were
similar in all cell lines (Fig. 5A). The latter suggests that mutating
tyrosines 487 and 595 in GHR did not significantly alter the ability of
GHR to bind and activate JAK2. Levels of JAK2 protein did not change
with incubation time with GH nor did they differ reproducibly between
cell lines (data not shown). These data indicate that mutation of Y595F
of GHR prolongs the duration of tyrosyl phosphorylation of JAK2 in
response to GH.
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GHR and immunoblotted
with PY (Fig. 6A). The level of
tyrosyl phosphorylation of GHR was quantified for several experiments
and was graphed as percent maximum vs. time of GH
stimulation in Fig. 6B. The time course of tyrosyl phosphorylation of
GHR is prolonged in cells expressing Y595F GHR, but not in cells
expressing Y487F GHR. After 15 min of GH treatment, the level of
tyrosyl phosphorylation of GHR in cells expressing wild-type GHR or
Y487F GHR was 47% and 41% maximal, respectively (Fig. 6B). By 1
h of GH treatment, the levels of tyrosyl-phosphorylated GHR diminished
to less than 10% maximal in both cell lines. In contrast, in cells
expressing Y595F GHR, the level of tyrosyl phosphorylation of GHR
remains near 100% of the maximal level after 15 min and only
diminishes to 60% of maximal after 1 h of GH stimulation. In all
three cell lines, the level of tyrosyl-phosphorylated GHR is reduced to
less than 10% maximal after 4 h of GH treatment, the longest time
point examined. Reprobing these membranes with GHR revealed that the
levels of wild-type or mutant GHR protein progressively diminish with
time of GH treatment (Fig. 6A, bottom panel), with the
amount of receptor protein paralleling the amount of tyrosyl
phosphorylation of GHR. These data therefore indicate that mutation of
Y595 prolongs the time course of GHR tyrosyl phosphorylation and delays
degradation of GHR in response to GH. This prolongation of both
tyrosine phosphorylation of GHR and GHR number is consistent with the
hypothesis of Gebert et al. (32) that dephosphorylation of
GHR signals its internalization and degradation.
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). In CHO
cells expressing Y487F GHR, GH induction of Spi tkluc activity was on
the average greater than the GH-induced activity in cells with
wild-type GHR (increase of 26 ± 32%, n = 3;
P 
0.1); however the increase did not achieve
statistical significance. These results are consistent with prolonged
tyrosyl phosphorylation of GHR, JAK2, and STAT5B in cells expressing
Y595F GHR, resulting in enhanced STAT5B-mediated transcription.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Mutation of Tyrosine 595 of GHR Prolongs GH-Promoted Tyrosyl
Phosphorylation of GHR and JAK2
To gain insight into the role of SHP-2 in GH signaling, we
examined the effect of mutating the SHP-2 binding sites in GHR on
tyrosyl phosphorylation of GH signaling molecules. Blocking the binding
of SHP-2 to GHR by a point mutation of GHR has the advantage over
overexpressing phosphatase-inactive SHP-2 of allowing us to examine the
consequences of removing only that SHP-2 that binds to GHR. The results
are not complicated by the effects of removing any SHP-2 that binds to
SIRP, IRS-1/2, STAT5, or any other cellular protein in response to
GH or other modulators of cell function. Our results indicate that
tyrosine-to-phenylalanine mutation of tyrosine 595 of GHR dramatically
prolongs tyrosyl phosphorylation of GHR in response to GH. Consistent
with prolonged tyrosyl phosphorylation of GHR resulting from mutation
of the SHP-2 binding site in GHR, mutation of the SHP-2 binding site in
the gp130 subunit of the leukemia-inhibitory factor (LIF) receptor
prolongs tyrosyl phosphorylation of gp130 in response to ligand (34).
These results suggest a model in which GH binds to a dimer of GHR and
activates JAK2. The activated JAK2 phosphorylates tyrosine 595 in GHR.
