(Received for publication, November 16, 1995; and in revised form, January 2, 1996)
From the
Activation of early response genes by interferons (IFNs)
requires tyrosine phosphorylation of the Stat transcription factors and
is mediated by the Jak family of tyrosine kinases. Recent evidence
suggests that ERK2 serine/threonine kinase modulates the IFN-stimulated
Jak/Stat pathway. In this report we show that in the myeloma cell line
U266 protein kinase A specifically interacts with the cytoplasmic
domain of the IFN/
receptor. Treatment of cells with the
adenylate cyclase activator forskolin inhibits IFN
-, IFN
-,
and hydrogen peroxide/vanadate-induced formation of complexes that bind
to enhancers known to stimulate the expression of IFN-regulated genes.
Immunoprecipitations followed by anti-phosphotyrosine immunoblots
indicate that tyrosine phosphorylation of the
chain of the
IFN
/
receptor, Jak1, Tyk2, as well as Stat1 and Stat2 is
reduced as a consequence of incubation of cells with forskolin. In
contrast, dideoxyforskolin, which fails to activate adenylate cyclase,
has no effect on IFN induction of the Jak/Stat pathway. These results
indicate a novel regulatory mechanism by which protein kinase A can
modulate the Jak/Stat signaling cascade.
The Janus family of tyrosine kinases (Jak1, Jak2, Jak3, and
Tyk2) is an integral component of many cytokine-activated signaling
cascades, which regulate tyrosine phosphorylation of the Stat proteins
such that they can translocate to the nucleus and bind DNA(1) .
These kinases are associated with one or more chains of a given
cytokine receptor. For instance, Tyk2 interacts with the subunit
of the type I IFN (
)receptor. Along with Tyk2, we have
determined that Jak1 and the SH2 domain containing tyrosine phosphatase
PTP1C, which acts as a negative regulator of the Jak/Stat pathway, also
selectively bind to a GST fusion protein, which consists of the
membrane proximal 50 amino acids of the
subunit of the
IFN
/
receptor(2) . During the course of these
experiments we showed that the 42-kDa form of MAPK (ERK2) binds the
IFN
/
receptor and is enzymatically activated by IFN
,
probably leading to the phosphorylation of Stat1 on serine
727(3) . This serine phosphorylation has been proven crucial
for the transactivation of IFN
-regulated genes without being
necessary for DNA binding of the Stat proteins(4) . As it has
been previously shown that in some cells the activation of MAPK through
various growth factors can be inhibited by increased levels of
intracellular cAMP(5, 6, 7, 8) , we
wanted to explore the possible involvement of protein kinase A, one of
the main targets of elevated cAMP, in the regulation of the
IFN
-induced Jak/Stat pathway.
The antagonistic actions of cAMP on growth factor activation
of MAPK have been shown to be cell-specific(12) . We therefore
wanted to determine whether forskolin, a direct stimulator of adenylate
cyclase, could inhibit IFN activation of MAPK in the myeloma cell
line U266. Cells were pretreated with forskolin for 20 min prior to the
addition of IFN
for 5 min. Extracts were prepared and incubated
with the IFN
/
receptor GST fusion protein as
described(2, 3) . MAPK activity was then assayed with
myelin basic protein and [
-
P]ATP to GST
fusion protein beads that had been incubated with the extracts. After
SDS-PAGE, the proteins were blotted to Immobilon and either exposed for
P incorporation into MBP (Fig. 1, upper
panel), probed with anti-phosphotyrosine antibody (middle
panel) or with anti-MAPK antibody (lower panel).
Incorporation of
P into MBP was enhanced about 5-fold when
cells were treated with IFN
(lanes 1 and 2).
Prior treatment of U266 cells with forskolin inhibited activation of
MAPK bound to the GST fusion protein by IFN
, as well as
IFN
-stimulated tyrosine phosphorylation of MAPK (compare lanes
2 and 4 of the upper and middle
panels). The amount of MAPK bound to the beads was not altered by
either forskolin or IFN
treatment of cells (lower panel).
Figure 1:
Forskolin
inhibits IFN-stimulated MAPK activity in U266 cells. U266 cells
were incubated without (lanes 1 and 3) or with
10
units/ml recombinant human IFN
(lanes 2 and 4) for 10 min at 37 °C. Forskolin (Forsk, 30 µM) was added to cells for 20 min at
37 °C prior to the addition of IFN
(lanes 3 and 4). MAPK activity was visualized by autoradiography of
P incorporated into MBP (top panel), and the blot
was then probed either with anti-phosphotyrosine (middle
panel) or pan-MAPK antibody (lower panel). The membranes
were developed using enhanced chemiluminescence (ECL). All monoclonal
antibodies except PY20 (ICN) were obtained from Transduction
Laboratories. WB, Western blot; CTL,
control.
