(Received for publication, September 9, 1994)
From the
tyk2 belongs to the JAK family of nonreceptor protein tyrosine
kinases recently found implicated in signaling through a large number
of cytokine receptors. These proteins are characterized by a large
amino-terminal region and two tandemly arranged kinase domains, a
kinase-like and a tyrosine kinase domain. Genetic and biochemical
evidence supports the requirement for tyk2 in interferon-/
binding and signaling. To study the role of the distinct domains of
tyk2, constructs lacking one or both kinase domains were stably
transfected in recipient cells lacking the endogenous protein. Removal
of either or both kinase domains resulted in loss of the in vitro kinase activity. The mutant form truncated of the tyrosine kinase
domain was found to reconstitute binding of interferon-
8 and
partial signaling. While no contribution of this protein toward
interferon-
binding was evident, increased signaling could be
measured. The mutant form lacking both kinase domains did not exhibit
any detectable activity. Altogether, these results show that a
sequential deletion of domains engenders a sequential loss of function
and that the different domains of tyk2 have distinct functions, all
essential for full interferon-
and -
binding and signaling.
The JAK family of protein tyrosine kinases presently consists of
four members, tyk2, JAK1, JAK2, and
JAK3(1, 2, 3, 4, 5, 6) .
These 120-130-kDa proteins are characterized by the presence of
(i) a carboxyl kinase domain, (ii) an adjacent kinase-like domain, and
(iii) five conserved domains extending toward the amino terminus and
the absence of SH2 or SH3 domains (reviewed in (1) and (2) ). The COOH-terminal kinase domain (or TK ()domain) contains all the conserved residues associated
with tyrosine kinases(7) . The kinase-like domain (or KL
domain) contains the subdomains shared by protein kinases, but lacks
several residues thought to be essential for protein kinase activity,
and thus it would not be predicted to exhibit kinase activity. The
function of this domain has yet to be established. The remaining five
blocks of homology exhibit varying degrees of conservation among the
family members and their functional role is to be elucidated. It is
likely that some of them might be responsible for the association of
the JAKs with members of the cytokine receptor superfamily. Recent
studies have in fact established the involvement of these enzymes in
signaling through interferon (IFN) receptors and a large number of
cytokine/growth factor
receptors(1, 2, 4, 5) . Physical
interaction between JAKs and the membrane-proximal region of some of
these receptor chains has also been demonstrated (reviewed in (2) ) (8, 9, 10, 11, 12, 13, 14) .
As most cytokine receptors, the IFN-/
receptor has a
multichain structure composed of the chain IFNAR1, interacting with at
least another membrane component(15, 16) . Although no
formal evidence exists yet, it is likely that the IFN-
/
binding protein of 51 kDa recently identified by Novick et al.(17) represents a component of the receptor unit possibly
interacting with IFNAR1. The signaling cascade initiated by binding of
IFN-
/
to its receptor relies on the activity of tyk2 and
JAK1, as was first demonstrated through the study of the IFN-resistant
cell lines 11,1 (or U1A) and U4A, deficient in one or the other of
these protein tyrosine kinases (18, 19) . The absence
of either protein prevents high affinity binding of IFN-
,
phosphorylation of the downstream transcriptional components (p113 or
STAT2 and p91/p84 or STAT1
/
) and activation of inducible
genes(20) . Thus, complementation of 11,1 cells with a cosmid
or the cDNA encoding tyk2 was shown to restore IFN-
/
responsiveness(18, 21) , whereas complementation of
mutant U4A with JAK1 cDNA restored both IFN-
/
and IFN-
signaling(19) . In an initial study of the tyk2-deficient cell
line 11,1 we found a residual sensitivity of these cells to IFN-
,
that suggested the existence of a minor IFN-
-specific pathway and
possibly a different utilization of tyk2 by the two IFN
species(22, 23) .
