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Department of Internal Medicine, Division of Endocrinology, University of Virginia (D.W.L., C.M.S.), Charlottesville, Virginia 22908; and the Endocrine Sciences Research Division, Department of Medicine, University of Manchester (P.E.C., A.J.W.), Manchester, United Kingdom M13 9PT
Address all correspondence and requests for reprints to: Dr. Corinne M. Silva, Box 511, Division of Endocrinology, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908. E-mail: cms3e{at}virginia.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although the general intracellular signaling pathways that are activated by GH have been characterized, our studies in the human IM-9 lymphocyte have demonstrated apparent differences in the pathways activated by GH compared with those reported in the well characterized 3T3-F442A mouse preadipocyte. As in 3T3-F442A cells, GH activates the tyrosine phosphorylation of JAK2 in the IM-9 lymphocyte (3). However, only STAT5 (not STAT1 or STAT3) is activated by GH in the IM-9 lymphocyte (4). Furthermore, activation of DNA binding, as seen by electrophoretic mobility shift assay analysis with the c-fos m67 sis-inducible element, is different in the two cell types (4). In the data presented here, we demonstrate by Western blotting and in vitro kinase assay that GH does not activate MAP kinase (MAPK) in the IM-9 lymphocyte (as it does in the 3T3-F442A preadipocyte). The adapter protein Shc and the Raf kinase (known to be involved in GH signaling to the MAPK pathway) are also investigated.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(IFN) were purchased from Genentech (San
Francisco, CA). The enhanced chemiluminescence kit (ECL) was purchased
from Amersham (Arlington Heights, IL). Polyclonal antiphosphotyrosine
antibody has been described previously (5). Horseradish
peroxidase-conjugated, recombinant antiphosphotyrosine (RC20H), and
polyclonal anti-Shc were obtained from Transduction Laboratories
(Lexington, KY). Anti-MAPK (Erk2) was purchased from Upstate
Biotechnology (Lake Placid, NY), and anti-phospho-MAPK was obtained
from New England Biolabs (Beverley, MA). Anti-STAT5 (sc-835) and
polyclonal anti-Raf (anti-A-Raf and anti-C-Raf-1) were obtained from
Santa Cruz Biotechnology (Santa Cruz, CA). Phorbol 12-myristate
13-acetate (PMA) was purchased from Sigma Chemical Co. (St. Louis, MO).
Acrylamide, bisacrylamide, SDS, and prestained molecular mass standards
as well as all tissue culture reagents were obtained from Life
Technologies (Gaithersburg, MD). Except where noted, other reagents
were either reagent or molecular biological grade from Sigma Chemical
Co.
Cell culture and preparation of cell lysates
IM-9 cells were obtained from the American Type Culture
Collection (Rockville, MD), and 3T3-F442A cells were provided by Dr.
P. J. Bertics (University of Wisconsin). IM-9 cells were grown as
suspension cultures in Corning T-150 flasks in a 37 C, 5%
CO2 incubator in MEM supplemented with nonessential amino
acids, glutamine, and 10% FCS. 3T3-F442A cells were grown in DMEM-10%
FCS on Corning T-75 flasks (Corning, Corning, NY). Cells were passaged
every 3 days at 1:10 (IM-9) or 1:20 (3T3-F442A) dilution. After 3 days
of growth, cells were preincubated in either MEM or DMEM containing
0.1% BSA, overnight or for 1 h at 37 C depending on the
particular experiment. The next day, cells were pelleted and washed in
0.1% BSA-containing medium in the case of IM-9s, or medium was removed
and new 0.1% BSA-containing medium was added in the case of 3T3-F442A
cells. Cells were then treated with medium alone (control), 200 ng/ml
rhGH, or 5 ng/ml PMA for various times at 37 C. At the end of this
incubation, cells were washed in PBS and then lysed in lysis buffer
which contained 150 mM NaCl, 5 mM EDTA, and 50
mM Tris, pH 7.4 and one of the following detergents (see
details of each experiment): 1% Nonidet P-40, 1% Triton X-100, or 1%
Triton X-100 and 1% deoxycholate. All lysis buffers contained the
following protease and phosphatase inhibitors: 25 µg/ml leupeptin,
0.076 trypsin inhibitor unit/ml aprotinin, 10 mM vanadate,
and 0.2 mM phenylmethylsulfonylfluoride. Lysates were
stored at -70 C until use. Upon thawing, lysates were microfuged
(100,000 x g, 10 min, 4 C), and resulting supernatants
were analyzed as described below.
