(Received for publication, October 5, 1995; and in revised form, January 9, 1996)
From the
The binding of growth hormone leads to dimerization of its receptor, accompanied by phosphorylation and activation of intracellular tyrosine kinases (JAKs) and the latent cytoplasmic transcriptions factors STAT1, STAT3, and STAT5. Both JAK1 and JAK2 are phosphorylated in response to growth hormone in mouse 3T3 F442A and human HT1080 cells. The roles of JAKs in growth hormone signal transduction were examined by using mutant HT1080 cells missing either JAK1 or JAK2. JAK2 is absolutely required for growth hormone-dependent phosphorylation of the receptor, STAT1 and STAT3, JAK1, and the SH2-containing adaptor molecule Shc. In contrast, JAK1 is not required for any of the above functions. These data indicate that JAK2 is both necessary and sufficient for the growth hormone-dependent phosphorylation events required to couple the receptor both to STAT-dependent signaling pathways and to pathways involving Shc. Furthermore, STAT5 is activated by growth hormone in 3T3 F442A cells, but not in HT1080 cells, revealing that the set of STATs activated by growth hormone can vary, possibly contributing to the specificity of the growth hormone response in different cell types.
GH ()regulates important physiological processes,
such as fat metabolism and growth of the long bones(1) . Its
effects are mediated through activation of the GHR, a 134-kDa
glycoprotein belonging to the cytokine/hematopoietin receptor
superfamily(2, 3, 4, 5, 6) .
In response to GH, the GHR itself (7) and members of the STAT
family of transcription factors (8) are phosphorylated
rapidly(9, 10, 11) . These events are
accompanied by phosphorylation on tyrosine and activation of the
receptor-associated tyrosine kinase JAK2 (12) . Although JAK
and STAT activations correlate with the GH-dependent proliferation of
lymphoid cell lines(13) , other signaling pathways also
contribute to the full GH response. GH has been shown to activate
mitogen-activated protein kinases and to promote phosphorylation on
tyrosine of Shc and the association of Shc with Grb2(14) ,
implicating Ras-dependent modes of signaling that may or may not be
linked to the JAK-STAT pathway.
JAKs and STATs are now known to
transduce signals initiated by many growth factors and
cytokines(6) . Although many of these ligands activate
overlapping sets of STATs, some, such as IFN- and IL-4, activate
unique family members (STAT2 and STAT6, respectively) not known to be
involved in other pathways(6) . Furthermore, activated STAT
monomers associate to form homo- and heterodimers, which can interact
with other DNA-binding proteins to produce complex transcription
factors with different sequence specificities(15) , providing
an additional level of regulation. Finally, it is clear that some of
the extracellular signaling proteins that activate JAK-STAT pathways
also trigger Ras-dependent pathways, which can activate the
transcription of distinct sets of genes(16) . At least three
different family members (STAT1, STAT3 (acute-phase response factor),
and STAT5) are activated by
GH(9, 17, 18, 19, 20) .
JAK2 is the only family member yet implicated in GH
signaling(12) .
We have used a genetic approach to isolate
cell lines defective in IFN-- or IFN-
-dependent
signaling(21, 22, 23) . Mutant cells have
been isolated in eight complementation groups, five of which lack
individual JAK or STAT proteins (reviewed in (8) ). These cells
have been extremely useful in characterizing the roles of individual
components in IFN-dependent signaling and in evaluating the roles of
these molecules in transducing signals initiated by other factors, such
as IL-6 (24) and EGF(25) . In this report, we have used
some of the mutant cell lines to assess the roles of individual JAKs
and STATs in GH-dependent signaling.
