(Received for publication, March 8, 1995; and in revised form, May 30, 1995)
From the
Many signaling pathways initiated by ligands that activate
receptor tyrosine kinases have been shown to involve the binding of SH2
domain-containing proteins to specific phosphorylated tyrosines in the
receptor. Although the receptor for growth hormone (GH) does not
contain intrinsic tyrosine kinase activity, GH has recently been shown
to promote the association of its receptor with JAK2 tyrosine kinase,
to activate JAK2, and to promote the tyrosyl phosphorylation of both GH
receptor (GHR) and JAK2. In this work, we examined whether tyrosines
333 and/or 338 in GHR are phosphorylated by JAK2 in response to GH.
Tyrosines 333 and 338 in rat full-length (GHR)
and truncated (GHR
) receptor were replaced with
phenylalanines and the mutated GHRs expressed in Chinese hamster ovary
cells. These substitutions caused a loss of GH-dependent tyrosyl
phosphorylation of truncated receptor and a reduction of GH-dependent
phosphorylation of the full-length receptor. Consistent with
Tyr
and/or Tyr
serving as substrates of
JAK2, these substitutions resulted in a loss of tyrosyl phosphorylation
of truncated receptor in an in vitro kinase assay using
substantially purified GH
GHR
JAK2 complexes. The Tyr to Phe
substitutions did not substantially alter GH-dependent JAK2 association
with GHR or tyrosyl phosphorylation of JAK2. These results suggest that
Tyr
and/or Tyr
in GHR are phosphorylated in
response to GH and may therefore serve as binding sites for SH2
domain-containing proteins in GH signal transduction pathways.
Ligand binding to membrane receptors with intrinsic tyrosine
kinase activity has been shown to result in the phosphorylation of
multiple tyrosines in the receptors themselves (reviewed in (1) ). Once phosphorylated, these tyrosines have been shown to
bind the Src homology 2 (SH2) domain (a region of 100 amino acids)
of both enzymes and noncatalytic proteins involved in signal
transduction (2, 3, 4, 5) . Thus,
phosphorylated tyrosines in receptors are believed to serve a vital
role linking extracellular stimuli and cellular responses. Although GHR (
)itself is not a tyrosine kinase, GH has recently been
shown to promote the association of GHR with the tyrosine kinase JAK2,
to activate JAK2, and to promote phosphorylation of tyrosyl residues in
both GHR and JAK2(6, 7) . Since JAK2 is activated (and
thus phosphorylated) in response to multiple ligands that bind to
members of the cytokine receptor
superfamily(6, 8, 9, 10) , it has
been presumed that specificity in ligand response resides at least in
part in the tyrosines that are phosphorylated in individual receptors.
We were therefore interested in determining which tyrosines in GHR are
phosphorylated in response to GH and thereby could potentially serve as
binding sites for SH2 domain-containing signaling molecules that
mediate responses to GH.
Previous studies showed that rat
GHR (numbering system of (11) ), which
lacks approximately half of the cytoplasmic domain of GHR, is
phosphorylated on tyrosines in response to GH in intact
cells(7) . Furthermore, this truncated receptor is
phosphorylated on tyrosines when GH
GHR
JAK2 complexes are
substantially purified from GH-treated cells and incubated with
ATP. These results indicate that one or
more of the 4 intracellular tyrosines (Tyr
,
Tyr
, Tyr
, Tyr
) present in
this truncated GHR is phosphorylated in response to GH, presumably by
JAK2. Based upon predictions of secondary structure, hydrophilicity,
chain flexibility, and surface
probability(12, 13, 14, 15, 16, 17) ,
Tyr
of GHR was judged to be the most likely of the 10
cytoplasmic tyrosines in the receptor to be phosphorylated. It is also
the only tyrosine in the truncated receptor that is conserved among
species (cow, human, rabbit, sheep, rat, mouse, chicken,
pig)(18, 19, 20, 21, 22, 23, 24, 25) ,
suggesting that Tyr
may play an important role in GH
action. In this work, we use site-directed mutagenesis to examine
whether Tyr
and/or its close neighbor Tyr
is phosphorylated in response to GH. Our findings provide strong
evidence that one or both of these tyrosines is phosphorylated in
response to GH, serves as a substrate for JAK2, and is not required for
association of the receptor with JAK2 or GH activation of JAK2. The
accompanying paper (42) provides evidence that Tyr
and/or Tyr
may be involved in at least some actions
of GH.
