(Received for publication, October 18, 1994; and in revised form, December 27, 1994)
From the
Growth hormone (GH) has been shown to stimulate the
mitogen-activated protein (MAP) kinases designated ERKs (extracellular
signal regulated kinases) 1 and 2. One pathway by which ERKs 1 and 2
are activated by tyrosine kinases involves the Src homology (SH)-2
containing proteins SHC and Grb2. To gain insight into pathways
coupling GH receptor (GHR) to MAP kinase activation and signaling
molecules that might interact with GHR and its associated tyrosine
kinase JAK2, we examined whether SHC and Grb2 proteins serve as
signaling molecules for GH. Human GH was shown to promote the rapid
tyrosyl phosphorylation of 66-, 52-, and 46-kDa SHC proteins in
3T3-F442A fibroblasts. GH also promoted binding of GHR and JAK2 to the
SH2 domain of 46/52-kDa SHC protein fused to glutathione S-transferase (GST). Constitutively phosphorylated JAK2, from
COS-7 cells transiently transfected with murine JAK2 cDNA, bound to SHC
SH2-GST fusion protein, demonstrating that the SHC SH2 domain can bind
tyrosyl-phosphorylated JAK2 in the absence of GHR. Regions of GHR
required for GH-dependent tyrosyl phosphorylation of SHC were examined
using Chinese hamster ovary cells expressing mutated rat GHR. In cells
expressing GHR1-638 and GHR1-638(Y333,338F), GH stimulated
phosphorylation of all 3 SHC proteins whereas GH stimulated
phosphorylation of only the 66- and 52-kDa SHC proteins in cells
expressing GHR1-454. GH had no effect on SHC phosphorylation in
cells expressing GHR1-294 or GHRP, the latter lacking amino
acids 297-311 containing the proline-rich motif required for JAK2
activation by GH. In contrast to SHC, Grb2 appeared not to interact
directly with GHR or JAK2. However, Grb2 was shown to associate rapidly
with SHC proteins in a GH-dependent manner. These findings suggest that
GH stimulates: 1) the association of SHC proteins with JAK2
GHR
complexes via the SHC-SH2 domain, 2) tyrosyl phosphorylation of SHC
proteins, and 3) subsequent Grb2 association with SHC proteins. These
events are likely to be early events in GH activation of MAP kinases
and possibly of other responses to GH.
Our laboratory has shown that GH ()binds to the GH
receptor (GHR) and activates the GHR-associated tyrosine kinase JAK2,
whereupon both GHR and JAK2 become tyrosyl phosphorylated(1) .
However, the signaling pathways initiated by the activated
GHR
JAK2 complex that lead to GH-dependent changes in gene
expression, metabolism, cellular differentiation, and body growth are
poorly understood(2, 3) . One class of signaling
molecules known to be activated by GH (4, 5, 6, 7) is the
mitogen-activated protein kinases, commonly referred to as MAP kinases
or extracellular signal regulated kinases (ERKs). MAP kinases are
believed to play a pivotal role in the regulation of cellular growth
and differentiation (8) and have been shown to phosphorylate a
number of proteins, including other protein kinases, cytoplasmic
phospholipase A
, cytoskeletal proteins, and transcription
factors (9, 10, 11, 12, 13) .
Therefore, MAP kinases are likely to represent important signaling
molecules for GH.
A well studied pathway leading from membrane receptor tyrosine kinases to MAP kinase activation involves SHC and Grb2 proteins(14, 15, 16) . Two SHC proteins, p46 and p52, have been cloned and found to be differential splice products. A third protein (p66) is recognized by SHC antibodies made to a C-terminal peptide of the 46/52-kDa proteins. It is translated from a distinct transcript. SHC proteins contain a src homology (SH)-2 and a collagen-like domain(17) . Like other SH2-containing proteins(18, 19, 20) , SHC proteins have been shown to bind to phosphotyrosine-containing sequences, including sequences in ligand-activated tyrosine kinase receptors. SHC proteins themselves are substrates for tyrosine kinases(21) . Upon tyrosyl phosphorylation, they serve as linker, or adaptor, proteins for other SH2-containing proteins in tyrosine kinase signal transduction pathways.
Grb2 is a 23-25-kDa protein containing an SH2 domain between two SH3 domains(22) . Unlike SHC proteins, Grb2 is not tyrosyl phosphorylated in response to growth factor stimulation. Some tyrosyl-phosphorylated membrane receptor tyrosine kinases have been shown to interact directly with Grb2, while others have been shown to bind and tyrosyl phosphorylate SHC which in turn binds Grb2(15, 23, 24, 25) . Recruitment of Grb2 in response to ligand binding is believed to initiate one pathway that leads to the activation of MAP kinases. In essence, Grb2 has been shown to interact with the mammalian homolog of the Drosophila gene product son of sevenless, a guanine nucleotide exchange factor which activates the small GTP binding protein, RAS. RAS, in turn, activates the serine/threonine kinase Raf. Raf phosphorylates and activates the mixed function serine/threonine/tyrosine kinase MEK which then phosphorylates and activates the MAP kinases designated ERKs 1 and 2(26) . It has recently been suggested that Grb2 may also serve as a linker protein in other signaling pathways(25, 27) .