We have recently reported on the
biochemical characterization of tyk2 as a 134-kDa protein mostly
cytosolic, with a minor membrane-associated fraction. The protein is
transiently phosphorylated on tyrosine in response to IFN-/
treatment and possesses an inducible tyrosine kinase activity
measurable in vitro(21) . To understand the role of
the kinase domains of tyk2, here we have constructed deleted forms of
the protein lacking either or both kinase domains and stably expressed
them in the tyk2-deficient cell line. The binding of IFN-
and
IFN-
and the signaling activities induced in these transfectants
were analyzed and compared.
Figure 1:
Structure
and expression of the deleted tyk2 proteins. A, Schematic
representation of the tyk2 deleted forms. Names of the proteins are on
the left and their predicted molecular masses in kilodaltons (kDa) are on the right. w.t., wild-type; KL,
kinase-like domain; TK, tyrosine kinase domain; N,
NH-terminal region. The striped box at the C
termini corresponds to the VSV-G epitope. B, anti-tyk2
immunoblot to compare the level of protein in 2fTGH cells and in clones
transfected with the indicated cDNAs. Fifteen micrograms of total
protein obtained from crude cell extracts were loaded on a SDS-7%
polyacrylamide gel. Minor forms of higher mobility were routinely
detected in cells overexpressing tyk2.
We tested the ability of the four
neo transfectants to grow in the selective medium (HAT) in
the presence of different concentrations of IFN-
. This medium
allows survival of 11,1 derivatives which have reverted to IFN
sensitivity (22) and constitutes our biological assay for tyk2
activity. Wild-type tyk2-expressing cells showed IFN-dependent growth
with as little as 10 IU/ml IFN-
(Wellferon, a mixture of purified
human
subtypes).
TK-expressing cells survived in HAT medium
only when a higher concentration of IFN-
was used. In contrast,
11,1 cells and
KL- and N-expressing cells failed to grow at any
given concentration of IFN-
tested (Fig. 2A). To
rule out the possibility that this behavior was unique to 11,1
derivatives, we generated
TK and
KL neo
transfectants from cell lines U1B and U1C, two independent
mutants of the same complementation group as 11,1(24) . The IFN
responsiveness of each transfectant gave comparable results (data not
shown).
Figure 2:
Biological assay of the activity of
wild-type and deleted tyk2 forms. A, The sensitivity to
IFN- of the indicated cell lines was tested by seeding 3
10
cells/well in a 24-well plate in HAT medium without or
with the indicated concentrations of IFN-
(Wellferon). After
6-7 days, cells were fixed and stained. B, comparison of
the dose-response range for IFN-
8 and IFN-
of mutant 11,1
cells and its derivative transfectants expressing wild-type tyk2 or the
deleted
TK form. The IFN response was assayed as described in A. After fixation, staining intensity was visually compared
for the three cell lines. Boxes are closed on the left
side at
1% growth response, and on the right side at
100% growth response. A half-log difference is regarded as
significant. 1 nM corresponds approximately to 4000 IU/ml. The
same profile of sensitivity was obtained for the 11,1 cell line and the
clones expressing
TK and N.
We next tested the sensitivities of the 11,1 transfectants
to recombinant IFN-8 and -
. These results are summarized in Fig. 2B. The specific activity of IFN-
was higher
than that of IFN-
8 on all clones tested. There was a
30-100-fold difference in the sensitivities of
TK-expressing
cells and 11,1 to either IFN. Interestingly, the sensitivity of
TK-expressing cells to IFN-
8 attained the level of
sensitivity of 11,1 cells to IFN-
. The analysis of the
KL-
and N-expressing clones gave results essentially identical to 11,1.
These data indicate that both tyk2 kinase domains are required to
restore a wild-type response to both IFN species. The truncated
TK
form can, however, partially reconstitute sensitivity to both IFNs.