Figure 1: Wild-type and mutated GHRs expressed in CHO cells. Denoted are the extracellular domain, the transmembrane (hatched area), and the cytoplasmic domain of the mutated rat liver GHR. Tyrosyl residues in the cytoplasmic domain are denoted by Y. Phenylalanine residues that were substituted for tyrosyl residues are denoted by F. Binding data were determined as described under ``Experimental Procedures.''
Figure 3:
Ability of mutated GHR to be
phosphorylated in an in vitro kinase assay. a, CHO
cells expressing GHR (lanes A and B) and GHR
Y333F,Y338F (lanes
C-F) (four 100-mm dishes/condition) were incubated for 1 h
at 25 °C with 4.5 nM (100 ng/ml) hGH (+GH, lanes
B, D, and F) or without hGH (-GH, lanes A,
C, and E) and lysed with HVTDP buffer. Solubilized
proteins were immunoprecipitated using
GH (1:10,000).
Immunoprecipitates were incubated with
[
-
P]ATP for 10 min and analyzed by
SDS-PAGE. The migration of molecular weight standards (
10
) are indicated on the right. The
migration of JAK2 and GHR
are indicated on the left. b, CHO cells expressing GHR
(lanes A-C) or
GHR
Y333F,Y338F (lanes D-I) were
incubated at 37 °C for 15 min with 4.5 nM hGH (+GH, lanes B, C, E, F, H, and I) or
without hGH (-GH, lanes A, D, and G).
Solubilized proteins were immunoprecipitated with
GH (1:10,000).
Samples were incubated without (-ATP, lanes A, B, D, E,
G, and H) or with (+ATP) (lanes C,
F, and I) 5 µM unlabeled ATP for 10 min.
Samples were eluted with SDS-PAGE sample buffer and Western-blotted
with
PY. The migration of molecular weight standards (
10
) is indicated on the right. The
migration of JAK2 and truncated receptor is indicated on the left.
Figure 4:
125I-hGH affinity labeling of mutated GHRs
expressed in CHO cells. One 100-mm dish each of CHO cells expressing
GHR (lanes A, B, and G),
GHR
(lanes C and D),
GHR
Y333F,Y338F (lanes E and F), and GHR
Y333F,Y338F (lane H)
were incubated for 1 h at 25 °C with
I-hGH in the
absence (TOT, lanes B, D, F, G, and H) or presence (NS, lanes A, C, and E) of 1 µg/ml unlabeled hGH.
Disuccinimidyl suberate (0.4 mM) was then added and the
incubation continued for 15 min at 8 °C. Samples were analyzed by
SDS-PAGE followed by autoradiography. The migration of molecular weight
standards (
10
) is indicated between lanes F and G. The migration of
GHR
, GHR
, and degraded
GHR
Y333F, Y338F is
indicated.
Figure 2:
GH-dependent tyrosyl phosphorylation of
mutated GHR. CHO cells expressing various GHR as noted were incubated
for 5 min at 37 °C without (-GH) (lanes A, C,
E, and G) or with 23 nM (500 ng/ml) hGH (+GH) (lanes B, D, F, and H). Cellular
proteins were immunoprecipitated with GH (1:8000) and
Western-blotted with
PY (1:7500). The migration of JAK2 and GHR
are indicated on the right and prestained molecular weight
standards (
10
) on the left.
