Synthetic Biology Reveals the Uniqueness of
the RIP Kinase Domain
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Steven M. Chirieleison, Sylvia B. Kertesy and Derek W.
Abbott
Published April 4, 2016, doi:10.4049/jimmunol.1502631
The Journal of Immunology
Synthetic Biology Reveals the Uniqueness of the RIP Kinase
Domain
Steven M. Chirieleison, Sylvia B. Kertesy, and Derek W. Abbott
T
he RIP kinases (RIPKs) play an essential role in inflammatory signaling and cell death (1, 2). RIPK1 is required
for TNF-induced NF-kB activation and helps regulate the
switch between TNF-induced apoptosis and necroptosis (1–3),
partnering with RIPK3 to induce necroptosis (1, 2, 4). RIPK2 is an
essential kinase regulating signaling downstream of the Crohn
disease susceptibility protein nucleotide-binding oligomerization
domain 2 (NOD2) (5, 6). In this role, RIPK2 is part of the protein
complex that recognizes intracellular bacterial infection and helps
tailor the cytokine response to eradicate an offending pathogen (7,
8). Although less well studied, RIPK4 is the causative gene in
popliteal pterygium syndrome, a disease characterized by early
lethality with multiple developmental abnormalities (9). Given the
collective influence of the RIPKs on innate immune and inflammatory signaling, there has been intense interest in manipulating
these kinases pharmacologically for clinical gain. Pharmacologic
RIPK1, RIPK2, and RIPK3 inhibitors have all been described and
Department of Pathology, Case Western Reserve University School of Medicine,
Cleveland, OH 44106
ORCIDs: 0000-0002-3997-5652 (S.M.C.); 0000-0003-4387-8094 (D.W.A.).
Received for publication December 18, 2015. Accepted for publication March 6,
2016.
This work was supported by National Institutes of Health Grants R01 GM086550 and
P01 DK091222 (to D.W.A.). S.M.C. is supported by the Case Western Reserve
University National Institutes of Health Medical Scientist Training Program
(T32GM007250).
S.M.C. generated the novel lentiviral vector, interpreted results, and edited the manuscript; S.B.K. provided technical assistance in preparing and performing the experimentation; and D.W.A. generated the reagents, performed the experimentation,
interpreted the results, and wrote the manuscript.
Address correspondence and reprint requests to Dr. Derek W. Abbott, Department of
Pathology, Case Western Reserve University School of Medicine, Room 6531 Wolstein Research Building, 2103 Cornell Road, Cleveland, OH 44106. E-mail address:
[email protected]
Abbreviations used in this article: CARD, caspase activation recruitment domain; F,
forward; HA, hemagglutinin; HygR, hygromycin resistance gene; m, murine; MDP,
muramyl dipeptide; NEMO, NF-kB essential modulator; NOD2, nucleotide-binding
oligomerization domain 2; R, reverse; RIPK, RIP kinase; WT, wild-type.
Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1502631
are in various states of clinical development for disorders as diverse as sepsis, inflammatory bowel disease, and multiple sclerosis
(10–19).
Despite this pharmaceutical interest, the function of the RIPKs’
kinase domains has been enigmatic with few bone fide substrates
identified (1, 2, 20). In no case is this truer than in the case of
RIPK2. Initial Basic Local Alignment Search Tool searches suggested that RIPK2 was a serine-threonine kinase, and indeed,
RIPK2 was shown to autophosphorylate (6, 21, 22). In these initial
descriptions, which were based largely on overexpression studies,
RIPK2’s kinase activity was shown to be dispensable for signaling
such that although the RIPK2 protein was essential for NOD1/2
signaling, its kinase activity was unnecessary (6, 21, 22). Hints
to RIPK2’s kinase function began to emerge when it was shown
that the joint p38 and RIPK2 inhibitor, SB203580, could cause
decreased expression of RIPK2, presumably through a loss of
protein stability (23). Although this work was also supported by
the fact that a genetic knockin of kinase-dead RIPK2 showed
decreased expression, this feature is shared by many kinases in
which a kinase-dead variant shows decreased expression (24). In
fact, additional pharmacologic studies using a more diverse and
specific panel of RIPK2 inhibitors have shown that inhibition of
RIPK2 kinase activity does not have a universal role in RIPK2
protein stability (11, 12, 19, 25); thus, the role of the kinase
activity in RIPK2 protein stability still remains unanswered. A
last mystery surrounding the RIPK family of kinases centers on
which phosphoacceptor they prefer to phosphorylate. RIPK2 was
initially misclassified as a serine-threonine kinase when in fact
it is a dual-specificity kinase, capable of phosphorylating serines, threonines, and tyrosines (11). Despite this advance in the
NOD–RIPK2 field, the preferred phosphoacceptors of the other
RIPKs remains unstudied.