The following antisera were used: anti-JAK1 from A. Ziemiecki (University of Berne, Berne, Switzerland); anti-JAK2 and anti-STAT6 from J. Ihle (St. Jude's, Memphis, TN); anti-STAT1 and anti-STAT2 from J. Darnell, Jr. (Rockefeller University, New York); anti-STAT3 from D. Levy (New York University); and anti-STAT5, raised against a synthetic peptide corresponding to amino acids 5-107 of sheep STAT5 (31) . Also used were anti-phosphotyrosine monoclonal antibodies PY20 (Transduction Laboratories, Lexington, KY) and 4G10 (Upstate Biotechnology, Inc., Lake Placid, NY), anti-GHR monoclonal antibodies 263 (Agen Corp., Parsippany, NJ) and 3B7 (Genentech, Inc.), polyclonal Shc antiserum (Upstate Biotechnology, Inc.), and fluorescein-conjugated goat anti-mouse serum for cell sorting (Dako Corp., Carpinteria, CA).
Figure 1: Expression of GHR in parental and mutant cell lines. FACScan analysis of GHR expression in parental 2C4 cells was compared with the level of expression in 2C4 and mutant cell lines stably transfected with a human GHR expression plasmid and sorted for high cell-surface GHR expression (filled profiles). The open profiles represent the background fluorescence of unstained 2C4 cells.
GH has been shown previously to promote
phosphorylation of the receptor-associated tyrosine kinase JAK2 in a
ligand-dependent manner(12) . As shown in Fig. 2A, JAK2 (130 kDa) did become phosphorylated
on tyrosine in 2C4/GHR cells following treatment with GH or IFN-
,
but not IFN-
. JAK1 was also phosphorylated on tyrosine following
GH stimulation, about as strongly as in cells treated with IFN-
or
IFN-
(Fig. 2B). These data indicate that JAK1 and
JAK2 might both be involved in GH signaling in 2C4/GHR cells. Similar
results were obtained with 3T3 F442A cells (Fig. 2, C and D). Treatment of cells with either IFN-
or
IFN-
leads to interdependent activation of two JAK family
members(26, 27) . To test whether the GH-dependent
JAK1 and JAK2 phosphorylations were also interdependent, the
phosphorylation of each JAK kinase was examined in mutant cells
defective in the other. As shown in Fig. 2E, JAK2
activation in response to GH was normal in U4C/GHR cells (missing
JAK1), in contrast to the result with IFN-
, where JAK2
phosphorylation required JAK1(27) . The reciprocal experiment
was performed in
2A/GHR cells (missing JAK2). In this case, JAK1
was not phosphorylated on tyrosine in response to either GH or
IFN-
(Fig. 2F). Therefore, the phosphorylation of
JAK1 by GH depends upon JAK2, but not vice versa.
Figure 2:
Phosphorylation of JAK kinases in response
to GH, IFN-, or IFN-
. 2C4/GHR (A and B),
U4C/GHR (E), or
2A/GHR (F) cells were
untreated(-) or treated with 500 ng/ml GH, 500 IU/ml IFN-
,
or 1000 IU/ml IFN-
for 15 min at 37 °C. 3T3 F442A cells (C and D) were left untreated(-) or were treated for
15 min with 500 ng/ml GH. Cell lysates were immunoprecipitated using
anti-JAK1 (B, D, and F) or anti-JAK2 (A, C, and E). Immunoprecipitates were
analyzed by Western blotting with anti-phosphotyrosine antibodies 4G10
and PY20 (a panels). The same blots were stripped and reprobed
with anti-JAK1 or anti-JAK2 to normalize for protein loading (b
panels). rhGH, recombinant human
GH.
Figure 3:
Stimulation of STAT phosphorylation by GH,
IFN-, or IFN-
. 2C4/GHR, U3A/GHR, U4C/GHR, or
2A/GHR
cells were treated as described for Fig. 2, and cell lysates
were immunoprecipitated with anti-STAT1 (A, D, and F) or anti-STAT3 (B, C, E, and G). Immunoprecipitates were analyzed by Western blotting using
anti-phosphotyrosine antibodies 4G10 and PY20 (a panels). The
blots were stripped and reprobed with STAT antisera (b
panels). rhGH, recombinant human
GH.
Mutant U4C/GHR and 2A/GHR
cells (missing JAK1 and JAK2, respectively) were used to examine the
requirement for each JAK protein in GH-induced STAT activation.