Figure 6:
The ability of mutated GHR to elicit
GH-dependent tyrosyl phosphorylation of JAK2. CHO cells expressing
various GHR as noted were incubated for 5 min at 37 °C without
(-GH) (lanes A, C, E, G, I, and K) or with 23
nM (500 ng/ml) hGH (+GH) (lanes B, D, F, H,
J, and L) as described for Fig. 2. Proteins were
immunoprecipitated with JAK2 (1:1000) and Western-blotted with
PY (1:7500). The migration of JAK2 is indicated. Lanes
A-H are directly comparable with lanes A-H in Fig. 2, since samples were prepared using aliquots from the same
cells and were separated on the same gel and Western-blotted
together.
Figure 7:
The ability of GH to stimulate tyrosyl
phosphorylation of cellular proteins in CHO cells expressing mutated
GHR. CHO cells expressing various GHR as noted were incubated at 37
°C with 23 nM (500 ng/ml) hGH for the times indicated and
then lysed with boiling SDS sample buffer diluted (20:80) with lysis
buffer. Proteins were separated by SDS-PAGE and Western-blotted with
PY. The migration of prestained molecular weight standards
(
10
) are indicated between lanes F and G. The migration of p121, p97, p42, and p39 is
indicated by arrows on the left.
Figure 5:
The ability of mutated GHR to associate
with JAK2. CHO cells expressing GHR as noted were incubated for 5 min
at 37 °C without (lanes B, D, F, and H) or with
23 nM (500 ng/ml) hGH (lanes A, C, E, and G). Cellular proteins were immunoprecipitated with GH
(1:8000) and Western-blotted with
JAK2 (1:7500). The migration of
JAK2 is indicated.
When
GHGHR
JAK2 complexes are precipitated using
GH from
GH-treated CHO cells expressing wild-type receptor and Western-blotted
with
PY, a broad band migrating with M
120,000-130,000 is observed (Fig. 2, lane
B), as reported previously(7) . Western blotting with
GHR and
JAK2 indicates that this band contains both GHR and
JAK2, with JAK2 migrating as a rather narrow band (M
130,000) (see Fig. 6) and GHR migrating as a diffuse
band (M
120,000) with and just below
JAK2(6, 7) . In
PY blots of
GH precipitates
from GH-treated CHO cells expressing Y333F,Y338F full-length receptor,
a diffuse band migrating with a M
appropriate for
both GHR and JAK2 is also observed (Fig. 2, lane D).
The diffuseness of the band indicates that the mutated receptor is
phosphorylated, suggesting that tyrosines other than 333 and/or 338 in
GHR are phosphorylated in response to GH. However, the significantly
reduced intensity of this band (by 76 ± 2%, n =
3) compared with that obtained with wild-type receptor suggests that
phosphorylation of Tyr
and/or Tyr
contributes to the level of GHR phosphorylation observed in
wild-type receptor.