As a measure of the response of the transfectants to IFN-, we
studied the transcriptional induction of the endogenous IFN-responsive
6-16 gene by Northern blot. Analysis of total RNA from wild-type
tyk2-expressing cells treated for 4 h showed accumulation of the 6-16
transcript with as little as 10 IU/ml IFN-
(Fig. 3). A
comparable level of transcript accumulated in
TK-expressing cells
treated with 5000 IU/ml IFN-
. In contrast,
KL-expressing
cells failed to induce the 6-16 message, behaving as mutant 11,1. These
results confirm the fine correlation between the transcriptional
activation of the 6-16 gene promoter by IFN and the ability of the
cells to grow in HAT medium. The steady-state mRNA level of three other
IFN-responsive genes (ISG-54, GBP, and IRF-1) was analyzed in the
various cell lines. None of these transcripts accumulated in cells
expressing the deleted tyk2 forms, treated with up to 5000 IU/ml
IFN-
(data not shown).
Figure 3:
Induction of the 6-16 mRNA by
IFN- in cells expressing wild-type or deleted tyk2 forms. Northern
transfers of 10 µg of total RNA from untreated cells(-) or
cells treated with the indicated amounts of IFN-
(Wellferon) for 4
h were hybridized to a 6-16-specific cDNA probe. Comparable signals in
each lane were obtained with a
-actin probe (data not
shown).
Figure 4:
Binding of human IFN-8 and IFN-
to cells expressing wild-type or deleted tyk2 forms. A,
Kinetics of uptake of
I-labeled human IFN-
(280
pM) at 37 °C by
, 2fTGH cells;
; 11,1 cells;
; wild-type tyk2-expressing cells; and
,
TK-expressing cells. Values are femtomoles of IFN taken up per
million cells. B, kinetics of uptake of
I-labeled human IFN-
8 (350 pM) at 37 °C
by
11,1 cells;
, wild-type tyk2-expressing cells;
,
TK-expressing cells. Values are femtomoles of IFN taken up per
million cells. C, Scatchard plots of direct binding of
I-labeled human IFN-
8 (
,
) and IFN-
(
,
) for 6 h at 4 °C to
,
,
TK-expressing cells;
,
,
KL-expressing cells.
Values are for femtomoles of IFN bound per million
cells.
To investigate this further and to
eliminate the dynamic aspects of cellular uptake, we compared the
binding of IFN-8 and IFN-
at 4 °C on the partially
sensitive
TK-expressing cells and on the IFN-insensitive
KL-expressing cells. The results, in the form of Scatchard graphs,
are shown in Fig. 4C. The binding of IFN-
was
nearly the same on the two cell lines, showing that, for IFN-
,
receptor expression appears to be unaffected by the status of tyk2. The
binding of IFN-
8 on the
TK-expressing cells was greater and
shows a Scatchard plot with a slope 2.4 times greater than that
obtained on the
KL-expressing cells. This suggests that the mutant
protein
TK contributes to the affinity of binding of IFN-
over the concentration range used. The Scatchard coefficient gave K
equivalent to 0.54 nM and 0.62 nM for IFN-
, 0.87 nM and >2.1 nM for
IFN-
8, on
TK- and
KL-expressing cells, respectively (the K
of 2.1 nM falls outside the range of
experimental points). The linear regressions extrapolated to infinite
ligand concentration meet the intercept at values close enough to
suggest that the main reason for the reduced uptake of IFN-
8 on
KL cells is a lower binding affinity.