MAPK activity is presumably only required for Stat1 serine
phosphorylation, which augments the transactivation of IFN
-induced
genes, but not for the tyrosine phosphorylation that mediates DNA
binding. In this context it also has been shown that the
serine/threonine kinase inhibitor H7 can block the transcription of
IFN
-regulated genes without affecting the formation of the ISGF3
complex(13, 14) . To determine whether the inhibitory
actions of forskolin were limited to MAPK, activation of Stat proteins
was examined by their ability to bind known enhancers using EMSA.
IFN-activated Stat1
binds specifically to the GRR, an enhancer
present in promoter of the high affinity Fc
R1 gene(15) . Stat1
and Stat2 together with the DNA
binding component ISGF3
form the multiprotein complex ISGF3, which
interacts with the ISRE present in many IFN
/
-activated early
response genes(1) .
Surprisingly, forskolin treatment of
U266 cells abrogated IFN-stimulated formation of DNA binding
complexes interacting with both the GRR (Fig. 2A, lanes 2 and 3) and the ISRE enhancers (lanes 10 and 11) by 70-90%. Furthermore, forskolin also
prevented the activation of GRR binding complexes by treatment of cells
with either IFN
(lanes 5 and 6) or with
H
O
and vanadate (lanes 7 and 8). In order to exclude other possible effects of forskolin
besides activation of adenylate cyclase as the mechanism of inhibition
of the Jak/Stat pathway, U266 cells were also exposed to
dideoxyforskolin, a derivative of forskolin that fails to activate
adenylate cyclase. As expected, this compound had no effect on the
activation of the Jak/Stat pathway (compare lanes 2, 3, and 4).
Figure 2:
Forskolin treatment of U266 cells inhibits
IFN activation of the Jak/Stat pathway. A, extracts were
prepared from U266 cells incubated with IFN
or with combinations
of forskolin (Forsk) and IFN
. Electrophoretic mobility
shift assays were performed using equal amounts of protein and a
P-labeled oligonucleotide probe corresponding to the
response region in the high affinity Fc
R1 gene (lanes
1-8) or the ISRE (lanes 9-11). The
IFN
-induced complex which binds to the GRR is labeled IFN-SF and that which binds to the ISRE is termed ISGF3. CTL, control; VAN, vanadate; DDO,
dideoxyforskolin. B, forskolin-induced intracellular cAMP
correlates with inhibition of IFN-induced IFN-SF. U266 cells were
either untreated or incubated with 0, 1, 3, 30, or 100 µM forskolin for 15 min prior to the addition of IFN
for 15 min.
Cell extracts were prepared, and cAMP was assayed (right
abscissa) or IFN-SF formation was quantitated using a
PhosphorImager (Molecular Dynamics) (left abscissa). C and D, forskolin inhibits IFN
-stimulated tyrosine
phosphorylation of Stat1
and Stat2. Lysates from U266 cells were
incubated with anti-Stat1
or anti-Stat2 antiserum for 2 h. Immune
complexes were collected on protein G beads, subjected to SDS-PAGE, and
immunoblotted with PY20 anti-phosphotyrosine antibody and detected with
ECL. Membranes were reprobed with anti-Stat1
or anti-Stat2
antibodies (lower panel). WB, Western blot; IP, immunoprecipitate;
-PY,
anti-phosphotyrosine. E, F, and G, forskolin inhibits
IFN
-stimulated tyrosine phosphorylation of Jak1, Tyk2, and the
IFN
/
receptor. Treatment of U266 cells, preparation of cell
lysates, immunoprecipitations, and Western blots with PY20 were
identical to B and C. Membranes were reprobed with
anti-Tyk2, anti-Jak1, or anti-IFN
/
receptor monoclonal
antibodies (lower panels). The antibodies used to
immunoprecipitate Tyk2, Jak1, and the IFN
/
receptor have been
previously described(2) .
To determine whether there was a
correlation between forskolin-stimulated accumulation of intracellular
cAMP and inhibition of IFN-stimulated GRR binding activity, cells
were incubated with increasing concentrations of forskolin prior to
treatment with IFN
. Cell extracts were prepared and intracellular
cAMP concentrations were measured, and IFN
-stimulated GRR binding
complexes were assayed by EMSA and subsequently quantitated on the
PhosphorImager (Fig. 2B). Inhibition in
IFN
-stimulated GRR binding correlated with dose-dependent
forskolin-induced increases in intracellular cAMP, confirming that
increases in cAMP in these cells could be inhibiting IFN
activation of the Jak/Stat pathway.
To determine directly whether
IFN-stimulated tyrosine phosphorylation of Stat1
and Stat2
was affected by forskolin treatment of U266 cells, cellular extracts
were subjected to immunoprecipitation with anti-Stat1
and
anti-Stat2 antisera, and immunoprecipitates were analyzed using
anti-phosphotyrosine antibodies (Fig. 2, C and D, upper panels). These results clearly demonstrated
that IFN
-stimulated tyrosine phosphorylation of these proteins is
drastically decreased in U266 cells incubated with forskolin (compare lanes 2 and 3). Reprobing of the blots with
Stat1
or Stat2 antisera indicated that equal amounts of protein
were immunoprecipitated from all samples (lower panels).