In this work, we examined whether GH recruits SHC and Grb2. The mechanism of interaction of these proteins with GHR and JAK2 was also assessed. The results of this study provide insight into signaling molecules that interact with GHR and its associated tyrosine kinase JAK2, and signaling pathway(s) leading from GHR/JAK2 to MAP kinase activation. Involvement of JAK2 suggests that other ligands that activate JAK2 are likely to activate MAP kinases by a pathway including SHC and Grb2 proteins.
COS-7 cells were
transiently transfected with the cDNA for murine JAK2 or JAK2(K882E)
using calcium phosphate precipitation. Briefly, cells were plated in
60-mm dishes and grown to 50% confluence. Expression vector containing
the full-length cDNA for JAK2 or JAK2(K882E) (0.1 µg/60-mm plate)
was mixed with 0.25 ml of 0.25 M CaCl and 0.25 ml
of 50 mM HEPES, 280 mM NaCl, and 1.5 mM Na
HPO
(pH 7.05). The COS-7 cells were
incubated with the DNA mixture for 18 h at 37 °C under 5%
CO
, 95% air. Medium was removed, and Dulbecco's
modified Eagle's medium and 10% fetal calf serum were added to
the cells for an additional 36 h.
Figure 1:
GH
stimulates tyrosyl phosphorylation of SHC. 3T3-F442A cells were
incubated with hGH for the times and concentrations indicated. Cellular
proteins were immunoprecipitated with SHC (1:100) and Western
blotted with
PY (1:7500). The migration of the three antigenically
related SHC proteins are indicated on the left.
Figure 2:
GH stimulates binding of 130-kDa
phosphoprotein(s) to the SH2 domain of SHC. 3T3-F442A cells were
incubated with hGH for the times and concentrations indicated. Cellular
proteins were precipitated using SHC SH2-GST fusion protein and bound
proteins were Western blotted with PY (1:7500). The migration of
the 130-kDa protein(s) is indicated on the left. The migration
of the 105-kDa standard is indicated on the right.
To determine whether JAK2 and/or GHR binds the SH2 domain of
SHC in a GH-dependent manner, the blot used in Fig. 2was probed
with JAK2 instead of
PY (Fig. 3, lanes
A-K) and duplicate 0 and 5-min samples were Western blotted
with
GHBP (Fig. 3, lanes L and M). The
results shown in Fig. 3confirm that both JAK2 and GHR bind to
the SH2 domain of SHC in a GH-dependent manner. Taken together, the
results of Fig. 1Fig. 2Fig. 3indicate that the
SH2 domain of SHC proteins is capable of binding to the activated
tyrosyl-phosphorylated GHR
JAK2 complex and that SHC proteins are
tyrosyl phosphorylated in response to GH.
Figure 3:
GH stimulates binding of JAK2 and GHR to
the SH2 domain of SHC. The Western blot used for Fig. 2was
probed with JAK2 (1:7500) (lanes A-K). Duplicate 0
and 5-min (500 ng/ml hGH) samples on the same original blot as lanes A-K were probed with
GHBP (1:10,000) (lanes L and M). The migration of JAK2 and GHR are
indicated.
Figure 4:
JAK2 associates with SHC SH2 domain when
transfected in COS-7 cells. COS-7 cells were transfected with JAK2 (lanes A, B, and E), JAK2(K882E) (lane C),
and control (lane D) cDNAs. Cellular proteins were incubated
with JAK2 (1:500, lanes A and B) or SHC SH2-GST
fusion protein (lanes C-E) and bound proteins were
Western blotted with
PY (1:7500, lane A) or
JAK2
(1:7500, lanes B-E). The migration of JAK2 and the
105-kDa standard is indicated.
