Download Binding of a Growth Hormone- Inducible Nuclear Factor Is Mediated

Binding of a Growth HormoneInducible Nuclear Factor Is Mediated
by Tyrosine Phosphorylation
Susan A. Berry, Pearl
Howard
C. Towle
L. Bergad,
Carmella
DeRose
Whaley,
and
Departments of Pediatrics (S.A.B., P.L.B., C.D.W.) and Biochemistry
(H.C.T.)
Institute of Human Genetics (S.A.B., H.C.T.)
University of Minnesota
Minneapolis, Minnesota 55455
achieving normalsomaticgrowth and fuel homeostasis.
GH action is initiated by bindingto a specific GH receptor on the cell surface, followed by activation of the
receptor-associated tyrosine kinase JAK2 (1). The
mode of signaltransduction that is elaborated by this
kinaseactivation in responseto GH is unknown.
One of the principaleffects of GH binding is to alter
specific gene expression in target cells. A number of
messengerRNA specieshave beenfound to be induced
by GH treatment. Two hepatic genesbelongingto this
class are the serine protease inhibitor genes Spi 2.1
and 2.3 (2). We have previously characterized an element in the 5’-flanking region of Spi 2.1 that is capable
of conferring GH responsivenessto a heterologous
promotor (GHRE). This elementbindsa hepatic nuclear
protein in a GH state-specific manner and does not
requirede nova protein synthesisfor activation of binding (3). This observation suggestedthat GH-responsive
bindingto the element requiresa reversibleposttranslationalmodification.Consideringthe activation of JAK2
tyrosine kinase activity upon GH binding, it seemed
logical to consider phosphorylation as a candidate
mechanismfor this modification.We examinedthe possible role of phosphorylationin the binding of the GHinducible nuclear factor (GHINF) and investigated
whether p91, a protein recently implicated in JAKB
mediatedsignaltransduction(4, 5) might participate in
GHINF bindingto the Spi 2.1 promotor elementin viva.
The nuclear mechanism
by which GH acts to induce
gene expression
after binding to its receptor on the
cell surface is not defined. We have characterized
an element in the 5’-flanking
region of the rat GHresponsive
serine protease inhibitor @pi) 2.1 gene
responsible
for its induction
by GH. This element
binds a hepatic nuclear protein(s)
in a GH statespecific manner. Activation
of binding by GH does
not require de nova protein synthesis,
suggesting
that a reversible
posttranslational
process
is required for binding to the element.
To define the
mechanism of this process, hepatic nuclear extracts
were analyzed by electrophoretic
mobility shift assays using a DNA fragment (-147 to -103) of the
Spi 2.1 gene. Treatment
of extracts with phosphatases resulted in a marked reduction
of GH statespecific binding. Addition of phosphatase
inhibitors
antagonized
the reduction in binding after phosphatase treatment.
The specific nature of the phosphorylation
event involved in binding was explored
using phosphotyrosine
antibodies
and a protein tyrosine phosphatase.
Treatment
of nuclear extracts
with either of these reagents ablated binding to the
response
element.
Because
the tyrosine-phosphorylated
transcription
factor protein p91 has recently been implicated
in cytokine signal transduction mediated by JAKS, we sought evidence that p91
was part of the GH-responsive
binding
complex.
Analysis of an enriched preparation
of GH-inducible
binding complexes
by Western blots using anti-p91
demonstrated
no immunoreactivity.
We conclude
that tyrosine phosphorylation
of a nuclear factor is
required
for GH state-specific
binding to this GH
response element in wivo, but that p91 is not present
in the binding complex.