Structural studies have also recently highlighted the differences
between, and the importance of, the kinase domains of this family
of proteins. RIPK2 contains an extended, deep ATP binding pocket,
which allows a pharmacologic manipulation likely not afforded
by the other RIPKs (11, 16, 18). Although molecular modeling
and crystal structures have shown largely superimposable kinase
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The RIP kinases (RIPKs) play an essential role in inflammatory signaling and inflammatory cell death. However, the function of
their kinase activity has been enigmatic, and only recently has kinase domain activity been shown to be crucial for their signal
transduction capacity. Despite this uncertainty, the RIPKs have been the subject of intense pharmaceutical development with a
number of compounds currently in preclinical testing. In this work, we seek to determine the functional redundancy between
the kinase domains of the four major RIPK family members. We find that although RIPK1, RIPK2, and RIPK4 are similar in
that they can all activate NF-kB and induce NF-kB essential modulator ubiquitination, only RIPK2 is a dual-specificity kinase.
Domain swapping experiments showed that the RIPK4 kinase domain could be converted to a dual-specificity kinase and is
essentially indistinct from RIPK2 in biochemical and molecular activity. Surprisingly, however, replacement of RIPK2’s kinase
domain with RIPK4’s did not complement a nucleotide-binding oligomerization domain 2 signaling or gene expression induction
defect in RIPK22/2 macrophages. These findings suggest that RIPK2’s kinase domain is functionally unique compared with other
RIPK family members and that pharmacologic targeting of RIPK2 can be separated from the other RIPKs. The Journal of
Immunology, 2016, 196: 000–000.
2
domains among RIPK1, -2, and -3, there are subtle structural
differences among these three kinases, which can help explain
pharmacologic specificity (18). Lastly, pharmacologic inhibitors
for RIPK1, RIPK2, and RIPK3 have been developed that independently target these three kinases (10–19). Although structural
studies have elucidated subtle differences among the kinase domains in this family of proteins, they provide only a snapshot of
the protein in the lowest energy state at a single point in time. In
contrast, little functional work has been done to determine potential in vivo cellular redundancy among the RIPKs. How specific
are the RIPK kinase domains for their cellular function? Can one
RIPK domain substitute for another, and does the signal transduction specificity of the RIPKs rely on the kinase domain or their
C-terminal effector domains? In this work, we study these central
questions in the field and show that RIPK2’s kinase domain is
uniquely required for innate immune signaling and NOD2-driven
gene expression.