The two phosphorylated tyrosines at 595 on the GHR monomers
simultaneously bind to the N- and C-terminal SH2 domains of SHP-2 and
activate the phosphatase activity of SHP-2 (12, 35, 36). The activated
phosphatase then dephosphorylates the SHP-2-associated GHR. In support
of this, tandem phosphopeptides corresponding to the binding site for
the SH2 domain of SHP-2 in platelet-derived growth factor receptor
potently stimulate SHP-2 phosphatase activity in vitro, and
SHP-2 dephosphorylates platelet-derived growth factor receptor in
vivo (12, 37).
Our results indicate that mutation of the SHP-2 binding site at Y595 of GHR also prolongs tyrosyl phosphorylation of JAK2. SHP-2 bound to Y595 could potentially dephosphorylate the GHR-associated JAK2, in addition to GHR. Depending upon the tyrosine being dephosphorylated, dephosphorylation of JAK2 could inactivate it or remove the binding site(s) for specific signaling molecules. Prolonged tyrosyl phosphorylation of JAK2 has also been reported in cells expressing mutated EPO receptor lacking the tyrosine that binds the related tyrosine phosphatase SHP-1 (13). Even if JAK2 is a substrate of the GHR-associated SHP-2, it seems unlikely that GHR-associated SHP-2 is the only determinant of the phosphorylation and activation state of JAK2 because the tyrosine phosphatase inhibitor orthovanadate prolongs JAK2 phosphorylation to a much greater extent (L. Argetsinger and C. Carter-Su, unpublished observation). This suggests that other phosphatases may contribute to the dephosphorylation of JAK2. One candidate for such a phosphatase is SHP-2 that binds directly to JAK2. Although SHP-2 has been shown to bind JAK2 (27), our data predict that it does not interact via its SH2 domains and therefore would not be activated. In support of this, coexpression of SHP-2 and JAK2 in COS or Sf9 cells does not result in dephosphorylation of JAK2 (13, 27). SHP-1, which is expressed in the CHO cell lines used in these experiments, is another candidate for a JAK2-binding phosphatase. SHP-1 has been demonstrated to dephosphorylate JAK2 (38) and to associate with JAK2 in response to GH (17). Consistent with SHP-1 dephosphorylating JAK2 in response to GH, the time course of JAK2 tyrosyl phosphorylation in mutant liver cells, which lack functional SHP-1, is prolonged (17). However, SHP-1 is reported to bind to and be modestly activated by JAK2 independent of the SH2 domains of SHP-1 (38).
Other factors, including other phosphatases or members of the SOCS/CIS family of cytokine-inducible proteins (39, 40, 41), may also be involved in terminating GH activation of JAK2. GH induces expression of several members of the SOCS family (42, 43), which inhibit GH signaling by inhibiting the kinase activity of JAK2 either directly (e.g. SOCS-1) or via binding to the tyrosine-phosphorylated GHR (e.g. SOCS-3 and CIS) (44, 45). The regions of GHR involved in binding SOCS-3 and CIS in response to GH are unclear. Using portions of GHR phosphorylated in bacteria by a tyrosine kinase other than JAK2, binding of SOCS-3 to membrane-proximal phosphotyrosine residues 333/338 (45) or to phosphotyrosines in the C terminus (amino acids 455–638) (44) of GHR has been reported. CIS is thought to bind to the C-terminal portion of GHR (45). Hence, it is possible that some of the prolongation in GH signaling we observe with Y487F GHR and Y595F GHR, especially at longer periods of GH stimulation, may result from an inability of SOCS proteins to bind to GHR. However, studies with phosphatase inhibitors have demonstrated a clear role for tyrosyl dephosphorylation in the decay of GH signaling (32, 47).
In the above model, mutating Y595 to phenylalanine is hypothesized to prevent binding of SHP-2, thereby prolonging tyrosyl phosphorylation of GHR. Because GHR number decreases at a rate similar to the decrease in tyrosyl phosphorylation of GHR, this model would predict that dephosphorylation of GHR precedes GHR degradation as hypothesized by Gebert et al. (32). While we think it less likely, we cannot rule out the alternative possibility that mutation of Y595 in GHR directly interferes with GH-induced internalization and/or degradation of GHR. Arguing against tyrosines 487 and 595 being required for normal rates of internalization is the finding that COS cells expressing truncated GHR that lack tyrosines 487 and 595 exhibit normal rates of internalization of 125I-hGH (46). Whether the Y595F mutation of GHR affects GH-induced degradation is not known.