(Molecular
Endocrinology
8:1714-1719,1994)
RESULTS
INTRODUCTION
GH is one of the principal developmental hormones
elaborated by the pituitary. Its action is critical for
o&la8E10/94$03.00/0
Mdecular
Endocrirology
Copyright CD 1994 by The Endoaine
Society
1714
AND DISCUSSION
We have previously demonstrated GH state-specific
binding of a hepatic nuclear factor to a DNA fragment
(-147 to -103) of the Spi 2.1 gene in electrophoretic
mobility shift assays.This site correlated with GHstatespecific deoxyribonuclease-l-hypersensitive sites located in proximity to this fragment of Spi 2.1 flanking
DNA. A concatamer of this fragment coupled to the
thymidine kinasepromotor and bacterial chloramphenicol acetyltransferasetransfected into isolated hepato-
GHINF Binding Is Mediated by Y Phosphorylation
1715
cytes conferred GH responsivenessto chloramphenicol
acetyltransferaseexpression.We concludedfrom these
observationsthat this fragment contains a GHRE that
acts by binding to a GH-inducible nuclear factor. GH
activation of GHINF bindingwas not blocked by cycloheximide pretreatment, suggesting that a posttranslational process was required
for activation
(3).
We sought information concerning the modification
that might be responsiblefor the alteration in binding
after GH treatment. As a number of nuclear binding
proteins are modulated by alterations in phosphorylation of specific amino acids, we initially examined the
effects of phosphatasetreatment on the binding reaction. Treatment of hepatic nuclear extracts from GHtreated rats with either acid or alkaline phosphatases
resulted in ablation of GH state-specific binding to the
GHRE(Fig. 1, left panel). The specificity of the response
was assessed
by using the same treated
extracts
to
bind to an oligonucleotide including the USF/MLTFbinding site of the adenovirus major late promotor, a
bindingreaction that is insensitiveto phosphatasetreatment (6). Binding
of upstream
stimulatory
factor/major
late transcriptionfactor (USF/MLTF) was unaffected by
phosphatasetreatment (Fig. 1, right panel). These data
suggest that phosphorylation of a nuclear protein is
GH
alk
ac
alk
ac
GH
critical for GH state-specific binding to the Spi 2.1
GHRE.
We examined whether inhibitors of phosphataseactivity would
with 1 mM sodium
of binding
by phospha-
vanadate
(Fig. 2). The marked
de-
crease in binding after phosphatasetreatment of extracts containing
GHINF without
the inhibitors
was sub-
stantially altered by the presence of these inhibitors.
Nonspecific inhibition of phosphatase
prevented the
reduction of bindingafter phosphatasetreatment.
To determine whether tyrosine phosphorylationwas
critical to GH-induciblenuclearfactor binding,we tested
the effects of a phosphotyrosineantibody on nuclear
protein binding to the Spi 2.1 GHRE. When nuclear
extracts were incubated with a monoclonalphosphotyrosine antibody, binding by the GH-induciblenuclear
factor was ablated(Fig. 3A). The useof other antibodies
failed to alter GHINF binding (data not shown). Thus,
tyrosine phosphorylationappears to be critical to GHmediatedfactor activation. To confirm this observation,
nuclearextracts were subjectedto treatment with protein tyrosine phosphatase-1B (PTPase),a phosphatase
specific for phosphorylated tyrosine residues.PTPase
alsoeliminatedbinding,and this ablationcould be inhibMEND-12
Fig. 1. Effect of Acid or Alkaline Phosphatase Treatment ot
Nuclear Extracts on Binding to Labeled Spi 2.1 GHRE (lefi) or
USF/MLTF (fight) Probes
Hepatic nuclear extracts from hypophysectomized rats without (Hx) or treated with human GH (150 pg/lOO g BW) 1 h
before liver harvest (all others) were used in electrophoretic
mobility shii assays after no phosphatase treatment (GH) or
after exposure to 0.3 U calf intestinal phosphatase (alk) or
0.01 U potato acid phosphatase (ac). There are also smaller
nonspecific complexes present in gel shifts with crude
extracts.
block the ablation
tase treatment. The addition of 10 mMpara-nitrophenol
phosphate, a phosphatase substrate, to the alkaline
phosphatasetreatment led to the reappearanceof GH
state-specific binding. Similar results were obtained
231141
FIG 2 18 X 17.9 (3614)
Fig. 2. Effect of Phosphatase Inhibitors on Diminution of
Binding after Phosphatase Treatment of Nuclear Extracts
Extracts from hypophysectomized rats treated with GH
were used in electrophoretic mobility shift assays with the Spi
2.1 GHRE probe after no alkaline phosphatase (0) or after
treatment with 0.1 or 0.3 U alkaline phosphatase (Alk Phos).