Materials and Methods
Transient transfection assays were performed using calcium phosphate
transfection of HEK293 cells (CRL-1573; American Type Culture Collection), which were grown in 10% FBS and 1% penicillin/streptomycin,
Myc-K399R NF-kB essential modulator (NEMO), and hemagglutinin
(HA)-ubiquitin, generated as previously described (7, 26). cDNA expression constructs for RIPK1 and RIPK3 were obtained from Vishva Dixit
(Genentech), and a cDNA expression construct for RIPK4 was obtained
from Shiv Pillai (Massachusetts General Hospital). The template for
RIPK2 was used as described (7). Gibson subcloning technology was used
to insert each of the RIPKs into the NTAP expression construct (Stratagene) (27). The NTAP expression construct contains an N-terminal
calmodulin binding domain and a streptavidin-binding domain. For
immunoprecipitation and pulldown assays, cell lysates were prepared
with a buffer containing 50 mmol Tris (pH 7.4), 150 mmol NaCl, 1%
Triton X-100, 1 mmol EDTA, 1 mmol EGTA, 2.5 mmol sodium pyrophosphate, 1 mmol b-glycerophosphate, 5 mmol iodoacetimide, 5 mmol
N-ethylmaleimide, 1 mmol PMSF, 1 mmol sodium orthovanadate, and protease inhibitor mixture. Streptavidin beads (Sigma-Aldrich) were blocked
with 1% BSA and added to the lysate overnight when an RIPK was precipitated. Immunoprecipitates were washed five times in lysis buffer before
boiling in an equal volume of 23 Laemmli sample buffer. Western blotting
was performed as described previously (7). For NEMO precipitation assays
assessing ubiquitination, lysates were boiled before immunoprecipitation
to denature the lysate and allow direct assessment of NEMO ubiquitination.
NEMO was precipitated via its N-terminal 3Xmyc tag (Ab 9E10 clone;
Santa Cruz Biotechnology). The K399R NEMO construct was used as
this limits background ubiquitination. For signaling experiments, 10 mg/ml
L-18 muramyl dipeptide (MDP; Invivogen) was added to the media for
the given amount of time before lysates were generated using the above
buffer. Protein concentrations were standardized by the Bio-Rad protein
assay (Bio-Rad), and Western blots were performed as described. The
HA Ab (16B12) was obtained from Covance. The phosphotyrosine Ab
(p-Tyr-100) was obtained from Cell Signaling Technology, as were the
p–IkB kinase, total IkB kinase, inhibitor of k L-chain gene enhancer
in B cells, and p-inhibitor of k L-chain gene enhancer in B cells. The
GADPH Ab was obtained from GenScript. The RIPK2 Ab (H-300) was
obtained from Santa Cruz Biotechnologies, recognizes the C terminus of
RIPK2, and is thus able to blot the chimeric constructs.
Viral production and stable cell line generation
Immortalized RIPK22/2 macrophages were obtained from Michelle
Kelliher (University of Massachusetts Medical School) and grown in 10%
FBS and 1% penicillin/streptomycin. Lentiviral Crispr V2 (Addgene) was
used as a Gibson subcloning template to generate the empty lentiviral
construct outlined in Fig. 3A. Gibson subcloning was then used to generate
the retroviral constructs containing full-length NTAP-tagged RIPK2 or the
NTAP-tagged RIPK3/2 and RIPK4/2 chimeric constructs. HEK293 cells
were transfected via calcium phosphate with pMD.2 (Addgene), psPAX
(Addgene), and the RIPK lentivirus in a 1:3:4 molar ratio. Two days later,
supernatant was harvested, centrifuged at 1200 rpm for 5 min, and filtered
through a 0.45-mm filter. Polybrene (8 mg/ml) was added to the viral supernatant, and this mixture was added to the RIPK22/2 macrophages. Two
days later, cells were selected in 500 mg/ml Hygromycin-Gold (Invivogen).
Selection continued for .2 wk. Greater than 10,000 individual colonies
were pooled, and Western blotting showed roughly equal expression levels
of the transduced construct.