To examine
JAK2-SHC association, lysates from the transfected cells were incubated
with the SH2 domain of p46/p52 SHC fused to GST. Bound proteins were
eluted and Western blotted with JAK2. Fig. 4, lane
E, shows that a protein that co-migrates with JAK2 binds to the
SH2 domain of SHC. Consistent with this band being JAK2, it was not
present when COS-7 cells were transfected with a control plasmid (Fig. 4, lane D). Probing the blot with
PY
indicated that the JAK2 bound to the SHC SH2 fusion protein is
tyrosyl-phosphorylated (data not shown). SHC SH2 domain appeared
capable of binding only to tyrosyl-phosphorylated JAK2, since JAK2
association was greatly reduced when the cells were transfected with a
cDNA encoding kinase-dead JAK2(K882E) in which the critical lysine in
the ATP binding region (34, 38) was mutated to
glutamic acid (Fig. 4, lane C). Lysines in the
equivalent position in other kinases have been shown to be involved in
the phosphotransfer reaction in the ATP binding site. Mutation of this
lysine in other kinases to any other amino acid has resulted in the
loss of kinase activity(39) . These data suggest that
constitutively active JAK2 in the absence of GHR can associate with the
SH2 domain of SHC. Tyrosyl phosphorylation appears to be required for
this interaction.
Figure 5: Wild-type and mutated GHRs expressed in CHO cells. Denoted are the extracellular domain, the transmembrane domain (hatched area), and the cytoplasmic domain of the various mutated rat liver GHR used in these studies. Tyrosyl residues in the cytoplasmic domain are denoted by ``Y.'' Phenylalanine residues that were substituted for tyrosyl residues are denoted by ``F.''
Figure 6:
Ability of mutated GHR to elicit
GH-dependent tyrosyl phosphorylation of SHC. A, CHO cells
expressing wild type or mutated GHR (see Fig. 5) as indicated
were stimulated for 5 min at 37 °C with 500 ng/ml hGH (lanes B,
D, F, H, J, and L) or vehicle (lanes A, C, E, G, I, and K). Cellular proteins were immunoprecipitated with
SHC (1:100) and Western blotted with
PY (1:7500). The
migration of the three antigenically related SHC proteins is indicated
on the left. Lanes A-H are from one experiment; lanes I-L are from a second experiment. B, the
amount of tyrosyl phosphorylation of the different SHC proteins was
quantified using either scanning densitometry (BioMed Instruments laser
scanning densitometer) or phosphoimaging (Bio-Rad GS-250 Molecular
Imager, Molecular Analyst/Macintosh Image Analysis software). Results
are expressed as a percentage of control (no GH) values for each cell
line. The means ± S.E. are shown. An asterisk denotes
that the mean is statistically different at the 95% confidence level
from 100% using a one-tailed, paired Student's t test.
The values for 52-kDa SHC for CHO1-454 versus CHO1-638 were not statistically different. N = 6, 5, 3, 3, and 4, for GHR1-638, GHR1-454,
GHR1-294, GHR
P, and GHR1-638(Y333,338F),
respectively.
A proline-rich motif (in GHR, amino acids 298-305) is the
only known motif present in the cytoplasmic domain of all members of
the cytokine receptor superfamily(40) . Deletion of this motif
has been shown to eliminate the ability of GHR to bind JAK2 and of GH
to activate JAK2(31, 41) . We therefore examined
whether deleting this motif would eliminate GH induction of SHC
phosphorylation. In CHO cells expressing GHRP, which lacks amino
acids 297-311, no GH-dependent increase in tyrosyl
phosphorylation of SHC proteins was detected (Fig. 6, A,
lanes G and H, and panel B). This finding
indicates that the proline-rich region of the GHR is required for
GH-dependent SHC phosphorylation, suggesting that JAK2 association with
GHR and activation is required for SHC phosphorylation.
Four
tyrosines are present in the cytoplasmic domain of GHR1-454. Of
these, tyrosines 333 and/or 338 is believed to be phosphorylated in
response to GH. ()To determine if either of these two
tyrosines is the major site of SHC protein association with GHR,
tyrosines 333 and 338 were mutated to phenylalanines. When
GHR1-638(Y333,338F) was expressed in CHO cells, a GH-dependent
increase in tyrosyl phosphorylation of the three SHC proteins similar
in magnitude to what was observed using cells expressing GHR
1-638 was observed (Fig. 6, A, lane L, and panel B), indicating that tyrosines 333 and 338 are not
required for GH-dependent SHC phosphorylation.
Figure 7:
GH promotes association of Grb2 with SHC.
3T3-F442A cells were incubated with hGH for the times and
concentrations indicated. Cellular proteins were immunoprecipitated
with SHC (1:100) and Western blotted with
Grb2 (1:250). The
migration of Grb2 is indicated.