Figure 5:
In vivo and in vitro tyrosine phosphorylation of tyk2 in response to IFN- in
wild-type and deleted tyk2-expressing cells. A, Anti-tyk2
immunoprecipitates from wild-type, deleted tyk2-expressing, and mutant
11,1 cells (5
10
cells) were immuno-blotted with
either anti-phosphotyrosine 4G10 (upper panel) or anti-tyk2 (lower panel) antibody. IFN treatment (1000 IU/ml of
Wellferon) was as follows: lanes 1, 3, 5, 7, and 9, no treatment; lanes 2, 4, 6, 8, and 10, 5 min with IFN-
. The
position of wild-type and mutant tyk2 forms is marked by an arrow. Faster migrating bands are routinely detected in cells
overexpressing tyk2. B, anti-tyk2 immunoprecipitates from
post-nuclear lysates (600 µg) of untreated or IFN-
treated
(1000 IU/ml Wellferon) wild-type, 11,1,
KL, and
TK-expressing
cells were assayed in an in vitro kinase assay. Acid-denatured
enolase was added to the reaction as an exogenous substrate. Kinase
reaction products were fractionated on a 7% SDS-polyacrylamide gel
electrophoresis and transferred to Hybond-C super membrane. Following
autoradiography, the filter was probed with anti-tyk2 (bottom
panel) antibodies. The expected positions of tyk2, enolase (En), and mutant
KL and
TK forms are
marked.
IFN- induces
tyk2 kinase activity and this can be measured in vitro in an
anti-tyk2 immunocomplex(21) . To determine whether the
deletions had an effect on the kinase activity of the protein, in
vitro kinase assays were performed on the wild-type tyk2 and the
TK and
KL mutant forms. As shown in Fig. 5B,
wild-type tyk2 exhibited increased autophosphorylating activity upon
IFN-
treatment. In contrast,
TK and
KL did not exhibit
autophosphorylating activity. A similar result was obtained when the
two deletion constructs were co-expressed in 11,1 cells (data not
shown). To investigate the ability of these proteins to phosphorylate
an exogenous substrate, the in vitro kinase assay was
performed in the presence of acid-denatured enolase. Upon IFN-
treatment, enolase was markedly phosphorylated by wild-type tyk2 (Fig. 5B, lane 2). In contrast, the exogenous
substrate was not phosphorylated in
KL and
TK-expressing
cells treated with IFN (Fig. 5B, lanes 4 and 6). The abundance of the immunoprecipitated proteins was
visualized by blotting the filter with anti-tyk2 antibodies (Fig. 5B, lower panel). These results indicate
that both kinase domains appear necessary for the enzymatic activity of
the protein since deletion of either domain generates a kinase-inactive
protein.
Figure 6:
Tyrosine phosphorylation of JAK1, p113,
and p91 in cells expressing the wild-type and the deleted tyk2 forms. A, cells were left untreated (-, lanes 1, 3, 5, and 7) or were treated (+, lanes 2, 4, 6, and 8) for the
indicated times (minutes) with IFN- (1000 IU/ml Wellferon). Cell
lysates were immunoprecipitated with anti-JAK1 and immunoblotted with
anti-phosphotyrosine (upper panel) or anti-JAK1 (lower
panel) antibodies. B and C, cells were left
untreated (-, lanes 1, 3, 5, and 7) or were treated (+, lanes 2, 4, 6, and 8) for 20 min with IFN-
(1000 IU/ml of
Wellferon). Cell lysates were immunoprecipitated with anti-p113 (B) or anti-p91C (C) antibodies, followed by
immunoblotting with anti-phosphotyrosine 4G10 (B, upper
panel) or a mixture of anti-phosphotyrosine 4G10 and PY20
antibodies (C, upper panel). The blots were stripped
and reprobed with anti-p113 or/and anti-p91 as indicated (lower
panels). The positions of JAK1, p113, and p91 are
marked.