To confirm that in the in
vitro kinase assay, there is a difference between mutated and
unmutated receptor in the amount of phosphate incorporated into
tyrosyl, as opposed to seryl and threonyl, residues,
GHGHR
JAK2 complexes were prepared from CHO cells treated
with 100 ng/ml (4.5 nM) GH for 15 min at 37 °C and
incubated in the absence and presence of unlabeled ATP at the same
concentration of ATP (5 µM) used in the
[
-
P]ATP experiment. Kinase assay-dependent
changes in the amount of tyrosyl phosphorylation of GHR were assessed
by Western blotting with
PY. An ATP-dependent tyrosyl
phosphorylation of a protein migrating with appropriate M
was observed when GHR was prepared from CHO
cells expressing truncated receptor (Fig. 3b, compare lanes B and C), but not when it was prepared from
cells expressing truncated receptor with the Tyr to Phe substitution (Fig. 3b, compare lanes E and F). As
in Fig. 3a, the intensity of the JAK2 band from mutated versus unmutated cells was reduced approximately to the same
extent as binding of
I-hGH (Fig. 1). Even when lanes E and F were exposed to film for a
substantially longer period of time (Fig. 3b, lanes H and I) to compensate for the 40% decrease in GH binding
in the cells expressing mutated receptor and making the JAK2 signal
comparable for the mutated and unmutated receptors, no band
corresponding to the mutated receptor was detectable. (
)
To verify
that mutation of Tyr and Tyr
to Phe did not
result in adventitious proteolysis of full-length and truncated
receptors to the extent that potential alternative phosphorylation
sites were deleted, CHO cells expressing the various GHRs were
incubated with
I-hGH, followed by the cross-linking
reagent disuccinimidyl suberate. Fig. 4illustrates that when
the molecular weight of hGH (22,000, (37) ) is taken into
account, the various cross-linked
I-hGH
GHR
complexes migrate as proteins of the appropriate size (M
134,000 for full-length, M
95,000
for truncated receptor). In Fig. 4, a significantly greater
portion of the
I-hGH
GHR
Y333F,
Y338F complexes compared with other
I-hGH
GHR
complexes appeared to be degraded, migrating as if the receptor was
truncated at amino acid
415. However, this large difference was
not reproducible. In the three cross-linking experiments performed, the
amount of
I-hGH cross-linked to full-length mutated GHR
was only 26 ± 37% less than the amount of
I-hGH
cross-linked to full-length GHR. This suggests that the reduced (by
80%) phosphorylation observed for
GHR
Y333F,Y338F
JAK2 complexes compared with
GHR
JAK2 complexes cannot be attributed to a
comparable reduction in the amount of intact receptor. Despite the
presence of substantial amounts of degraded
I-hGH
GHR
complexes
corresponding in size to GHR
in Fig. 5,
one does not see in Fig. 2a phosphorylated band corresponding
in size to this truncated receptor. This provides additional evidence
that Tyr
(predicted to be the only tyrosine present in
Tyr
Phe mutated GHR
) is not phosphorylated
in response to GH, supporting our overall conclusion that Tyr
and/or Tyr
, but not Tyr
(or
Tyr
) are phosphorylated in response to GH.
To determine whether Tyr and/or Tyr
are required for activation of JAK2, we
compared the abilities of the mutated and unmutated GHR to mediate
GH-dependent tyrosyl phosphorylation of JAK2. Tyrosyl phosphorylation
of JAK kinases is thought to be due to autophosphorylation and thus to
reflect JAK activation(6, 8) , a hypothesis supported
by the finding that tyrosyl phosphorylation of JAK2 correlates well
with JAK2 activation by GH. (
)JAK2 was precipitated from CHO
cells expressing the various GHRs using
JAK2 and tyrosyl
phosphorylation of JAK2 was assessed by Western blotting with
PY. Fig. 6illustrates that for CHO cells expressing the four
different GHR, including two CHO cell lines expressing different levels
of GHR
used in the accompanying paper (42) (clone 23 used in all other experiments (relatively high)
and clone 3 (relatively low)), GH stimulates tyrosyl phosphorylation of
JAK2, roughly in proportion to the amount of GH binding detected in the
different cell lines. Phosphorylation of JAK2 was 129 ± 29% (n = 2) for mutated versus unmutated
full-length receptor and 48 ± 20% (n = 2) for
mutated versus unmutated truncated receptor. The corresponding
ratios for
I-hGH binding were 88 ± 2% and 60
± 6% (n = 10), respectively.
JAK2 Western
blots of the
JAK2 immunoprecipitates revealed similar amounts of
JAK2 expressed in the different cell lines (data not shown), indicating
that differences in JAK2 phosphorylation in response to GH cannot be
attributed to differences in levels of JAK2 expressed in the different
cell lines.