Western blotting
Lysates prepared as described above were mixed 1:1 with Laemmli
buffer (6) and fractionated through a 7.5% polyacrylamide gel,
electrophoretically transferred to nitrocellulose, and blotted as
described previously (3). Blocking buffers were 0.15 M
NaCl, 0.1% Tween-20, and 50 mM Tris, pH 8.0, and 3% BSA
for the antiphosphotyrosine antibody or 5% nonfat dry milk for all
other antibodies. Secondary antibodies were either donkey antirabbit or
sheep antimouse conjugated to horseradish peroxidase, and antibody
binding was detected using the ECL detection kit.
Immunoprecipitation
For Shc immunoprecipitation, cells were lysed in 1% Triton
X-100 and 0.5% Nonidet P-40-containing lysis buffer. For Raf
immunoprecipitation, cells were lysed in a 1% Nonidet P-40 lysis
buffer (see above). Lysates were incubated with antibody overnight at 4
C. Protein A-agarose (Boehringer Mannheim, Indianapolis, IN) was added
for an additional 1 h at 4 C. Agarose pellets were washed (three
times) in detergent buffer, and then bound proteins were removed by
boiling in 1 x Laemmli buffer.
MAPK activity assay
Cells were treated with GH (200 ng/ml) or PMA (5 ng/ml) for 5
min at 37 C before incubations were terminated by aspiration of the
medium. Cells were washed twice with 10 ml PBS (4 C) before being
harvested in extraction buffer (50 mM ß-glycerophosphate,
pH 7.3; 1.5 mM EDTA; 1 mM benzamidine; 0.5
mM Na3VO4; 1 mM
dithiothreitol; 0.1 mM phenylmethylsulfonylfluoride; and 1
µg/ml each of leupeptin, aprotinin, and pepstatin A). Samples were
quickly frozen in liquid nitrogen and stored at -70 C. MAPK activity
was measured in triplicate by the ability of cell extracts to
phosphorylate threonine 669 of a synthetic peptide substrate, as
reported previously (7). Briefly, 5–20 µl extract were incubated
with 50 µM ATP (containing 200 µCi/ml
[-32P]ATP) in the presence and absence of 0.2
mM T669 for 10 min at 30 C. Reactions were terminated by
spotting 25-µl aliquots onto 2-cm2 squares of P-81 paper
and immersion in 150 mM phosphoric acid. Samples were
washed four times with phosphoric acid and once with ethanol before
being air-dried and counted for radioactivity. The protein content of
lysates was determined using the Bio-Rad protein assay kit, and MAPK
activity was calculated as picomoles of phosphate incorporated per mg
protein. Results are presented as the median and range. Differences in
MAPK activity between control and treated cells were examined by
nonparametric analysis using the Mann-Whitney U test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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demonstrates that
tyrosine phosphorylation is activated in response to GH in both cell
types (4, 8, 9, 10). GH stimulates the tyrosine phosphorylation of JAK2
(
120 kDa), the GH receptor (the diffuse band above JAK2 in IM-9
cells and below JAK2 in the 3T3-F442A cells), and STAT proteins (93
kDa). In addition to the tyrosine-phosphorylated proteins discussed
above, other laboratories have reported that GH treatment of 3T3-F442A
cells results in the tyrosine phosphorylation and activation of MAPK
(11, 12, 13). To determine whether MAPK was activated by GH in IM-9 cells
as well, lysates from IM-9 and 3T3-F442A cells were immunoblotted with
an antibody specific for MAPK, which, at the concentration used,
detects the 42-kDa form of MAPK. As seen in the control lanes of Fig. 1B, MAPK is detected in both IM-9 and 3T3-F442A cells. However, an
upshift in molecular mass (indicating activation) occurs only in
3T3-F442A cells and not in IM-9 cells. This result is consistent with
antiphosphotyrosine blotting from our original work in IM-9 cells,
which failed to show a tyrosine-phosphorylated protein in the MAPK
molecular mass range (14).