RNA isolation and quantitative RT-PCR
The stably transduced RIPK macrophages were treated with 10 mg/ml MDP
for the indicated time. Cells were then harvested and RNA extracted using
a Qiagen RNeasy kit using the manufacturer’s instructions. RNA was reverse transcribed using a Quantitect reverse transcription kit (Qiagen). The
following primer pairs were used for amplification: murine (m)CXCL10forward (F) 59-TCCTTGTCCTCCCTAGCTCA-39 and mCXCL10-reverse
(R), 59-ATAACCCCTTGGGAAGATGG-39; mGPR84-F, 59-GGGAACCTCAGTCTCCAT-39 and mGPR84-R, 59-TGCCACGCCCCAGATAATG-39;
mIRG1-F, 59-GTTTGGGGTCGACCAGACTT-39 and mIRG1-R, 59CAGGTCGAGGCCAGAAAACT-39; mIL-6-F, 59-GCCTTCTTGGGACTGATGCT-39 and mIL-6-R, 59-TGCCATTGCACAACTCTTTTCT-39;
and mGAPDH-F, 59-AGGCCGGTGCTGAGTATGTC-39 and mGAPDH-R,
59-TGCCTGCTTCACCACCTTCT-39. SYBR Green was obtained from
Bio-Rad, and the real-time PCR reactions were carried out using a
CFX96 C1000 Real-Time Thermal Cycler from Bio-Rad. RT-PCR data
are presented as the mean 6 SEM. RT-PCR experiments were performed
in duplicate and repeated three times. Significance of comparisons shown
was assessed by Student two-tailed t test. Significance levels are shown
in each graph.
Results
Despite the homology within the kinase domains, the RIPKs
show differential molecular abilities
The RIPKs have been classified into a family of kinases based on
homology within the kinase domains. All of the kinase domains lie
in the N terminus of the protein, C-terminal to the kinase domain;
however, their domain architecture differs significantly. Although
both RIPK1 and RIPK3 contain RIP homotypic interaction motif
domains to allow for homotypic protein–protein interactions (28),
only RIPK1 also contains a death domain (29). RIPK4 contains
Ankyrin repeats (30), and RIPK2 contains a caspase activation
recruitment domain (CARD) (21, 22), which allows it to interact
with NOD2 and serve as a sensor of intracellular bacterial exposure (Fig. 1A) (6). Given that there is widespread interest in targeting this family of kinases pharmacologically for diseases as
diverse as autoinflammation, sepsis, and autoimmunity (10–19),
we sought to formally compare the molecular and biochemical
activities of the RIPKs to determine unique features and functional
redundancy of this kinase family. NF-kΒ luciferase studies
showed that RIPK1, RIPK2, and RIPK4 could all activate NF-kB,
whereas RIPK3 could not (Fig. 1B). Surprisingly, only RIPK2 was
confirmed as a dual specificity kinase as only RIPK2 could
autophosphorylate on tyrosine (Fig. 1C). Lastly, every RIPK except RIPK3 could induce the ubiquitination of NEMO, a key
feature of NF-kB activation (31) (Fig. 1D). These findings suggest
that RIPK1, -2, and -4 share similar molecular abilities to activate
the NF-kB signaling pathway, whereas RIPK3 diverges. RIPK2
uniquely autophosphorylates on tyrosine, and under these biochemical conditions is the only dual-specificity kinase among this
family.
Domain switching reveals that RIPK2’s and RIPK4’s kinase
domains are functionally similar
Given that RIPK2’s tyrosine autophosphorylation is required for
downstream NOD2 signaling (11), we were surprised that the
other RIPKs did not show tyrosine autophosphorylation activity.