The results presented in this work provide strong evidence
that Tyr and Tyr
are not required for JAK2
association with GHR or for JAK2 activation. This is consistent with
the finding using human GHR expressed in FDC-P1 cells (published while
the present paper was under review) that no tyrosines in GHR are
required for JAK2 phosphorylation in response to GH(41) . The
results of this study also provide evidence that Tyr
and/or Tyr
are phosphorylated in response to GH,
since receptors lacking these tyrosines are phosphorylated to a
significantly reduced extent (full-length) or not at all (truncated
receptor) compared with the same sized receptors retaining these
tyrosines. Presumably, it is the GHR-associated JAK2 tyrosine kinase
that phosphorylates these tyrosine(s), since the truncated receptor is
phosphorylated when GH
GHR
JAK2 complexes are precipitated
with
GH and incubated with [
-
P]ATP,
whereas the truncated receptor lacking Tyr
and
Tyr
is not. Although Tyr
seems the most
likely candidate based upon sequence analysis, additional studies will
be required to determine which of the two tyrosines (333 or 338) is
phosphorylated. Whether or not these are the only tyrosines in
GHR
that are phosphorylated is not known, since
it is possible that phosphorylation of Tyr
and/or
Tyr
is required for the subsequent phosphorylation of
Tyr
and Tyr
. Although it seems unlikely
given the conservative nature of the amino acid substitution, our
results cannot rule out the alternative possibility that Tyr
and Tyr
are not themselves phosphorylated but
rather, mutating Tyr
and Tyr
to Phe alters
the ability of Tyr
and/or Tyr
to be
phosphorylated.
Tyr and/or Tyr
appear
not to be the only tyrosines phosphorylated in response to GH, since GH
appears to stimulate the tyrosyl phosphorylation of the full-length,
Tyr
Phe mutated receptor. The 6 tyrosines between amino acids
454-638 are the most likely candidates because tyrosines other
than 333 and/or 338 present in GHR
appear not to
be phosphorylated to any great extent. Multiple phosphorylated
tyrosines in GHR would be consistent with multiple sites of
phosphorylation in receptors with intrinsic tyrosine kinase activity (e.g. receptors for insulin, epidermal growth factor,
platelet-derived growth factor) (reviewed in (1) ). Multiple
sites of phosphorylation with differing affinities for various SH2
domains would provide a mechanism by which GH could initiate several
signaling pathways simultaneously.
The finding that Tyr and/or Tyr
are likely to be phosphorylated in
response to GH raises the question of whether either or both serve as
binding sites for specific SH2 domains. Neither tyrosine, with its
surrounding amino acids, closely resembles a high affinity binding site
(as currently defined) of the SH2 domains of Csk, SHC, Syk, Vav, Grb2,
3BP2, HCP, or fps/fes(5) . Consistent with this, neither
tyrosine appears to be required for GH-dependent SHC
phosphorylation(40) . Since SHC lies downstream of GHR and
upstream of mitogen-activated protein kinases, the SHC data are
consistent with the data presented here and in the accompanying paper (42) that Tyr
and Tyr
are not
required for GH activation of the mitogen-activated protein kinases
ERKs 1 and 2. Tyr
and Tyr
also appear not
be required for GH stimulation of insulin receptor substrate-1 (
)or for activation of signal transducer and activator of
transcriptions (Stats) 1 and 3 , (
)although the data do not
exclude the possibility that one or more of these SH2 domain-containing
proteins binds to Tyr
and/or Tyr
. However,
as described in the accompanying paper(42) , Tyr
and/or Tyr
do seem to be required for other actions
of GH (lipid synthesis, protein synthesis), cellular responses for
which the signaling pathways are less well defined. Verification that
Tyr
and/or Tyr
are tyrosyl-phosphorylated
using phosphopeptide analysis and identification of the signaling
molecules that bind to the corresponding phosphorylated tyrosine(s) is
likely to provide important information about signaling pathways used
not only by GH but by other ligands that signal via tyrosine kinases.