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show Western blot analysis of
lysates from GH-treated IM-9 and 3T3-F442A cells. A band was detected
only in GH-treated lysates from 3T3-F442A cells, but not in GH-treated
IM-9 lysates, again indicating that MAPK is activated by GH in
3T3-F442A preadipocytes, but not in IM-9 lymphocytes. To determine
whether MAPK could be activated under any conditions in IM-9
lymphocytes, cells were treated with IFN, which we have shown
previously activates the JAK/STAT pathway in IM-9 cells (3), and with
the phorbol ester PMA, which has been shown to activate MAPK,
presumably through protein kinase C (PKC) activation of Ras and/or Raf
(15, 16, 17). Antiphospho-MAPK blotting of lysates from GH-, IFN-, and
PMA-treated IM-9 cells demonstrated again that no phosphorylated MAPK
band was present in the GH-treated lysates (Fig. 2, bottom
panel). Furthermore, there was no band indicative of
phosphorylated MAPK in the IFN-treated lysates, indicating that in
these cells, this cytokine does not activate MAPK despite its ability
to activate JAK1, JAK2, and STAT1, as shown by us previously (3). We
have also found, by the same techniques discussed here, that insulin
does not activate MAPK in IM-9 cells (data not shown). In contrast, a
phospho-MAPK band is clearly seen in the PMA-treated lysates,
indicating that this phorbol ester is able to activate the
Ras/Raf/MAPK/ERK kinase (MEK)/MAPK pathway in IM-9 cells.
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shows analysis of MAPK activation in
3T3-F442A preadipocytes using Western blotting with both
antiphosphotyrosine (Fig. 3A) and anti-MAPK (Fig. 3B). As reported by
others (11, 12, 13), activation of MAPK by GH was time dependent and, as
seen by both antiphosphotyrosine and anti-MAPK blots, was evident at 5
and 10 min, but decreased by 30 min in the 3T3-F442A cells.
Furthermore, a robust activation of MAPK occurred in response to PMA
and lasted throughout the 30-min treatment period; in fact, all of the
MAPK protein detectable by anti-MAPK blotting was activated to the
slower mobility form (see Fig. 3B).
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shows antiphosphotyrosine
blotting of lysates from IM-9 cells treated with GH for up to 60 min.
As shown previously, GH stimulated the tyrosine phosphoryl-ation of
the GH receptor, JAK, and STAT proteins, and this stimulation occurred
after 5 min of treatment and lasted for up to 30 min, but was obviously
decreased by 60 min. However, we found that there was no protein in the
MAPK molecular mass range that was tyrosine phosphorylated in response
to GH at any of these time points. This lack of activation was even
more obvious compared with that after treatment with PMA. A protein of
the correct molecular mass for MAPK was clearly activated at all times
of treatment with PMA (Fig. 4A, arrow). Figure 4B shows
immunoblotting of IM-9 lysates with the MAPK antibody; results
different from those seen with 3T3-F442A cells were obtained. No
shifted band indicative of activated MAPK was seen in any of the
GH-treated samples (Fig. 4B). These results demonstrate that GH does
not activate MAPK in the IM-9 cell during time points at which MAPK is
activated in the 3T3-F442A pre-adipocyte and under conditions in
which JAK2 and STAT5 are tyrosine phosphorylated. In the IM-9 samples
treated with PMA, an upper band resulting in a doublet upon MAPK
blotting indicated that MAPK was activated in IM-9 cells with this
treatment. However, the MAPK protein that was detected in IM-9 cells
was not completely activated to the phosphorylated form by PMA in the
IM-9 cells as it was in the 3T3-F442A cells (compare Fig. 4B with Fig. 3B
).
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, both GH and PMA activated MAPK
activity in 3T3-F442A cells when measured in this assay. In contrast,
although PMA clearly activated MAPK activity in IM-9 cells, GH did not.
In fact, the median activity was very comparable in the control and
GH-treated extracts. Assays over a time range of 5, 10, 30, and 60 min
of GH treatment showed no significant increase in MAPK activity,
whereas PMA increased activity, with a peak at 5 min (data not shown).
These activity results are consistent with the Western blotting results
shown in
Figs. 1–4, supporting the conclusion that GH does not
activate MAPK in the IM-9 lymphocyte, and that although PMA does
activate MAPK in IM-9 cells, it is not activated to the extent that it
is in 3T3-F442A cells.
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shows Western blotting and
immunoprecipitation with isoform-specific Raf antibodies (anti-A-Raf
and anti-C-Raf-1). Blotting indicated that both isoforms of Raf were
present in IM-9 lymphocytes (see Fig. 5, A and B, total lysates).