Inhibitors of phosphatase [lo mM para-nitrophenol phosphate
(pNPP) or 1 mu sodium vanadate] were added as indicated
above the lanes, as noted in Materials
and Methods.
Also
shown to the right of the vanadate lanes is an additional lane
with no phosphatase and no vanadate (No inhib).
MOL ENDO. 1994
1716
Vol8No.12
Fig. 3. Involvement of Phosphotyrosine in Binding
A, Effects of phosphotyrosine monoclonal antibody on binding. Extracts from GH-treated rats were incubated with increasing
quantities of phosphotyrosine antibody (anti-pY) before the addition of Spi 2.1 GHRE probe and electrophoresis. Hx, Extract from
hypophysectomized rats. B, Effects of PTPase on binding and inhibition of this effect by vanadate. Using hepatic nuclear extracts
from GH-treated rats, reaction mixtures had N&VO,, 1 mM (Van), or 500 ng (10 pl) PTPase (PTP) added according to the scheme
above the lanes, where (+) indicates that that reagent was added to the reaction mixture and (-) indicates that the reagent was
not added. C, Effects of phosphotyrosine antibody and PTPase on USF/MLTF binding. Quantities and conditions were identical to
those in A and B, except that the USF/MLTF probe was added to binding reactions.
ited by the addition of 1 mM vanadate (Fig. 38). Treatment of USF/MLTF-binding reactions with either phosphotyrosine antibody or PTPase resulted in no
alterationsin binding(Fig. 3C). The ablation of binding
by phosphotyrosineantibody and a phosphatasespecific for phosphotyrosineis strong evidencethat a phosphorylatedtyrosine residueis critical for GHINFbinding.
The understandingof signaltransduction by certain
polypeptide hormoneshas recently been facilitated by
observationsconcerningthe role of p91, a DNA-binding
factor phosphorylated at a critical tyrosine residue in
responseto cytokine signaltransduction(7,8). Because
the GH receptor-associatedtyrosine kinaseJAK2 has
been implicated in some instances of p91-mediated
geneactivation (4,5), and becausetyrosine phosphorylation is required for GHINF binding, we examined
whether p91 was present in the GHINF complex. We
useda commerciallyavailableantibody that recognizes
the N-terminalportion of p91 as well as the alternatively
splicedprotein ~84, which derives from the N-terminal
portion of p91 (9). This antibody has been used by
others to demonstrate the presence of p91 in complexes formed by y-interferon-treated extracts binding
to their regulatory element (10). Incubation of hepatic
nuclear extracts with this N-terminal p91 antibody did
not result in a change in the pattern
of GHINF binding
(data not shown). The failure to demonstrate a supershift suggeststhat neither p91 nor p84 is present in the
complex. To further investigate this possibility, we purified GHINF using affinity chromatography with the
GHRE and examinedthe highly enriched fraction using
Western blotting. The enrichedGHINF preparationcontains two polypeptides of about 93 and 70 kilodaltons
(kDa) that are immunoreactive with phosphotyrosine
antibodies(Fig. 4A) and are not seen in crude extracts
from the hepatic nuclei of hypophysectomized animals
(data not shown). A Western blot using p91 antibody
shows that although p91 is demonstrablein GH-activated crude nuclear extracts, there is no immunoreactivity in purified fractions containing GHINF (Fig. 48).
Although p91 is presentin GH-treated rat liver extracts,
the GHINF complex isolatedby affinity chromatography
doesnot contain proteinsrecognizableby an N-terminal
p91 antibody even when not bound to DNA.
Based on these observations, we conclude that tyrosine phosphorylationof GHINF is the mechanismby
which GH activates the bindingof GHINF.Alternatively,
a factor that activates GHINF binding could itself be
activated by tyrosine phosphorylation,so that the effect
on GHINF is indirect. We do not favor the latter possibility because the effect of phosphatasetreatment is
preserved in highly enrichedGHINF preparations(data
not shown). As yet, the candidate kinase responsible
for GHINF phosphotylationin responseto GHtreatment
remains unidentified. Our work suggests that neither
p91 nor p84 is involved in the GH responsivenessof
Spi 2.1. This is surprisingin view of the known participation of JAK2 in GH action and the observation that
JAK2 is required for some p91-mediated responses
(11). Clearly, a protein immunoreactivewith p91 antibody is present in our GH-treated crude nuclear extracts and in those of Gronowski and Rotwein (12).