To determine if this activity was unique to RIPK2’s kinase domain
or if it required the specific spacial proximity to the substrate
present in RIPK2’s C terminus [in which Y474 is phosphorylated
(11)], synthetic biology techniques were used to generate chimeric
RIPK constructs. In each of these constructs, the C terminus of
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Cell lines, plasmids, transfection, and Western blotting
UNIQUENESS OF THE RIP KINASE DOMAIN
The Journal of Immunology
3
RIPK2 (immediately downstream of the kinase domain) was held
constant, whereas the kinase domains were swapped. For example,
the RIPK1/2 chimera contained the N-terminal RIPK1 kinase
domain with RIPK2’s C terminus, although the RIPK3/2 chimera
contained RIPK3’s kinase domain with RIPK2’s C terminus
(Fig. 2A). We first determined if these chimeric molecules could
maintain the interaction with NOD2. NOD2 is known to interact
with RIPK2 through RIPK2’s C-terminal CARD domain (6) and
thus should interact with the chimeric kinases. Western blotting
following coimmunoprecipitation from transfected cells showed
that all three chimeric constructs as well as wild-type (WT)
RIPK2 could interact with NOD2, suggesting that the chimeric
proteins were folding correctly and could still interact with
RIPK2’s key signaling partner (Fig. 2B). To then test if kinase
domain swapping could biochemically function, in vitro kinase
assays were performed. Of the RIPKs, RIPK4 could autophosphorylate on tyrosine only when RIPK2’s C-terminal domain was
present (Fig. 2C). Neither the RIPK1/2 or RIPK3/2 chimeric kinases could autophosphorylate on tyrosine (Fig. 2C). Domain
swapping further revealed that the RIPK3/2 and RIPK4/2 chimeras could induce NEMO ubiquitination, albeit at lower levels
relative to WT RIPK2 (Fig. 2D). Lastly, ubiquitination of NEMO
was not sufficient for NF-kB activation, as only the RIPK4/2
chimeric protein, and not the RIPK3/2 chimeric protein, could
activate NF-kB (Fig. 2E). These findings suggest that, like RIPK2,
RIPK4 also possesses tyrosine kinase activity, as well as the
ability to induce NEMO ubiquitination and cause subsequent
NF-kB activation. The chimeric RIPK4/2 protein is therefore
functionally similar to WT RIP2 and gives us an important tool
to now dissect the uniqueness of RIPK2’s kinase domain in
signaling and gene expression systems. This line of research is
especially important as numerous pharmaceutical companies have
RIPK inhibitors in clinical development (10–19).
RIPK2’s kinase domain is uniquely required for NOD2
signaling
To then answer if RIPK2’s kinase domain is uniquely required for
NOD2 signaling, we used synthetic biology techniques to develop
a novel lentiviral expression construct [generated from the lentiCRISPR V2 construct (32)] and then made use of immortalized
RIPK2 2/2 macrophages. This lentiviral expression construct
contains standard lentiviral long terminal repeats; however,
the EF-1 promoter drives exogenous mRNA transcription. A
hygromycin resistance gene (HygR) was Gibson cloned in frame
to a C-terminal P2A self-cleaving peptide cassette. Finally, NTAPtagged RIPK2, RIPK3/2, and RIPK4/2 were Gibson cloned in to
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FIGURE 1. Comparison of the molecular activities of the RIPKs. (A) Schematic showing the RIPKs’ domain structure. Homology lies within the kinase
domain in the N terminus, whereas the C termini have differing domain architecture. (B) HEK293 cells were transfected with CMV-Renilla, NF-kB–driven
luciferase, and 1.5 mg of the indicated RIPK construct. Transfection efficiency was standardized to Renilla expression, and luciferase activities were
measured. RIPK1, RIPK2, and RIPK4 could activate NF-kB, but RIPK3 could not. (C) HEK293 cells were transfected as indicated, and streptavidin bead
association isolated the individual RIPK. In vitro kinase assays were performed in the presence or absence of ATP. Only RIPK2 was able to autophosphorylate on tyrosine. (D) HEK293 cells were transfected with HA-tagged ubiquitin, myc-tagged NEMO, and the indicated RIPK construct. NEMO was
isolated by immunoprecipitation under stringent conditions, and Western blotting was performed. RIPK1, RIPK2, and RIPK4 were all able to cause NEMO
ubiquitination, whereas RIPK3 was not. Each given experiment was performed in at least three biologic replicates with similar results in each. *p , 0.02.
IP, immunoprecipitation; RHIM, RIP homotypic interaction motif.