Treatments with IFN-
and IFN-
were performed in parallel as
controls. JAK2 was absolutely required for GH- and IFN-
-dependent
(but not IFN-
-dependent) STAT activation (Fig. 3, F and G). JAK1, on the other hand, although phosphorylated
in response to GH, was not required for GH-dependent STAT1 or STAT3
phosphorylation (Fig. 3, D and E). As
expected, however, it was required for STAT activation by both
IFN-
and IFN-
(Fig. 3, D and E).
To extend these studies, STAT activation by GH was also examined by
electrophoretic mobility shift assay in 2fTGH/GHR and mutant cells. As
shown in Fig. 4, three GH-induced complexes (A, B, and C) were
detected with a P-labeled Fc
GAS probe in 2fTGH/GHR,
2C4/GHR, and U4C/GHR cells, but not in
2A/GHR cells. When
2A/GHR cells were transfected with a murine JAK2 expression
plasmid, STAT binding to the Fc
GAS probe was restored (albeit not
to wild-type levels, probably due to poor JAK2 expression) (data not
shown). The intensity of induced bands obtained in U4C/GHR cells was
generally less than in other cell lines, but remained unchanged in
U4C/GHR cells complemented with JAK1 (data not shown). In U3A/GHR
cells, only complex A was observed, suggesting that complexes B and C
contain STAT1. Complexes A, B, and C were observed at normal levels in
cells lacking STAT2 or Tyk2 (data not shown).
Figure 4:
GH-induced binding to a Fc GAS probe.
Extracts were prepared from parental 2fTGH/GHR and 2C4/GHR cells and
from mutant cell lines U3A/GHR, U4C/GHR, and
2A/GHR
untreated(-) or treated (+) with GH for 15 min. Complexes
were identified by electrophoretic mobility shift assay. The positions
of the three major GH-inducible complexes are indicated. rhGH,
recombinant human GH.
To analyze the
complexes in more detail, antibody supershift experiments were
performed (Fig. 5). As expected from the results with U3A/GHR
cells, complexes B and C were lost when anti-STAT1 was included in the
binding reaction (Fig. 5A, lane 3). Bands A
and B were supershifted by anti-STAT3 (lane 5), suggesting
that band B is a STAT1-STAT3 heterodimer and showing that band A
contains STAT3; it is probably a homodimer. Antisera to STAT2, STAT5,
or STAT6 had no effect when used with extracts from GH-treated
2fTGH/GHR cells (lanes 4, 6, and 7). In
murine 3T3 F442A cells, four complexes were obtained with an Fc
GAS probe. The lower three behaved identically to the three complexes
observed in 2fTGH/GHR cells and represented STAT3 homodimer,
STAT1-STAT3 heterodimer, and STAT1 homodimer, respectively. The
uppermost band, however, was supershifted by anti-STAT5 (lane
14). To investigate further the possibility that STAT5 might be
induced by GH in these cells, a GAS-like DNA-binding element from the
promoter of the prolactin-responsive
-casein gene was used in band
shift experiments (Fig. 5B). This probe has been shown
to recognize STAT5 homodimers preferentially, but can also bind weakly
to other STATs (31) . The predominant complex obtained with
extracts of 3T3 F442A cells was supershifted by anti-STAT5 (lane
3), but none of the complexes from 2fTGH/GHR cells were affected
by this antibody (lane 6). These data demonstrate that, in 3T3
F442A but not 2fTGH/GHR cells, STAT5 or related proteins are activated
and may be involved in GH signaling.
Figure 5:
Antibody supershift analysis of GH-induced
STAT binding to a Fc GAS probe. A, whole cell extracts
from untreated (lanes 1 and 9) or GH-treated (lanes 2-8 and 11-14) 2fTGH/GHR cells (lanes 1-8) or 3T3 F442A cells (lanes
9-14) were analyzed. The STATs present in complexes were
evaluated with antisera to STAT1 (lanes 3 and 12),
STAT2 (lane 4), STAT3 (lanes 5 and 13),
STAT5 (lanes 6 and 14), or STAT6 (lane 7) or
with preimmune serum (PI; lane 8). A shorter exposure
of the 3T3 F442A experiment is included to show the separation of the
two upper complexes. The positions of the three GH-induced complexes
observed in 2fTGH/GHR cells are indicated, and the position of the
GH-dependent complex obtained only in 3T3 F442A cells is marked with arrows. B, GH-dependent complexes formed with a
casein GAS probe are shown. Binding assays were performed as described
for A, using whole cell extracts from untreated(-) or
GH-treated 3T3 F442A cells (lanes 1-3) or 2fTGH/GHR
cells (lanes 4-6). Supershift analysis was performed
with a STAT5 antiserum (lanes 3 and 6). AB,
antibody; rhGH, recombinant human
GH.