1717
GHINF Binding Is Mediated by Y Phosphorylation
A
B
anti -pY
antLp9m4
217
32.5
Fig. 4. Antibodies to p91/84 Do not Recognize the Tyrosine
Phosphorylated Proteins Obtained after GHRE-Specific Affinity
Chromatography Purification of GHINF
A, lmmunoblot of crudeGH-treatedrat livernuclearextract
(cr) and affinity-purifiednuclearextracts (aff) incubatedwith
monoclonal phosphotyrosine antibody (anti-pY). B, Immunoblot of crude GH-treated rat liver nuclear extract (cr) and
affinity-purified nuclear extracts (aff) incubated with N-terminal
ISGFB (p91/84) antibody (anti-p91 /84). Mol wt markers (xl 03)
are shown to the left.
This protein is activated in responseto GH treatment,
as p91 immunoreactivityis absent in our crude extracts
from hypophysectomized liver (data not shown) (12).
Further, in GH-activated cell lines,a protein comigrating
with p91 was induced in responseto GH (13, 14).
However, activation of gene expression by other
polypeptide hormones via tyrosine phosphorylation in
the absenceof immunoreactivep91 has bean demonstrated (1516) suggestingthat the GH responsecould
have features in common with activation of gene
expressionby these hormones.Thus, the GH-mediated
pathway of signal transduction may diverge into p91dependentand -independentpathways. There are other
proteinsimmunologicallyrelated to p91 that serve similar roles in signaling pathways for cytokines; for example,the acute phaseresponsefactor APRF(Stat3) in
the interleukin-6 pathway (17-19). This protein has
52.5% amino acid homology with p91 (18). Additional
proteins in this gene family have also been described
(20). There is a family of p91-like genes, presumably
with different binding and transcriptional activation
activities.
Our studies cannot determine whether GHINF is a
memberof such a p91 gene family, but immunologically
distinct from it. Finbloomet al. (21) demonstrated the
presenceof a 93-kDa tyrosine-phosphorylatedprotein
in GH-activated IM-9 cell nuclear extracts, which was
present in complexes bound to an oligonucleotideof
the r-response region of the FCy-receptor factor. A
p91 antiserumfailed to produce a supershift, but when
the extracts were purified by affinity chromatography,
both an internal and C-terminal p91 antibody recog-
nized a 93-kDa protein on Western blots of sodium
dodecyl sulfate-polyacrylamidegels of the purified extracts (21). The 93-kDa tyrosine-phosphorylatedprotein
seenin our affinity-purified fraction could be this protein
despite the absence of immunoreactivity to N-terminal
p91 antibodies.
The signalingpathways of growth factor activation of
Ras proteins, followed by activation of a kinase cascade, may also be activated by GH. It has been demonstrated that mitogen-activatedprotein (MAP) kinases
are part of the phosphorylationcascadein the response
after GH binding to GH receptor in 3T3-F442A preadipocytes (22, 23). This also was noted in Chinesehamster ovary cells transfected with GH receptor (24) and
in GH-treated liver nuclear extracts (12). Ultimately,
many aspectsof MAP kinase-activatedsignaltransduction are mediatedby Jun/Fos binding to Hela cell activator protein-l (AP-1) elements. As Fos and Jun both
demonstrate GH responsiveness(25, 26) this may be
an alternative pathway for GH-mediatedgene activation. The Spi 2.1 gene does not contain a sequence
related to the consensusAP-l-binding site. However,
Fos expressionitself is may be mediatedby a p91-like
protein in a complex with other phosphoproteins.Thus,
GH may activate both p91-type and MAP kinasepathways in affecting different metabolicresponses.