Furthermore, a shift of the C-Raf band in the PMA-treated lane upon
Western blotting of total lysates with anti-C-Raf indicated that it was
activated in response to this treatment (Fig. 5A). In contrast,
blotting with anti-A-Raf did not demonstrate a shift in this protein
with treatment, suggesting that A-Raf was not activated (Fig. 5B).
Immunoprecipitation with isoform-specific antibodies also showed a
shift of C-Raf (Fig. 5A), but not A-Raf (Fig. 5B), with PMA treatment,
supporting the hypothesis that Raf (probably C-Raf in particular) is
activated in response to PMA treatment (presumably through activation
of PKC). Neither C-Raf nor A-Raf underwent an apparent increase in
molecular mass in response to GH treatment.
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, immunoprecipitation followed by
blotting with Shc antibody demonstrated that three forms of Shc were
present in 3T3-F442A cells (66, 52, and 46 kDa), whereas IM-9 cells
contained predominantly the 52- and 46-kDa forms of Shc (on
overexposure, some signal at 66 kDa could be seen). Interestingly, in
response to PMA treatment of 3T3-F442A cells, the 66-kDa form of Shc
was shifted in apparent molecular mass, as seen by Shc blotting, but
was apparently not tyrosine phosphorylated (as seen by anti-pTyr in
Fig. 6B). This shift may correspond to serine phosphorylation of 66-kDa
Shc, as recently reported in response to insulin (see
Discussion). Anti-pTyr blotting (Fig. 6B) showed that the
66- and 52-kDa forms of Shc in 3T3-F442A cells were tyrosine
phosphorylated in response to GH, as described by others (18).
Furthermore, there were two tyrosine-phosphorylated bands seen at each
molecular mass, suggesting further GH-stimulated modification (possibly
a serine/threonine phosphorylation). Most importantly, neither the 52-
nor 46-kDa Shc isoforms present in IM-9 cells were tyrosine
phosphorylated in response to GH treatment, indicating that Shc is not
tyrosine phosphorylated or activated, corresponding with the lack of
MAPK activation by GH in these cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As a means toward characterizing the MAPK pathway in IM-9 cells, we have demonstrated that the phorbol ester PMA is able to activate MAPK in these cells. Phorbol ester stimulation of MAPK is thought to occur through PKC, which activates the pathway at the level of Ras and/or Raf (15, 16, 17, 24). Specific analysis of Raf isoforms in IM-9 cells indicated that C-Raf-1 was activated in response to PMA treatment. Therefore, these PMA studies suggest that the pathway from Ras/Raf to MAPK is functional and able to be activated in the IM-9 cell. We found that PMA activation of MAPK occurred to a greater extent in 3T3-F442A cells than in IM-9 cells. This difference could be seen both by Western blotting as well as by in vitro kinase assays and suggests that a negative regulatory mechanism may be constitutively active in the IM-9 cell. One such mechanism would involve a phosphatase acting on the MAPK pathway. Along these lines, we are currently investigating the potential role of the serine/threonine phosphatase PP2A in the MAPK pathway in IM-9 cells.