Figure 6:
Requirement of JAK2 for GH-dependent GHR
phosphorylation. 2C4, 2C4/GHR, U4C/GHR, or 2A/GHR cells were
analyzed either without treatment (lanes 2, 4, and 6) or after treatment for 15 min with 500 ng/ml GH at 37
°C (lanes 1, 3, 5, and 7). Cell
lysates were immunoprecipitated (IP) with a monoclonal
antibody to the human GHR, and tyrosine phosphorylation was determined
by Western blotting with anti-phosphotyrosine antibodies. The positions
of molecular mass standards and of the IgG heavy chain (HC)
are indicated. rhGH, recombinant human
GH.
In addition to the GHR and specific members of the JAK-STAT pathway, the SH2-containing adaptor molecule Shc becomes phosphorylated on tyrosine following treatment of cells with GH(14) . To examine the role of JAKs in this phosphorylation, Shc was immunoprecipitated from 2C4/GHR, U4C/GHR, and 2A/GHR cell lysates, and the phosphorylation status of the three Shc isoforms was examined by Western analysis with anti-phosphotyrosine antibodies (Fig. 7). Phosphorylation of the 66- and 52-kDa isoforms was observed in 2C4/GHR and U4C/GHR cells, but not in 2A/GHR cells, indicating that JAK2 is required for these phosphorylations following GH treatment. There was little phosphorylation of the 46-kDa isoform.
Figure 7:
Requirement of JAK2 for GH-dependent Shc
phosphorylation. 3T3 F442A, 2C4/GHR, U4C/GHR, or 2A/GHR cells were
analyzed either without treatment (lanes 1, 3, 5, and 7) or after treatment for 15 min with 500
ng/ml GH at 37 °C (lanes 2, 4, 6, and 8). Cell lysates were immunoprecipitated with a polyclonal
antibody to human Shc, and tyrosine phosphorylation was determined by
Western blotting with anti-phosphotyrosine antibodies (panel
a). The same blots were stripped and reprobed with Shc antiserum
to normalize for protein loading (panel
b).
Using cell lines missing individual JAKs or STATs to examine their roles in GH signaling, we have shown that JAK2 is required for phosphorylation on tyrosine residues of STAT1 and STAT3, GHR, JAK1, and Shc. JAK2 has been implicated previously in signal transduction by GH, based on its association with the GHR and its activation upon ligand binding(12) . However, it is clear that these criteria alone do not provide conclusive evidence that a given JAK has a functional role in a signal transduction pathway. For example, Tyk2, JAK1, and JAK2 are all phosphorylated in response to IL-6(42, 43) . However, experiments with mutant cells have demonstrated that only JAK1 is efficient in IL-6-dependent phosphorylation of the gp130 receptor subunit, STAT activation, and transcriptional induction of the interferon regulatory factor-1 gene(24) . Furthermore, JAK1 is also phosphorylated in response to EGF, but is not required for STAT activation by this ligand(25) . The data reported here suggest a similar situation for GH. JAK1 is phosphorylated on tyrosine in response to GH treatment of both 2C4/GHR and 3T3 F442A cells, a process that requires the presence of JAK2. However, STAT activation and ligand-dependent phosphorylation of the GHR and Shc were only slightly reduced in JAK1-minus U4C/GHR cells when compared with parental cells. It is unclear whether the reduced responsiveness to GH results from the JAK1 defect in these cells or whether other mutations contribute to this phenotype. Complementation with a murine JAK1 expression plasmid did not restore a wild-type response, and so it is assumed that JAK1 is dispensable for most, if not all, GH responses. Nonetheless, an involvement in some other aspect of GH signaling cannot be ruled out.