4
UNIQUENESS OF THE RIP KINASE DOMAIN
the vector in frame with the P2A cassette. The end result is an
expression vector that is driven by EF-1 (a promoter insensitive to
NF-kB activity) and generates a single mRNA containing the
resistance gene and our gene of interest. Upon translation, the
single mRNA product is generated as two individual proteins
(schematic shown in Fig. 3A). Although the RIPK4/2 chimeric
protein can both tyrosine autophosphorylate and activate NF-kB,
the RIPK3/2 protein can perform neither of these functions and
was therefore used as an additional negative control. Lentivirus
was produced and used to infect RIPK22/2 macrophages. Postinfection, cells were selected in hygromycin for 2 wk before .10,000
colonies were pooled. Western blotting showed that although
RIPK4/2 was initially expressed at a slightly lower level (Fig. 3B),
upon stronger hygromycin selection, levels of the exogenous proteins normalized (Fig. 3C, 3D). Signaling experiments were
performed. Although RIPK2 expression could rescue NOD2dependent signaling in the RIPK22/2 macrophages, expression of
empty vector (Fig. 3C), RIPK3/2, or RIPK4/2 could not (Fig. 3D),
suggesting that despite the biochemical similarities between
RIPK2 and RIPK4/2, RIPK4’s kinase domain could not replace
RIPK2 in NOD2 signaling. To then further determine the extent of
the signaling defect in a manner more quantifiable, NOD2-driven
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FIGURE 2. Domain swapping reveals similar molecular activities between RIPK2 and RIPK4. (A) Schematic showing the chimeric constructs generated
and used. The C terminus of the constructs is identical to the C terminus of RIPK2, whereas the kinase domains have been swapped as indicated. (B)
Cotransfection into HEK293s with the indicated constructs followed by immunoprecipitation (IP) and Western blotting shows that all chimeric RIPK
proteins can bind to NOD2. (C) HEK293 cells were transfected as indicated, and streptavidin bead association isolated the individual RIPK. In vitro kinase
assays were performed in the presence or absence of ATP. Although RIPK2 could autophosphorylate on tyrosines, only the RIPK4/2 chimera retained this
ability. (D) HEK293 cells were transfected with HA-tagged ubiquitin, myc-tagged NEMO, and the indicated RIPK chimera. NEMO was isolated by IP
under stringent conditions, and Western blotting was performed. RIPK2, RIPK3/2, and RIPK4/2 were all able to cause NEMO ubiquitination to a certain
degree, whereas RIPK1/2 was not. (E) HEK293 cells were transfected with CMV-Renilla, NF-kB–driven luciferase, and the indicated RIPK construct.
Transfection efficiency was standardized to Renilla expression, and luciferase activities were measured. Of the chimeric constructs, only the RIPK4/2
chimera could activate NF-kB. Each given experiment was performed in at least three biologic replicates with similar results in each. *p , 0.05.
The Journal of Immunology
5
gene expression was studied. Our laboratory has previously used
NextGen sequencing technologies to identify the NOD2-driven
genes most sensitive to RIPK2’s kinase activity (12, 25). We
used these genes as readouts for gene expression. In all cases, only
RIPK2 expression could rescue NOD2-driven gene expression.
This was true for IL-6 (Fig. 4A), CXCL10 (Fig. 4B), IRG-1
(Fig. 4C), and Gpr84 (Fig. 4D). Together, these data suggest
that despite molecular and biochemical similarities between
RIPK2 and RIPK4’s kinase domains, RIPK2’s kinase domain
functions uniquely, a key feature if one hopes to pharmaceutically
target RIPK2 for clinical gain.
