(Received for publication, October 11, 1995)
From the
We have shown previously that a four-amino acid block residing
at positions 266-269 (LPKS) in the intracellular domain of the
human interferon- (IFN-
) receptor
chain is critical for
IFN-
-dependent tyrosine kinase activation and biologic response
induction. Herein we show that this sequence is required for the
constitutive attachment of the tyrosine kinase JAK-1. Using a vaccinia
expression system, a receptor
chain-specific monoclonal antibody
coprecipitated JAK-1 from cells coexpressing JAK-1 and either (a) wild type IFN-
receptor
chain, (b) a
receptor
chain truncation mutant containing only the first 59
intracellular domain amino acids, or (c) a receptor mutant
containing alanine substitutions for the functionally irrelevant
residues 272-275. In contrast, JAK-1 was not coprecipitated when
coexpressed with a receptor
chain mutant containing alanine
substitutions for the functionally critical residues 266-269
(LPKS). Mutagenesis of the LPKS sequence revealed that Pro-267 is the
only residue obligatorily required for receptor function. In addition,
Pro-267 is required for JAK-1 binding. These results thus identify a
site in the IFN-
receptor
chain required for constitutive
JAK-1 association and establish that this association is critical for
IFN-
signal transduction.
Interferon- (IFN-
) (
)is a potent
immunomodulatory cytokine that exerts its pleiotropic biologic effects
by interacting with a single high affinity receptor expressed on the
surface of nearly all host-derived
cells(1, 2, 3, 4, 5) .
IFN-
receptors consist of two species of matched polypeptides: a
90-kDa
chain (6, 7, 8, 9, 10) that is
responsible for ligand binding, ligand trafficking through the cell,
and signal transduction and a 62-kDa
chain (11, 12) that plays only a minor role in ligand
binding (13) but which is obligatorily required for
function(5) . IFN-
signal transduction is known to require
at least three other components in addition to the receptor
polypeptides. Two of these are the tyrosine kinases, JAK-1 and
JAK-2(14, 15, 16, 17, 18) ,
which become rapidly activated following IFN-
receptor ligation
and effect the tyrosine phosphorylation of the third component, a
latent cytosolic transcription factor, Stat1, which subsequently forms
an activated homodimer, translocates to the nucleus, and initiates
transcription of IFN-
-inducible
genes(19, 20, 21, 22, 23, 24, 25) .
We have previously described a series of structure function analyses
performed on the human IFN- receptor
chain intracellular
domain. These studies involved replacing specific IFN-
receptor
amino acids with alanine residues and examining the capacity of the
resulting mutant human receptor
chains to confer human IFN-
responsiveness to a murine cell line containing the human receptor
chain (SCC16-5). Using this approach we and others
identified two topographically distinct regions in the receptor
chain intracellular domain that were required for receptor signaling (26, 27, 28) . The first was a block of four
amino acids (LPKS) situated 13 residues from the transmembrane domain
at positions 266-269 that was required for ligand-induced
activation of intracellular tyrosine kinase activity, phosphorylation
of the receptor
chain intracellular domain, and induction of
IFN-
-mediated cellular responses. The second was a membrane distal
sequence located at positions 440-444 consisting of the residues
YDKPH. The tyrosine residue within this sequence was found to be a
physiologically important substrate site for the IFN-
-activated
tyrosine kinase activity. Ligand-induced phosphorylation of Tyr-440 was
shown to form a Stat1 docking site on the receptor
chain, which
also contained the residues Asp-441 and His-444. Binding of Stat1 to
this site was shown to be a prerequisite for IFN-
-dependent Stat1
tyrosine phosphorylation and subsequent signal transduction
events(25) .
A key question that remains unanswered is the
precise function of the membrane proximal LPKS sequence in the
IFN- receptor intracellular domain. Recent coprecipitation
experiments performed by Igarashi et al.(29) suggest
that JAK-1 associates with the IFN-
receptor
chain.
Therefore it is possible that the LPKS sequence may play a role in
mediating receptor interaction with JAK-1.
In the current study we
document that JAK-1 indeed binds to the IFN- receptor
chain
in a constitutive manner and demonstrate that the LPKS sequence of the
receptor plays a key role in mediating the association. Furthermore, we
report the fine mapping of the LPKS sequence and show that the proline
residue at position 267 is required for JAK-1-receptor
chain
binding and receptor function. These results thus establish the
physiologic relevance of the molecular interactions that occur between
the IFN-
receptor and one of its two required tyrosine kinase
components.