Several lines of evidence are consistent with SHC
proteins binding to phosphorylated tyrosyl residues in JAK2. First,
full-length GHR1-638(Y333,338F) is capable of mediating
GH-dependent SHC phosphorylation. If SHC proteins bind only to
phosphorylated tyrosines and if Tyr and/or Tyr
are the only tyrosine is GHR1-454 that is phosphorylated in
response to GH, as hypothesized(17) ,
then this
finding suggests that GHR1-454, which is capable of mediating
GH-dependent tyrosyl phosphorylation of SHC proteins, does not itself
contain a SHC binding site. Rather, SHC must be binding to a
GHR-associated protein (e.g. JAK2). Second, when prepared from
GHR-deficient COS cells that express constitutively activated and
tyrosyl-phosphorylated murine JAK2 in the absence of GHR, JAK2 is
capable of binding the SH2 domain of SHC. Third, based upon amino acid
analysis(42, 43, 44) , murine JAK2 contains a
number of potential SHC binding sites. The JAK2 sequence
YLFV is similar to what is believed to be the highest
affinity SHC binding site in epidermal growth factor receptor (
YLRV) and the platelet-derived growth factor receptor (
YIYV)(17, 43) . Three additional
tyrosine-containing motifs in JAK2 (
YLKF,
YGQL, and
YGSL) fit the consensus sequence
for SHC binding (Y, hydrophobic, X, hydrophobic) derived from studies
of SHC binding to proteins and peptides containing phosphorylated
tyrosyl residues(21, 44, 45) .
While the results of our experiments discussed above
support the hypothesis that SHC proteins can bind to JAK2GHR
complexes, we have been unable to detect SHC proteins in
GHR,
GH, or
JAK2 immunoprecipitates nor have we been able to
detect GHR or JAK2 in
SHC immunoprecipitates (data not shown).
Potential explanations for this apparent discrepancy include: 1) the
antibodies used may be unable to immunoprecipitate
SHC
GHR
JAK2 complexes because their epitopes are either not
accessible when the antigens are present in a complex or antibody
binding disrupts pre-existing complexes; 2) SHC
GHR
JAK2
complexes may adventitiously dissociate during the cell lysis and
immunoprecipitation steps; 3) the antibodies may be unable to detect in
Western blots the potentially small amounts of proteins expected to be
co-precipitated. Alternatively, it is possible that while JAK2
GHR
complexes can bind to the SH2 domain of SHC proteins in vitro,
they do not readily do so in intact 3T3-F442A cells where the
concentration of SHC proteins is lower and the environment is
different. Others have hypothesized the existence of an adaptor protein
of M
150,000 that facilitates SHC binding to
certain receptors(46) . Arguing against the possibility that
SHC association with GHR
JAK2 complex occurs indirectly via this
or another adaptor protein is our inability to detect any GH-dependent
phosphoprotein other than SHC proteins in
SHC immunoprecipitates
(data not shown). Thus, the simplest interpretation of our findings is
that SHC proteins bind directly to GHR
JAK2 complexes. However,
our data do not exclude the alternative possibility that an additional
adaptor protein facilitates binding of SHC to GHR
JAK2 complexes.
In conclusion, these and previous results (1) suggest that,
in 3T3-F442A cells, GH binds to GHR, promotes association of JAK2 with
GHR, and activation of JAK2. The activated JAK2 phosphorylates both
itself and GHR. SHC proteins then interact with GHRJAK2 complexes
via the SH2 domains of SHC and are phosphorylated by JAK2. The
tyrosyl-phosphorylated SHC proteins in turn bind Grb2. This pathway is
likely to be an initial response to GH which ultimately leads to MAP
kinase activation. Our results suggesting that SHC proteins associate
with phosphorylated JAK2 imply that SHC proteins will be recruited in
proportion to the amount of JAK2 activated by the numerous ligands
capable of activating JAK2 (e.g. GH, prolactin,
erythropoietin, interleukin-3, -5, and -6, granulocyte-macrophage
colony-stimulating factor, granulocyte colony-stimulating factor,
ciliary neurotrophic factor, leukemia inhibitory factor, oncostatin M,
and
interferon-
(1, 34, 47, 48, 49, 50) ).
However, our evidence suggesting that GHR may also contribute SHC
binding sites raises the possibility that the ligands that activate
JAK2 may not stimulate SHC phosphorylation simply in proportion to the
amount of JAK2 they activate. Those receptors that are more abundant
and have higher affinity SHC binding sites would be expected to recruit
SHC proteins to a greater extent than those that are less abundant and
have lower affinity SHC binding sites. In this regard, it has been
reported that of the cytokines known to activate JAK2, interleukins-3
and -5, erythropoietin, and granulocyte-macrophage colony-stimulating
factor stimulate tyrosyl phosphorylation of SHC
proteins(23, 51, 52) . These cytokines as
well as interleukin-6, leukemia inhibitory factor, and oncostatin M,
have been reported to activate MAP
kinases(53, 54, 55) . It will be interesting
to determine for these cytokines whether SHC proteins interact with
receptor, JAK2, or some other protein.