Our observations indicate that tyrosine phosphorylation of a nuclear protein is critical in the in vivo responseof rat liver to GH treatment. This is madeevident
by the observation of GH state-specific binding of
GHINF to the GHRE of Spi 2.1, which is altered by
antagonism of tyrosine phosphotylation. The exact
pathway(s) by which GH-responsive Spi 2.1 gene
expression could be modulated by reversibletyrosine
phosphorylation of a nuclear binding protein remains
undefined.Emergingevidence about GH responsecurrently supports the relatively simple mechanismof direct activation of a latent cytoplasmic DNA-binding
protein, but if GHINF is a p91-like protein, it is immunologicallydistinct from it, suggestingthat it is neither
p91 nor the immunologicallyrelated Stat3 protein. Provocative evidence supporting polypeptide activation of
the complex MAP kinase pathway in GH action also
exists, and we cannot rule out activation of Spi 2.1
gene expression by this signal transduction cascade.
The resolution of this uncertainty will be provided ultimately by analysis of GHINF and its phosphorylation
site(s).
MATERIALS
AND METHODS
Nuclear Extract Preparation
Male rats (Fischer strain) were hypophysectomized at lOO125 g by the supplier (Taconic Farms, Germantown, NY) and
observed for at least 3 weeks to confirm growth failure. All
animals were maintained in accordance with the NIH Guide for
the Care andUseof LaboratoryAnimalsunderthe supervision
of Research Animal Resources of the University of Minnesota.
They were given 150 pg human GH (Genentech, South San
MOL
1718
ENDO.
1994
Vol8
Francisco,
CA)/1 00 g BW, iv, and their livers were removed
1
h later. All subsequent
work was performed
at 4 C unless
otherwise
specified.
Nuclei were isolated
according
to the
method of Gorski et al. (27). Extracts
of nuclei were prepared
by resuspension
in nuclear lysis buffer [lo mM HEPES (pH
7.6). 0.1 M KCI, 3 mM MgCI2.0.1
mrv EDTA, 1 mM dithiothreitol,
0.1 mM phenylmethylsulfonylfluoride,
and 10% glycerol]
in a
Dounce homogenizer
(4 ml/O.5 ml nuclear vol). While stirring
gently, nuclear lysis buffer containing
1.2 M KCI was added
dropwise
to a final concentration
of 0.4 M. After 30 min, the
mixture
was centrifuged
at 35,000 rpm in a Beckman
Ti-75
rotor (Beckman,
Palo Alto, CA) for 60 min. The resultant
supematant
was either frozen
in aliquots
immediately
or dialyzed against 25 mM HEPES (pH 7.6) 0.1 M KCI, 0.1 mM
EDTA, 1 mM dithiothreitol,
and 10% glycerol
twice for 2 h
each, then frozen in aliquots.
All aliquots were stored at -80
C. Protein concentrations
were determined
using the Bio-Rad
Protein Assay Kit (Richmond,
CA).
Probe
Preparation
An oligonucleotide
corresponding
to the coding strand of the
GHRE located from -147
to -103
of the rat Spi 2.1 gene
promoter
sequence
(3) was synthesized,
including
Taql recognition
sites at both ends for cloning
purposes.
A short
oligonucleotide
complementary
to the 3’end of the oligonucleotide (13 basepairs)
was also synthesized.
The annealed
duplex was extended
using Escherichia
co/i polymerase-I
(Klenow) with deoxy-ATP
as the radioactive
nucleotide.
It was
purified through
a B&Spin
6 column (BioRad
Laboratories).
Its specific activity was 1 x 1 Og cpm/pg.
A synthetic
duplex corresponding
to the adenovirus
USF/
MLTF-binding
site (6) was labeled by fill-in using E. co/i polymerase-l (Klenow) with deoxy-CTP
as the radioactive
nucleotide.
It was also purified through
a Bio-Spin
6 column and had a
specific activity of 1.5 X 1 O8 cpm/pg.
Electrophoretic
Mobility
Shift
Assay
All electrophoretic
mobility shift assays were performed
in a
buffer containing
20 mM HEPES (pH 7.6) 10% glycerol,
2 mM
MgC12, 5 rnhf &Cl,,
0.1 mg/ml .BSA, 4% Ficoli 400, 1 mM
spermidine,
1 mM DlT, and 1 mM phenylmethylsulfonylfluoride.