Figure 1:
JAK-1 associates constitutively with
the IFN- receptor
chain but requires IFN-
stimulation
for activation. Left panel, fifty million Colo-205 cells were
stimulated for 5 min at 37 °C with either 10,000 units/ml IFN-
(lanes 2 and 4) or PBS (lanes 1 and 3). The cells were lysed in lysis buffer containing Brij 96
detergent and subjected to immunoprecipitation with either a mAb
specific for the human IFN-
receptor (IFN
R)
chain (lanes 1 and 2) or a mAb specific for the human
Type I TNF receptor (TNFR) (p55) (lanes 3 and 4). The immunoprecipitates (IP) were separated on a
6% SDS-polyacrylamide gel, transferred to nitrocellulose, and blotted
for the presence of JAK-1. Subsequently, the blot was stripped and
reblotted for the presence of the IFN-
receptor
chain (lower panel). Right panel, Colo-205 cells were
stimulated with PBS (lane 1) or IFN-
(lane 2) as
in the left panel. Cells were immunoprecipitated with mAbs to
the human IFN-
receptor
chain and blotted with the 4G10
anti-phosphotyrosine (p-Tyr) mAb. The blot was stripped and
reblotted for the presence of the IFN-
receptor
chain (lower panel).
Figure 2:
JAK-1 associates with the first 59 amino
acids of the IFN- receptor
chain and requires residues
L
PKS
. One-hundred thousand HeLa cells were
infected with a recombinant vaccinia virus expressing T7 RNA polymerase
for 45 min at room temperature. The cells were then transfected by
lipofection with cDNA constructs coding for JAK-1 and the full-length
human IFN-
receptor
chain (hgR, lane 1),
the IFN-
receptor
chain with residues
L
PKS
replaced by alanines (L266-S269A, lane 2), the IFN-
receptor
chain with S
VVR
replaced by alanines (S272-R275A, lane 3), or a IFN-
receptor
chain truncation expressing only the first 59 amino acids of the
intracellular domain (hgR
311, lane 4). Control
cells were also transfected with the wild type IFN-
receptor
chain alone (hgR, lane 5) and JAK-1 alone (lane
6). The cells were incubated for 14 h at 37 °C, lysed,
immunoprecipitated with a mAb to the IFN-
receptor (IFN
R)
chain and Western blotted for the presence
of JAK-1, as described under ``Experimental Procedures'' (upper panel). Total cell lysates were Western blotted for the
IFN-
receptor
chain (middle panel) and JAK-1 (lower panel) to control for levels of expression. IP, immunoprecipitate.
Since the LPKS sequence
resides in this region, we repeated the coprecipitations using two
IFN- receptor
chain block mutants in which alanines were
substituted for either the functionally important LPKS residues at
positions 266-269 or the functionally unimportant SVVR residues
at positions 272-275. Replacement of residues Ser-272 through
Arg-275 with alanine did not diminish the ability of JAK-1 to associate
with the receptor
chain. In contrast, alanine replacement of the
LPKS sequence resulted in almost total abrogation of the interaction
between JAK-1 and the receptor. When analyzed by receptor
chain-specific Western blotting, all receptor
chain derivatives
were expressed at similar levels and displayed the appropriate
molecular mass indicating that the alanine substitutions were not
causing an abnormal truncation of the mutant proteins. Thus, the JAK-1
binding site in the intracellular domain of the IFN-
receptor
chain is critically dependent on the functionally important LPKS
sequence residing near the membrane.
As
expected, SCC16-5 cells transfected with empty vector did not
respond to human IFN- while SCC16-5 cells expressing the
wild type human IFN-
receptor showed significant enhancement of
MHC class I expression following incubation with human IFN-
(Table 1). Cells expressing a mutant receptor
chain, which
contained a block replacement of four alanines for the entire LPKS
sequence (LPKS-A1.2), were unresponsive to human IFN-
. Cells
expressing receptor
chain mutants that contained alanine
substitutions for Leu-266 (LA266.11), Lys-268 (KA268.5), and all the
serine and threonine residues within the first 40 intracellular domain
amino acids including the serine residue at position 269 (S/T.4)
responded to human IFN-
. Based on dose-response experiments, the
response to human IFN-
of cells expressing these three mutant
receptors was indistinguishable from that of cells expressing the wild
type human IFN-
receptor
chain (data not shown). In
contrast, SCC16-5 cells expressing the receptor
chain with
an alanine substitution for Pro-267 (PA267.7) were unable to respond to
human IFN-
. This observation was validated by two controls. First,
all of the cell lines responded to homologous murine IFN-
indicating that expression of the human receptor
chain
derivatives did not affect the general ability of the cell to regulate
MHC class I expression. Second, identical results were obtained when
the cell lines were tested for the ability of human IFN-
(in
combination with lipopolysaccharide) to induce nitric oxide production
from the cells (data not shown). Thus Pro-267 is the only amino acid
located within the membrane proximal region of the
chain
intracellular domain that is required for induction of an
IFN-
-dependent biologic response.