Discussion
Despite their homology and familial grouping, the RIPKs participate in varied biologic functions. RIPK1 and RIPK3 are important
in dictating the mechanism of cell death in response to a variety of
innate immune and inflammatory signaling (1, 2, 33). RIPK2 is
critically required for NOD1/2 signaling in response to intracellular bacterial exposure (34), and RIPK4 is required for proper
development (9). Despite this, recent structural work has shown
that a number of broad-spectrum kinase inhibitors target RIPK1,
RIPK2, and RIPK3 with similar potency (16–18). This same
structural work has shown that although there are subtle structural
differences that may help direct medicinal chemistry toward
specific inhibitors, these three kinases overlap significantly in a
three-dimensional structural context, potentially making such
efforts futile (18). Additionally, work presented in this manuscript
shows that they have overlapping molecular and biochemical
activities. RIPK1, RIPK2, and RIPK4 all induce NEMO ubiquitination and subsequent NF-kB activation. Despite this, only
RIPK2 autophosphorylates on tyrosine and is the only RIPK
proven to be a dual-specificity kinase. Given the interest in
pharmacologically targeting a family of kinases with both similar
and divergent molecular activities (10–19), it was important to
determine the functional redundancy of the kinase domain between the RIPK family members. To this end, domain-swapping
synthetic biology approaches were used. In this context, the only
kinase domain that could replicate RIPK2 kinase domain function
in an in vitro system was the RIPK4 kinase domain. Substituting
RIPK4’s kinase domain for RIPK2’s allowed NOD2 binding,
tyrosine autophosphorylation, NEMO ubiquitination, and NF-kB
activation, all key molecular events in which RIPK2 is required
downstream of NOD2 activation. Surprisingly, despite the molecular similarities between the two kinase domains, the RIPK4
kinase domain could not substitute for RIPK2 in an endogenous
setting. It could not support NOD2-induced signaling in RIPK22/2
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FIGURE 3. The kinase domain of RIPK2 is uniquely required for NOD2 signaling. (A) Schematic showing novel lentiviral construct designed to express
the RIPK chimeras. HygR is cloned in frame with the self-cleaving peptide, P2A, and the NTAP-tagged RIPK chimera. A single mRNA is generated under
the EF-1 promoter and upon translation; the P2A sequence allows a translational skip such that during translation, two proteins (HygR [HygroR] and the
NTAP-tagged RIPK) are generated from a single mRNA. (B) Immortalized RIPK22/2 macrophages were transduced with lentivirus containing no RIPK
(empty), RIPK2, RIPK3/2, and RIPK4/2. Two days after transduction, cells were selected with hygromycin. After 2 wk of selection, .10,000 individual
cell colonies were pooled. Streptavidin bead isolation and Western blotting showed that the stable cell lines expressed the gene of interest. (C and D) The
indicated RIPK cell line was treated with 10 mg/ml of the NOD2 agonist L-18 MDP for the indicated time period. Lysates were generated, and Western
blotting was performed. Although the empty vector line showed no signaling (consistent with RIPK2 being genetically absent), cells reconstituted with
RIPK2 show a strong signaling response. Neither RIPK3/2 nor RIPK4/2 reconstituted cells showed a NOD2-dependent signaling response. In (D), the final
two lanes are RIPK2 reconstituted such that a positive control is present on those blots. Each given experiment was performed in at least three biologic
replicates with similar results in each. cPPT, central polypurine tract; IKK, IkB kinase; LTR, long terminal repeat; WPRE, woodchuck posttranscriptional
regulatory element.
6
UNIQUENESS OF THE RIP KINASE DOMAIN
macrophages and could not replace RIPK2’s role in driving
NOD2-induced gene expression. These findings suggest that
RIPK2’s kinase domain is uniquely required for NOD2 signaling
and cannot be replaced by even its closest homologs, implying
that unique pharmacologic targeting of the RIPK family members
is readily achievable.
RIPK2’s role in innate immune signaling has largely centered
on its scaffolding function. RIPK2 clearly helps nucleate signaling
complexes to transduce signals from NOD1 and NOD2, and genetic loss of RIPK2 does not allow signaling through the NOD1
and NOD2 receptors (5, 33, 34). The fact that overexpression
of kinase-dead RIPK2 could activate NF-kB suggested that the
kinase domain might be dispensable for RIPK2’s major known
function (6, 21, 22). Despite this, recent work uncovering specific
inhibitors of RIPK2 suggested that although initial and acute
NF-kB signaling did not require kinase activity, optimal NODstimulated cytokine and gene expression absolutely require it
(11, 19). This finding is supported by our prior study using
NextGen RNAseq methods showing that a significant subset of
NOD2-induced genes require RIPK2’s kinase activity for optimal
expression (12, 25). A key question that remains centers on the
scaffolding function of RIPK2’s kinase domain versus its actual
kinase activity. To answer this question, we used domainswapping experiments in which we replaced RIPK2’s kinase domain with its closest structural homologs (RIPK4 and RIPK3).