Tyk2 and Jak1 are required for IFN/
-stimulated tyrosine
phosphorylation of Stat1
and Stat2 and become activated and
tyrosine-phosphorylated as a result of incubation of cells with
IFN
/
. In order to determine whether forskolin was exerting
its inhibitory effects on activation of Tyk2 and Jak1, U266 cells were
incubated with IFN
in the presence or absence of forskolin, and
Tyk2 and Jak1 were immunoprecipitated from cell lysates. The
immunoblots from this experiment were probed with anti-phosphotyrosine
antibody (Fig. 2, E and F).
IFN
-stimulated tyrosine phosphorylation of both Tyk2 and Jak1 was
also significantly inhibited with forskolin treatment of U266 cells
(compare lanes 2 and 3) while the total amount of
these proteins in the immunoprecipitates is not altered (lower
panels).
It has been previously shown that the chain of
the IFN
/
receptor becomes tyrosine-phosphorylated in response
to IFN treatment (16) . In order to determine whether the
IFN
/
receptor itself was also a target for the inhibitory
effects of elevated cAMP levels, monoclonal antibodies were used to
immunoprecipitate the
chain of the IFN
/
receptor from
cells that had been exposed to IFN
with or without prior treatment
with forskolin(2, 3) . Proteins were resolved on
SDS-PAGE and transferred to Immobilon, and the blots were probed with
anti-phosphotyrosine antibody. The results shown in Fig. 2G clearly demonstrate that IFN-stimulated tyrosine phosphorylation
of the IFN
/
receptor is decreased in U266 cells incubated
with forskolin and IFN
(compare lanes 2 and 3)
compared with cells treated with IFN
alone.
It has been
previously shown that many of the key signaling components required for
IFN/
stimulation of the Jak/Stat pathway constitutively
associate with a GST fusion protein containing the membrane-proximal 50
amino acids of the
chain of the IFN
/
receptor(3, 11) . In order to explore a possible
association of protein kinase A with the receptor, GST fusion proteins
were expressed, which contained either the membrane-proximal 50 amino
acids of the cytoplasmic domain of the
subunit of the
IFN
/
receptor or GST alone. U266 cells were either left
untreated or treated with IFN
for 5 min, and cell lysates were
prepared and incubated with the GST or the GST fusion proteins coupled
to glutathione-agarose. Complexed proteins were resolved on SDS-PAGE
and transferred to Immobilon, and the blots were probed with a
monoclonal antibody to the type 1 regulatory subunit of protein kinase
A. Protein kinase A was found to associate specifically but in a
ligand-independent manner with the GST fusion protein representing the
membrane-proximal 50 amino acids of the cytoplasmic tail of the
IFN
/
receptor (Fig. 3, lanes 2 and 3) but not with GST alone (lane 1).
Figure 3:
Interaction of protein kinase A (PKA) with the cytoplasmic domain of the chain of the
IFN
/
receptor. Cellular extracts were prepared as described
above and incubated with either GST alone (lane 1) or a GST
fusion protein containing the membrane-proximal 50 amino acids of the
cytoplasmic domain of the
subunit of the IFN
/
receptor
(465) (lanes 2 and 3). Expression of the fusion
proteins has been previously described(3) . WB,
Western blot; CTL, control.
Although
cross-talk between signaling networks is a well described phenomenon,
until recently modulation of the Jak/Stat pathway has been restricted
to covalent modification of its components by tyrosine phosphorylation.
Recent reports indicate that serine phosphorylation of Stat1 and Stat3
presumably by activation of MAPK can enhance IFN/
and
IFN
induction of early response genes(3, 4) . The
results presented here implicate the serine/threonine kinase protein
kinase A as a regulator of Jak/Stat activation. The effects of
forskolin described here are also cell-specific with regard to
inhibition of tyrosine phosphorylation of the Jak/Stat proteins (data
not shown). Protein kinase A may directly or indirectly be responsible
for modification of one or several of the Jak tyrosine kinases such
that they cannot become tyrosine-phosphorylated (and presumably
activated). Alternatively, the target for the actions of protein kinase
A is upstream of Tyk2 and Jak1. The fact that forskolin also inhibits
both IFN
- and vanadate-induced GRR binding complexes in U266 cells
suggests that the IFN
/
receptor is not the primary target for
the inhibitory actions of forskolin. There are other key control points
in the Jak/Stat pathway though, which could also be regulated by
protein kinase A, such as the protein tyrosine phosphatase activity,
which appears to be required to initiate tyrosine phosphorylation of
Stat1 and Stat2 by IFN
/
but not to maintain the activation
cascade (17) . Other signaling proteins such as insulin
substrate 1 or protein kinase C, which are known to be regulated by IFN
treatment of cells but not at present implicated in IFN stimulation of
the Jak/Stat pathway, must also be considered targets for the actions
of protein kinase A (18, 19) . Definition of the
cell-specific substrates modified by protein kinase A, which inhibit
Jak/Stat activation, is clearly an important piece of information to
understand the mechanisms by which these distinct signaling networks
influence each other's activities.