Poly(dldC~poly(dldC)
(1.5 pg) and poly(dAdT)-poly(dAdT)
(0.5 pg; Pharmacia-LKB,
Piscataway,
NJ) were added to each
reaction. For assays with the USF/MLTF
probe, all conditions
were identical, except that MgC12 and CaC12 were omitted from
the binding buffer.
For every 19 ~1 reaction
mixture,
6 fig
nuclear orotein extract were added. and the final KCI concentration was adjusted to 50 mM if necessary.
Where indicated,
para-nitrophenol
phosphate
at a final concentration
of 10 mM
or sodium vanadate
(N&VO,)
at a final concentration
of 1 rnt+t
was also added.
In examination
of the effects of phosphatase
on binding to
the GHRE. alkaline phosphatase
or potato acid phosphatase
(both from Boehringer
Mannheim
Biochemicals,
Indianapolis, IN) was appropriately
diluted in 0.1 mg/ml BSA and added
to the binding mixture.
After a brief mixing, the mixture
was
incubated
at 30 C for 20 min. For PTPase (Upstate
Biotechnology, Lake Placid, NY) studies, 500 ng PTPase on agarose
beads were added, and the mixture was incubated
at 37 C for
30 min. After phosphatase
treatments
of the extracts,
1 ~1
containing
20 fmol labeled GHRE was added to the treated
extracts,
and the mixture was further incubated
at 30 C for an
additional
20 min.
For examination
of alterations
of binding by antibodies,
the
monoclonal
phosphotyrosine
antibody 4GlO (Upstate Biotechnology) or anti-ISGF3
(p91/84)
polyclonal
antibody
(Transduction Laboratories,
Lexington,
KY) was added to the reaction
mixture and incubated
at 4 C for 1 h. One microliter
containing
20 fmol labeled GHRE was added, and the mixture was further
incubated
at 30 C for an additional
20 min.
The reaction mixtures
were loaded onto a nondenaturing,
No. 12
3.2% glycerol,
4% polyacrylamide
gel (39.51
acrylamide-bisacrylamide)
in a Bio-Rad
Protean
II system in 0.5 x TBE (45
mM Tris, 45 mM boric acid, and 1 mM EDTA, pH 8.3) and
electrophoresed
at 150 V for 2.5 h. The gels were dried and
exposed
to film.
Affinity
Purification
and lmmunoblotting
of GHINF
GHINF was purified from crude nuclear extracts
by chromatography
on separation,
using heparin agarose
followed
by
calf thymus-DNA
Sepharose.
The active fractions were affinity
purified by chromatography,
using an octomer
of the Spi 2.1
GHRE fragment
from -147 to -102 (3) coupled to cyanogen
bromide-activated
Sepharose4B,
as previously
described
(28). All elutions
were monitored
by electrophoretic
mobility
shift assay with the GHRE probe. Details of the purification
will be published elsewhere
(Bergad,
P. L. , H. C. Towle, S. J.
Schwarzenberg,
H.-M. Shih, and S. A. Berry, manuscript
in
preparation).
Proteins were subjected
to sodium dodecyl sulfate-polyacrylamide
gel electrophoresis
in a 7.5% gel, followed
by transfer
to BA-S nitrocellulose.
The blots were incubated
with either phosphotyrosine
antibody
or anti-ISGFB
and developed
with alkaline phosphatase-conjugated
secondary
antibodies according
to manufacturers’
protocols.
Acknowledgments
Received
March 3, 1994. Revision received
July 28, 1994.
Accepted
August 23, 1994.
Address
requests
for reprints
to: Susan A. Berry, M.D.,
Department
of Pediatrics,
University
of Minnesota,
420 Delaware Street
SE, Box 75 UMHC,
Minneapolis.
Minnesota
55455.
This work was performed
with the support
of the Vikings
Children’s
Fund. and the Deoartment
of Pediatrics
and the
Institute for Disabilities
Studies, University
of Minnesota.
Portions of the work were supported
by NIH Grant DK-32817.
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