Mutation of Tyrosine 595 Prolongs GH-Promoted Tyrosyl
Phosphorylation of STAT5B
To gain additional insight into the role of SHP-2 in GH signaling,
we examined the effects of mutation of the SHP-2 binding sites in GHR
on tyrosyl phosphorylation of STAT5B. STAT5B is rapidly and transiently
tyrosyl phosphorylated in a variety of cell lines and tissues in
response to GH, but how STAT5B or any other member of the STAT family
is dephosphorylated and inactivated in response to ligand is still
unclear (4, 47). Our data indicate that mutation of Y595 of GHR
substantially prolongs GH-promoted tyrosyl phosphorylation of STAT5B
and enhances STAT5B-dependent activation of a spi 2.1
reporter gene. One model consistent with our data is the following.
After GH treatment, tyrosyl-phosphorylated STAT5B translocates to the
nucleus, binds DNA, transactivates genes, is dephosphorylated, and then
returns to the cytosol. The dephosphorylated STAT5B is recruited again
to tyrosyl-phosphorylated GHR and retyrosyl phosphorylated by JAK2. A
second cycle of STAT5B tyrosyl phosphorylation and activation of target
genes is thereby initiated. In this model, mutating Y595 to
phenylalanine would prolong the tyrosyl phosphorylation and the
half-life of GHR, and the phosphorylation and activation of JAK2,
allowing for more cycles of activation of STAT5B. Consistent with JAK2
initiating multiple STAT5B activation cycles before deactivation of
GHR/JAK2, the lifetime of the activated GHR/JAK2 complex in
liver-derived cells is longer than that of activated STAT5B (32).
Furthermore, addition of genistein, an inhibitor of JAK2, hastens the
decline in tyrosyl phosphorylation of STAT5B in wild-type and Y595F GHR
cells (data not shown), the predicted result if STAT5B is continuously
being dephosphorylated, recycling back to GHR/JAK2 complexes, and being
rephosphorylated by activated JAK2/GHR complexes. The tyrosine
phosphatase that dephosphorylates cytokine-activated STAT5B has not
been definitively identified although both SHP-1 and SHP-2 have been
suggested (48, 49). It is conceivable that dephosphorylation of STAT5B
involves in part a phosphatase whose association with or ability to
dephosphorylate STAT5B is dependent upon Y595.
While the stimulatory effects of Y595F on the tyrosyl phosphorylation of GHR, JAK2, and STAT5B are pronounced and consistent with the inability of Y595F GHR to bind SHP-2, statistically significant effects of mutating Y487F GHR on GHR and JAK2 phosphorylation were not detected. It may be that only a small amount of binding of SHP-2 to Y487F GHR is sufficient to prolong the tyrosyl phosphorylation of STAT5B but is insufficient to detect substantial prolongation of the tyrosyl phosphorylation of GHR or JAK2. Alternatively, Y487F GHR may cause prolonged tyrosyl phosphorylation of STAT5B by a different mechanism than Y595F. For example, mutating Y595 to phenylalanine may prolong tyrosyl phosphorylation of STAT5B primarily by preventing the binding of SHP-2 to GHR. In contrast, mutating Y487 to phenylalanine may prolong tyrosyl phosphorylation of STAT5B primarily by preventing the binding of a phosphatase or a protein required for recruiting a STAT5B phosphatase.
Roles of SHP-2 in Signaling by GH and Other Cytokine Receptors
Our results suggest that SHP-2 negatively regulates GH signaling,
because mutation of the SHP-2 binding site in GHR substantially
prolongs tyrosyl phosphorylation of GHR, JAK2, and STAT5B in response
to GH. These data are the first indications that a single tyrosine in
GHR regulates the duration of tyrosyl phosphorylation of signaling
molecules for GH. Our results are consistent with previous studies,
which demonstrate that mutation of the SHP-2 binding site in the
receptor for CNTF or leptin enhances STAT3 DNA binding or reporter gene
activity in response to ligand (21, 22, 23, 34). Taken together, these
results suggest that SHP-2 may negatively regulate STAT activation in
response to ligands for multiple members of the cytokine receptor
superfamily.