STAT5 has recently been shown to be activated in response to GH in GHR-transfected COS cells(19) . That STAT5 or a related factor is involved in GH signaling in some cell types is also demonstrated by our studies. STAT5, initially identified as a mediator of prolactin signaling in sheep(31) , has since been implicated in erythropoietin (19) and IL-3 (44, 45) signal transduction. In the mouse, STAT5 is encoded by two genes that give rise to at least four different isoforms of STAT5 protein(44, 45) . It is likely that the STAT5-like factor(s) that we have observed in 3T3 F442A cells represents one or more of these isoforms. Curiously, activation of STAT5 by GH was not observed in the human fibrosarcoma cells examined here, even though these cells express the STAT5 protein. It is unclear at this time whether the activation of STAT5 by GH is cell type-specific or whether species differences account for the difference between murine 3T3 F442A and human HT1080 cells. Cell type-restricted STAT activation has been described by others studying GH signaling. STAT1 is activated by GH in preadipocyte cells ( (9) and this report) and rat liver(17) , but not in IM9 lymphoblastoid cells(10, 11) . Thus, it appears that selectivity involving specific STAT subsets is a general feature of GH signal transduction.
The mechanism of STAT activation by GH is not well
understood. Elimination of all sites for tyrosine phosphorylation from
the intracellular domain of the GHR did not prevent signaling or STAT
activation(13) , arguing against a direct, SH2-mediated
interaction between STATs and the GHR. In contrast, STAT1 interacts
with the specific phosphotyrosine Tyr(P) of the IFN-
receptor, which lies in the sequence GpYDKPH(46) , and STAT3
binds to the intracellular domain of the gp130 receptor, where the
sequence motif pYXXQ (X is any amino acid) appears to
be both necessary and sufficient for STAT-receptor
interaction(43) . Neither of these sequences is present in the
GHR. Therefore, STAT interaction with the GHR may be mediated either
through adaptor molecules or through a JAK2-STAT association.
Alternatively, the GHR may require additional receptor subunits, as yet
unidentified, that function in STAT binding.
Our data indicate that,
in addition to its important role in coupling the GHR to STAT signaling
pathways, JAK2 is required to couple the receptor to pathways involving
Shc. Shc is thought to function as an adaptor molecule to recruit
Grb2-mSos1 complexes to the activated receptor(47) . The
nucleotide exchange factor mSos1 then promotes formation of
p21, initiating a cascade of phosphorylation events
that culminate with phosphorylation of specific transcription factors
in the nucleus(16, 47) . In EGF signaling, Shc
interacts with the EGF receptor by binding to specific phosphotyrosine
residues within its cytoplasmic tail(48, 49) . In the
case of erythropoietin, it has been reported that receptor
phosphorylation is not required for ligand-dependent Shc-receptor
association or for Shc phosphorylation(50) . Instead, Shc
appears to associate directly with JAK2 following erythropoietin
treatment(50) . A similar situation may exist for GH since JAK2
and Shc become associated following GH stimulation(14) . These
data suggest that Shc may be a direct target for phosphorylation by
JAK2 following GH and erythropoietin stimulation, although in the case
of GH, it is currently unknown whether receptor phosphorylation is
required for Shc-GHR interaction.
Further insight into GH signaling should be obtained when more genes that are targets for GH activation are identified. Although several early response genes, such as c-fos, are known to be transcriptionally activated by GH(51) , the mechanism of induction is not known. The c-fos promoter contains the c-sis-inducible element, a canonical STAT binding sequence thought to play a role in induction by c-sis (platelet-derived growth factor)(37, 52) . However, the c-fos promoter is complex, and its transcriptional regulation involves coordinated activation through elements that respond to both Ras-dependent and Ras-independent pathways (16, 53) . Therefore, the identification of GH-responsive genes that respond exclusively through the JAK-STAT pathway, if they exist, would provide a better means to assess the roles of individual STATs in the transcriptional response to GH.