We next
investigated whether p113 and p91, components of the transcriptional
complex ISGF3, were phosphorylated upon IFN- treatment of cells
expressing the tyk2 deletion mutants. Phosphorylated p113 was detected
in wild-type tyk2-expressing cells after 20 min of IFN-
treatment (Fig. 6B, lanes 2), whereas no phosphorylated
p113 was detected in cells expressing the deleted tyk2 forms (Fig. 6B, lanes 4 and 6). Similarly,
neither phosphorylated p91 nor p113
p91 complex (Fig. 6C, lane 2) were observed in cells
expressing mutant tyk2 forms (Fig. 6C). Comparable
levels of proteins were present in all extracts (Fig. 6, B and C, bottom panels). We were not successful at
detecting phosphorylated JAK1, p91, or p113 in the
TK-expressing
cells even when different experimental conditions, such as increased
dose and time of IFN treatment or increased amount of cell extract
immunoprecipitated, were assayed.
In this study we have examined the effects of deletions on
the activity of tyk2 expressed in an IFN-resistant cell line lacking
the endogenous protein. Deletions of one or both kinase domains were
found to abrogate tyk2 catalytic activity when measured in vitro for autophosphorylation and phosphorylation of an exogenous
substrate. The absence of autophosphorylation could simply be
consequent to the removal of major phosphorylation sites. However, the
mutant KL protein retains two contiguous tyrosine residues,
recently identified as targets of in vitro IFN-induced
autophosphorylation in the wild-type tyk2. These sites
(Tyr
-Tyr
) map in the TK domain,
between subdomains VII and VIII, and, as shown for other protein
tyrosine kinases, their phosphorylation parallels activation of the
enzyme. (
)Thus, it appears that, when juxtaposed to the
NH
-terminal region, the TK domain of tyk2 is not active.
This could either reflect a conformational alteration of the protein
caused by the deletion, or be a consequence of the excision of a domain
playing an essential role in the activity of the enzyme. Despite the
fact that the
KL mutant protein is catalytically inactive, it is
phosphorylated in vivo, independently of the presence of IFN (Fig. 5A). When expressed in IFN-treated cells
containing endogenous tyk2, the mutant form
KL showed no evidence
of phosphorylation above the basal level (data not shown), suggesting
that IFN-induced trans-phosphorylation between the wild-type and the
mutant protein cannot take place. Although at this time the site and
the functional significance of this IFN-independent phosphorylation is
unknown, these observations argue for the existence of another cellular
protein tyrosine kinase capable of phosphorylating
KL. Tyk2
deleted of its TK domain (
TK) is catalytically inactive,
indicating that the KL domain lacks intrinsic kinase activity. Indeed,
no activity of the bacterially expressed JAK1 kinase-like domain could
be detected for a number of exogenous substrates(31) .
One fundamental question regarding tyk2 and the other JAK family members concerns their mode of regulation which might differ from that of cytoplasmic protein tyrosine kinases possessing an SH2 domain and a single kinase domain(32) , resembling more that of other bifunctional proteins bearing a kinase-related domain(33, 34, 35) . One such protein is the cell surface receptor for atrial natriuretic peptide containing in its intracellular region a kinase-like domain next to a guanylyl cyclase catalytic domain. Deletion of the kinase domain constitutively activates the cyclase(33, 36) . Thus, a negative regulatory role has been proposed for this kinase-like domain, repressing the guanylyl cyclase in absence of atrial natriuretic peptide. This is not the case for tyk2, since deletion of its KL domain does not create a constitutively active enzyme. It is likely that a more complex mode of regulation involving both kinase domains exists for the JAK family.
To study the biological activity of tyk2 mutant
forms, we have taken advantage of a unique property of 11,1 cells in
which the expression of the marker guanine phosphoribosyl transferase
and the consequent resistance to HAT is dependent on IFN and
tyk2(18) . Thus, we have found that the function of tyk2 is
completely abrogated by removal of the KL domain or of both kinase
domains: cells expressing the mutant tyk2 forms KL and N behaved
essentially as cells lacking tyk2. Conversely, expression of the
TK protein resulted in a partial reconstitution of sensitivity
toward IFN-
and in an increased sensitivity to IFN-
(Fig. 2). This effect could be appreciated only when
transfectants were expanded in G418, prior to testing their IFN
responsiveness. When transfected cells were directly seeded in HAT, no
survivors could be isolated in up to 10
IU/ml IFN, probably
due to the poor colony-forming ability of these cells.