However, other studies have implicated SHP-2 as a positive regulator of
GH, PRL, and IFN signaling, based on the ability of a catalytically
inactive SHP-2 mutant to diminish c-fos, ß-casein, or
IFN-stimulated response element reporter gene activity in response to
ligand (20, 23, 50). Classification of SHP-2 as a positive or negative
regulator of signaling may oversimplify the role(s) of SHP-2 in
signaling by GH, as well as in signaling by ligands for other members
of the cytokine receptor superfamily. In fact, SHP-2 has been shown to
have dual roles in CNTF signaling. Overexpression of SHP-2 diminishes
DNA binding of STAT3 and STAT3-dependent gene expression in response to
CNTF, while SHP-2 is required for full activation of c-fos
in response to ligand (23). Thus, SHP-2 may have distinct roles for
activation of mitogen-activated protein kinase (MAPK)-dependent (such
as c-fos expression) vs. JAK/STAT-dependent
signaling pathways for cytokine receptor superfamily members, including
GHR. The ability of SHP-2 to bind and dephosphorylate multiple
signaling molecules, such as cytokine receptors, SIRP1, STATs, or
JAKs may also provide the basis for the diverse effects of SHP-2 on
signaling by multiple cytokine receptors that utilize JAK2.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Antisera
Antiphosphotyrosine antibody 4G10 (PY) was purchased from
Upstate Biotechnology, Inc. (Lake Placid, NY) and was used
at 1:7500 for Western blotting. Antibody to GHR (GHR), raised
against recombinant rat GH-binding protein, was kindly provided by W.
Baumbach (American Cyanamid, Princeton, NJ) and was used
at dilutions of 1:500 and 1:7500 for immunoprecipitations and
immunoblotting, respectively. Antibody to JAK2 (JAK2), raised
against a peptide corresponding to amino acids 758–776 of murine JAK2,
was used at a dilution of 1:100 for immunoprecipitations and 1:15,000
for Western blotting. Antibody to STAT5B (STAT5B), raised against
amino acids 763–779 of human STAT5B, was purchased from Santa Cruz Biotechnology, Inc.
Transfection and Cell Culture
The stock of 3T3-F442A cells was kindly provided by H. Green
(Harvard University, Boston, MA). Mouse 3T3-F442A cells were cultured
in DMEM supplemented with 100 U of penicillin per ml, 100 µg of
streptomycin per ml, 0.25 µg amphotericin
(Life Technologies, Gaithersburg, MD) per ml and 10% calf
serum as described previously (51). The stock of CHO cells stably
expressing full-length rat GHR has been described previously (52). CHO
cells were cultured in F-12 medium supplemented with 1 mM
L-glutamine, 100 U of penicillin per ml, 100 µg of
streptomycin per ml, 0.25 µg of amphotericin per ml, and
10% FBS. The expression vectors encoding rat GHR in which tyrosine
487, 595, or 627 is mutated to phenylalanine were generated by PCR as
described previously (53). All mutations were confirmed by DNA
sequencing. Stable CHO cell lines expressing GHR Y487F or GHR Y595F
were created as described previously (54). CHO cells were transfected
with the plasmid pRSV neo encoding resistance for G418 and pLM108
encoding either GHR Y487F or GHR Y595F at a 1:4 ratio using Lipofectin
as described by the manufacturer. Transfected cells were subjected to
selection in medium containing 1 mg/ml of G418, and individual clones
were isolated by limiting dilution. Expression of mutated GHR in the
clonal cell lines was monitored by 125I
labeled-GH binding assays and the correct size verified by
cross-linking of 125I hGH to GHR as described
previously (28). COS cells were maintained in DMEM supplemented with
antibiotics, 1 mM L-glutamine, and 10% FBS.
COS cells were transfected with expression vectors encoding the
indicated cDNAs by calcium phosphate precipitation. After 24 h,
cells were washed twice with DMEM and incubated for an additional
24 h with DMEM containing 10% FBS, antibiotics, and
L-glutamine.