The adapter protein Shc has been shown to become activated by a number of cytokines. Shc, through binding to the cytokine receptor/JAK complex becomes tyrosine phosphorylated, leading to the binding of Grb2 and the activation of Sos, Ras, Raf, MEK, and MAPK (25, 26). Three isoforms of Shc have been identified (66, 52, and 46 kDa), and their regulation, expression, and activation are just now beginning to be elucidated (27). Both serine and tyrosine phosphorylations of Shc isoforms have been shown to be involved in their regulation. For example, serine as well as tyrosine phosphorylations of 52- and 46-kDa Shc isoforms have been reported in response to erythropoietin (28). Kao et al. have reported an insulin-stimulated serine phosphorylation of p66shc but not p52shc in CHO (Chinese hamster ovary) and 3T3-L1 adipocytes (29). A recent report suggests a negative regulatory role for the 66-kDa form of Shc in epidermal growth factor stimulation of the MAPK pathway (30). All three isoforms of Shc have been identified in the 3T3-F442A preadipocyte and have been shown to be tyrosine phosphorylated in response to GH treatment, leading to the binding of Grb2, which through Sos and Raf leads to activation of the MAPK pathway (18, 19). We found that the 52- and 46-kDa forms of Shc are the predominant forms present in the IM-9 cell, similar to other hematopoietic cell types (27). Furthermore, GH stimulation of these cells does not activate tyrosine phosphorylation of either of these forms as it does in 3T3-F442A cells. PMA treatment did not have any apparent effect on the 52- and 46-kDa Shc isoforms in IM-9 cells. A long exposure of the Shc blot from IM-9 lysates resulted in some detection of a 66-kDa band. The role of such a low level of 66-kDa Shc in IM-9 cells is not known; however, PMA did cause an increase in the apparent molecular mass of this band. Furthermore, PMA treatment resulted in an obvious shift in the molecular mass of the 66-kDa form in 3T3-F442A cells. This PMA-induced shift in the 66-kDa form was not due to tyrosine phosphorylation and, thus, may have been due to a serine phosphorylation similar to that reported in response to insulin in 3T3-L1 cells (see above). However, the potential role of a serine phosphorylation of p66shc in the PMA stimulation of MAPK is not known. Therefore, the Shc adapter protein is apparently differentially expressed and regulated in IM-9 and 3T3-F442A cells. The mechanism for this divergence is not understood, but may involve cell type-specific expression and/or activation of phosphatases and kinases involved in the tyrosine and/or serine phosphorylation of Shc or other, as yet unidentified, adapter proteins.
Recent studies have indicated that the Shc/Grb2/Sos/Ras/Raf pathway is
not the only pathway that leads to activation of MAPK in response to
GH. Like other cytokines, such as the IFNs (
and ) and the
interleukins (2, 4, 7, 15), GH has been shown to stimulate the
tyrosine phosphorylation of IRS-1, leading to the association of the
PI-3-kinase (31, 32, 33, 34). More recently, Anderson et al. have
shown that GH increases PI-3-kinase activity in 3T3-F442A cells and
that a PI-3-kinase inhibitor (wortmannin) attenuates the GH-stimulated
MAPK activity in these cells (35). Studies using antisense
oligodeoxynucleotides provide evidence for the role of a specific
isoform of PKC (PKC
) in the activation of the MAPK pathway by GH in
the 3T3-F442A preadipocyte (36). However, inhibition of either PKC or
PI-3 kinase only partially inhibits GH stimulation of MAPK (35, 36).
Thus, at least in the 3T3-F442A preadipocyte, there may be two pathways
by which GH activates MAPK, one involving Shc/Grb2 and one involving
IRS-1/PI-3-kinase/PKC. Whether these pathways converge and whether
they are involved in MAPK activation by GH in all cell types are not
clear. We are currently investigating the potential role of the
IRS-1/PI-3 kinase/PKC pathway in GH signaling in IM-9 cells.
In summary, our studies in the human IM-9 lymphocyte continue to demonstrate cell type-specific signaling by the GH receptor. Our initial work in IM-9 cells characterizing GH-stimulated tyrosine phosphorylation indicated that GH did not stimulate MAPK in these cells (14). The studies presented here specifically address the MAPK pathway and demonstrate that GH does not stimulate MAPK activity in these cells. Whether failure of GH to activate this pathway is a particular characteristic of IM-9 cells, a transformed cell line derived from a human myeloma, or whether it represents GH signaling in the lymphocyte cell type in general remains to be determined. Studies by others have demonstrated a stimulatory effect by GH on IM-9 cell growth under certain serum conditions (37, 38). Together with our studies, these results suggest that GH activation of growth in these cells occurs without activation of the MAPK pathway. Therefore, the IM-9 lymphocyte continues to provide a model for characterizing differential signaling from the GH receptor and the roles of individual signaling pathways.
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Acknowledgments |
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Footnotes |
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Received September 3, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in IM-9 cells. J Biol Chem 269:27532–27539 activates the MEK-ERK pathway in a
manner independent of Ras and dependent on Raf. J Biol Chem 271:23512–23519 engages the insulin receptor substrate-1 to
associate with the phosphatidylinositol 3'-kinase. J Biol Chem 270:15938–15941, and leukiemia
inhibitory factor promoted tyrosyl phosphorylation of insulin receptor
substrate-1. J Biol Chem 270:14685–14692This article has been cited by other articles:
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lane in the IPs indicates a negative control with
antibody but without sample. Arrows indicate Raf
proteins.