Surprisingly, we found that despite the fact that a RIPK4/2 chimera could largely replace RIPK2’s function in overexpression
systems, it could not replace RIPK2’s function in more endogenous, acute signaling experiments. This finding is surprising
because pharmacologic experiments have shown that although
RIPK2’s kinase activity is required for optimal gene expression, it
is largely dispensable for acute NF-kB signaling (19). This experiment shows that there must be structural elements of the
RIPK2 kinase domain independent of its kinase activity such that
RIPK4 could not replace RIPK2’s role in acute signaling. The
scaffolding function and acute NF-kB signaling of RIPK2 is
dependent on its kinase domain but not its kinase activity, and this
scaffolding activity cannot be replaced even by RIPK2’s closest
homolog.
Another interesting finding in this study centers on tyrosine
phosphorylation. RIPK2 is known to be a dual-specificity kinase
(11), but work in this manuscript shows that this feature is not
shared by the other RIPK family members. Given this, it is surprising that RIPK4 is able to autophosphorylate on tyrosine when
its C-terminal Ankyrin repeats are replaced by RIPK2’s intermediate and CARDs. Although native RIPK4 cannot autophosphorylate on tyrosine residues, the RIPK4/2 chimera can
autophosphorylate on tyrosines, and this activity matches RIPK2’s
tyrosine kinase activity. This surprising result suggests that
RIPK4’s kinase domain has the intrinsic ability to be a dualspecificity kinase; however, its ability to phosphorylate on tyrosine is substrate-restricted rather than kinase activity restricted.
To our knowledge, this substrate-driven dual-specificity kinase
activity is unique and has broader implications for the kinase
field as a whole, suggesting that phosphoacceptor preferences
can be altered by substrate selection rather than by intrinsic kinase structure.
Thus, in addition to categorizing and comparing the RIPKs to
one another in terms of their ability to activate NF-kB and perform
NEMO ubiquitination, this study illustrates two key features of the
RIPK family. First, RIPK2’s kinase domain is uniquely structured
in such a way as to nucleate signaling complexes independent of
its kinase activity. For this reason, its closest homologous kinase
domain, RIPK4, cannot replace it structurally despite having
similar kinase activity. Secondly, RIPK4 has substrate-restricted
dual-specificity kinase activity that can be induced by physically
fusing the substrate to its kinase domain. In the context of subsequent pharmacologic targeting of this family, the work suggests
that not only might a small molecule exclusively target RIPK2, but
also that by exclusively targeting RIPK2, the function of the other
RIPKs might not be affected. It also suggests that by developing
type III kinase inhibitors for RIPK2 and RIPK4, one might be able
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FIGURE 4. The kinase domain of RIPK2 is required for NOD2-driven gene expression. (A–D) The RIPK-reconstituted cells were treated with 10 mg/ml
MDP for 2.5 or 5 h. Quantitative RT-PCR was performed using expression of GADPH as an RNA quantification control. Only cells reconstituted with fulllength RIPK2 allowed NOD2-driven gene expression of IL-6 (A), CXCL10 (B), IRG-1 (C), and Gpr84 (D). Mu, macrophage.
The Journal of Immunology
to identify substrate-specific inhibitors and limit substrate phosphorylation rather than eliminate all RIPK2 or RIPK4 phosphorylation.
Acknowledgments
We thank Drs. George Dubyak, Tsan Xiao, and Parameswaran Ramakrishnan
(Case Western Reserve University School of Medicine) for helpful comments
and critiques on the manuscript. Constructs and reagents were obtained from
Vishva Dixit (Genentech), Michelle Kelliher (University of Massachusetts
Medical School), and Shiv Pillai (Massachusetts General Hospital).
Disclosures
The authors have no financial conflicts of interest.
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