Immunoprecipitation and Western Blotting
Confluent 3T3-F442A fibroblasts or CHO cells stably transfected
with rat GHR were incubated in serum-free medium overnight as described
previously (55). 3T3-F442A cells or CHO cells expressing GHR were
incubated at 37 C with GH for the indicated times at the indicated
concentrations. CHO cells, COS cells, or 3T3-F442A cells were washed
twice with ice-cold PBSV (10 mM sodium phosphate, pH 7.4,
150 mM NaCl, 1 mM
Na3VO4) and solubilized in
lysis buffer (50 mM Tris, pH 7.5, 0.1% Triton X-100, 150
mM NaCl, 2 mM EGTA, 1 mM
Na3VO4, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml
leupeptin). Cell lysates were centrifuged at 12,000 x
g for 10 min, and the supernatants were incubated on ice for
2 h with the indicated antibody. Immune complexes were collected
with protein A agarose for 1 h at 8 C and were washed three times
with 50 mM Tris, pH 7.5, 0.1% Triton X-100, 137
mM NaCl, 2 mM EGTA) and
boiled for 5 min in a mixture of lysis buffer and 5 x SDS-PAGE
sample buffer (250 mM Tris, pH 6.8, 10% SDS,
10% ß-mercaptoethanol, and 40% glycerol). Samples were resolved by
SDS-PAGE followed by Western blot analysis with the indicated
antibodies using the ECL detection system (56). As indicated, blots
were either directly reprobed with antibody or stripped in stripping
buffer (100 mM ß-mercaptoethanol, 2% SDS, 62.5
mM Tris-HCl) at 55 C for 30 min and then reprobed
with the indicated antibody. To quantify the levels of tyrosyl
phosphorylation of proteins, autoradiographs were scanned using an Agfa
scanner and Fotolook SA software and quantified using the Molecular
Analyst image software from Bio-Rad Laboratories, Inc.
(Richmond, CA). Means and SE values were calculated. The
one-tailed paired Student’s t test was used to determine
the statistical significance of differences in protein tyrosyl
phosphorylation in multiple independent experiments.
In Vitro Interaction Assay with GST Fusion Proteins
and Far Western Blotting.
For GST fusion protein interaction assays, immobilized fusion
protein containing the SH2 domains of human SHP-2 was incubated with
3T3-F442A cell lysates or transfected COS cell lysates for 2 h at
4 C, washed with lysis buffer, and boiled for 5 min in 5x SDS-PAGE
sample buffer. Samples were analyzed by Western blotting as described
above. For far Western blotting experiments, 3T3-F442A cell lysates
were immunoprecipitated with GHR or JAK2 and analyzed by SDS-PAGE
as described above. The membrane was incubated with GST fusion protein
encoding the two SH2 domains of SHP-2 (N + C SH2) at 1.5 µg/ml
overnight at 4 C. After extensive washing, the membrane was
immunoblotted with SHP-2, stripped, and reprobed with PY.
Luciferase Assay
CHO cells stably expressing wild-type or mutant GHR were
transfected with a GH-responsive luciferase reporter (Spi tkluc, 0.5
µg) and 0.1 µg RSV-ß-gal using Fugene (Stratagene,
La Jolla, CA) according to the protocol suggested by the manufacturer.
Spi tkluc contains eight copies of the spi 2.1 promoter
(-147/-102) upstream of a luciferase gene (33). Cells were
serum-deprived overnight, treated with GH for 24 h, or left
untreated, and cell lysates prepared in reporter lysis buffer (100
mM potassium phosphate, pH 7.8, 0.2% Triton
X-100, 1 mM dithiothreitol). Luciferase and
ß-galactosidase activity were measured in triplicate using a MGM
Opticomp luminometer. In each sample, luciferase activity was
normalized to ß-galactosidase activity and the mean fold change in
luciferase activity in response to GH for each cell line was
calculated. Data from three independent experiments expressed as the
ratio of fold GH-dependent luciferase activity in cells expressing
mutant GHR compared with activity in cells with wild-type receptor were
analyzed by one-tailed paired Student’s t test. Differences
were considered to be statistically significant at P <
0.05.
|
ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported by NIH Grants DK-48283 and DK-34171 (to C.C.-S.).
Received for publication April 7, 1999. Revision received May 9, 2000. Accepted for publication May 15, 2000.
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