Our binding
studies showed that the capacity for receptor-mediated IFN- uptake
is partially restored by the
TK mutant protein, probably through
an increase in binding affinity (Fig. 4). The inability of the N
and the
KL proteins to perform this function suggests that the KL
domain in its proper conformation contributes toward the binding
affinity of the receptor unit and indeed may be in contact with some
component of that unit. In relation to this, tyk2 has been recently
found to physically associate with the chain IFNAR of the receptor
complex, independently of the presence of the ligand (11) . (
)Despite an increase in the affinity of binding of
IFN-
on
TK-expressing cells, there are dynamic aspects of the
interaction that have not been reconstituted, and that must require the
TK domain. One hypothesis awaiting further investigation is that
ligand-induced tyk2 kinase activity could play a role in post-binding
events such as the internalization rate of the receptor unit, as
proposed for the intrinsic kinase activity of epidermal growth factor
and insulin receptors(37, 38, 39) .
The
contribution of the TK protein toward ligand binding is much less
evident for IFN-
than for IFN-
. While there is a partial
reconstitution of sensitivity towards IFN-
with the
TK
protein, the early receptor binding events appear to be unaffected.
Full reconstitution requires the tyrosine kinase domain. These data
suggest that tyk2 has both structural and enzymatic functions in the
IFN-
/
response, with a more direct structural relationship
with IFN-
than with IFN-
, and this has been schematized in Fig. 7. In wild-type cells, the different elements are obviously
well integrated. From the study of mutant cell lines, we would suggest
that IFN-
can utilize a minor or tyk2-independent binding
pathway(s), an option not available to IFN-
. Further work will be
needed to identify elements unique to the IFN-
response and the
recent report of a protein that can be coprecipitated with one ligand
binding protein and phosphorylated only following IFN-
treatment
goes into that direction(40) . If the apparently greater
flexibility of IFN-
is a reflection of its evolutionary seniority (41) , then the close dependence of IFN-
and tyk2 may have
its origin in the diversification of the mammalian IFNs.
Figure 7:
Tyk2
in the receptor-mediated pathways induced by IFN- and -
. The central box contains all known and unknown components, other
than tyk2, involved in signal transduction. Ifnar1 and Ifnar2 are ligand binding proteins(43) . Both binding
pathways are deemed to be open to IFN-
, whereas IFN-
is
restricted to the major binding pathway.
We have not
yet identified the signaling components involved in the minor
pathway(s). Phosphorylated JAK1 or STATs could not be detected in
TK-expressing cells in response to IFN-
, possibly due to the
low number of molecules activated and the limited sensitivity of this
assay. Recently, it has been shown that IFN-
/
induce tyrosine
phosphorylation of the p95
proto-oncogene in
hematopoietic cell lines, suggesting that vav may be involved
in an alternative IFN-
/
signaling pathway in addition to the
ISGF3 pathway(42) . It will be interesting to determine if
signaling proteins similar to vav are activated in response to
IFN-
/
in cells other than hematopoietic cell lines, as it may
provide a hint of a possible alternative IFN signaling pathway.
Certainly, more work will be needed to understand how tyk2 and its
deleted TK form contribute to receptor function. In parallel, the
study of the JAK1-deficient U4A mutant cell line, whose IFN-
binding activity is impaired(19, 20) , should help to
clarify the role of JAK1 in receptor function. In relation to this,
recently Novick et al.(17) have shown that the
IFN-
/
-binding chain that they have identified can be
co-precipitated with JAK1. Altogether, our studies support a model in
which heterodimerization of receptor chains and consequent
juxtaposition and activation of associated JAK1 and tyk2 all contribute
to the generation or stabilization of ligand-induced, conformationally
correct, and productive receptor units.