Figure 3:
JAK-1/IFN- receptor
chain
association requires residue Pro-267. One-hundred thousand HeLa cells
were infected with a recombinant vaccinia virus expressing T7 RNA
polymerase for 45 min at room temperature. The cells were then
transfected with cDNA constructs coding for JAK-1 and the full-length
human IFN-
receptor
chain (hgR, lane 1),
the IFN-
receptor
chain (IFN
R
) with
residues Pro-267 replaced by alanine (PA267, lane 2),
and the IFN-
receptor
chain with Lys-268 replaced by alanine (KA268, lane 3). Control cells were also transfected
with either the wild type IFN-
receptor
chain (hgR, lane 5) or JAK-1 alone (lane 4). The cells were
incubated for 14 h at 37 °C, lysed, immunoprecipitated with a mAb
to the IFN-
receptor
chain, and Western blotted for the
presence of JAK-1, as described under ``Experimental
Procedures'' (upper panel). Total cell lysates were
Western blotted for the IFN-
receptor
chain (middle
panel) and JAK-1 (lower panel) to control for levels of
expression. IP, immunoprecipitate.
Following receptor ligation, two temporally distinct events
must occur to achieve coupling of the IFN- receptor to its signal
transduction system: activation of the JAK-1 and JAK-2 tyrosine kinases
and recruitment of Stat1 to the receptor(25, 37) . Our
previous studies revealed that these events are mediated by
topographically distinct regions within the IFN-
receptor
chain intracellular domain (25, 26, 27) . It
is now clear that the membrane distal receptor sequence serves as the
Stat1 docking site following tyrosine phosphorylation. However, the
precise role of the membrane proximal residues required for kinase
activation remains unclear. The studies presented herein document that
the functionally important membrane proximal L
PKS
sequence identified in our previous studies plays a critical role
in mediating the constitutive binding of JAK-1 to the receptor
chain. Moreover, we show that within this sequence, the proline residue
at position 267 plays a dominant role in effecting both JAK-1 binding
and receptor function. These results thereby demonstrate that
JAK-1-receptor
chain association is a prerequisite for induction
of IFN-
-dependent biological responses in cells.
The
observation that JAK-1 attachment to the IFN- receptor
chain
intracellular domain requires the LPKS sequence suggests that these
residues function as a box 1-like motif. In Type I cytokine receptors
the box 1 motif contains a conserved PXP sequence and has been
suggested to mediate receptor association with JAK kinases (38, 39, 40, 41) . In contrast, in
the IFN-
receptor
chain LPKS sequence there is a only a
single proline required for JAK-1 attachment and receptor function.
Importantly, a similar sequence containing a single proline (LPKV)
occurs in the membrane proximal region of the IFN
receptor subunit
recently cloned by Novick et al.(42) . This receptor
subunit also associates with JAK-1. Thus, it is possible that formation
of a JAK-1 binding site on IFN receptor family members is critically
dependent on this modified box 1 motif.
It is also of interest that
JAK-2 was not found to associate with the receptor chain. Recent
studies by Sakatsume et al.(43) and Kotenko et
al. (44) show that JAK-2 associates with the IFN-
receptor
chain. Moreover, JAK-2-
chain association has been
mapped to a 12-amino acid proline-rich sequence within the
chain
intracellular domain. (
)Finally, recent studies by Ashkenazi
and co-workers (13) and Bach and Schreiber
clearly
demonstrate that the IFN-
receptor
and
subunits do not
constitutively associate with one another but rather become associated
upon exposure to ligand. Taken together these results demonstrate that
each receptor subunit associates with a specific JAK kinase. Thus
ligand-dependent association of the IFN-
receptor
subunits(45, 46)
brings into close
juxtaposition inactive forms of receptor subunit-associated JAK-1 and
JAK-2, which transactivate one another to initiate the IFN-
signaling response.