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PKK mediates Bcl10-independent NF-κB Activation Induced by Phorbol Ester*

Akihiro Mutoa, Jürgen Rulandc, Linda M. McAllister-Lucasab, Peter C. Lucasa, Shoji Yamaokad,

Felicia F. Chena, Amy Linc, Tak W. Makc, Gabriel Núñezae and Naohiro Inoharaae

From the aDepartment of Pathology and Comprehensive Cancer Center, bDepartment of

Pediatrics and Communicable Diseases, The University of Michigan Medical School, Ann Arbor,

Michigan 48109, USA, cthe Amgen Institute and Ontario Cancer Institute and Departments of

Medical Biophysics and Immunology, University of Toronto, Toronto, Ontario M5G 2C1,

Canada, and dthe Department of Microbiology, Tokyo Medical and Dental University, School of

Medicine, Yushima 1-5-45, Bunkyo-ku, Tokyo 113-8519, Japan,

e To whom correspondence should be addressed. Tel.: 734-764-8509; Fax 734-647-9654; E-mail:

ino@umich.edu or bclx@umich.edu

Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on June 28, 2002 as Manuscript M202222200 by guest on M

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Running Title: PKK is a phobol ester-responsive activator of NF-κB.

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ABSTRACT

Protein kinase C-associated kinase (PKK) is a recently described kinase of unknown

function that was identified on the basis of its specific interaction with PKCβ. PKK contains N-

terminal kinase and C-terminal ankyrin repeats domains linked to an intermediate region. Here

we report that the kinase domain of PKK is highly homologous to that of two mediators of

nuclear factor-κB (NF-κB) activation, RICK and RIP, but these related kinases have different C-

terminal domains for binding to upstream factors. We find that expression of PKK, like RICK

and RIP, induces NF-κB activation. Mutational analysis revealed that the kinase domain of PKK

is essential for NF-κB activation whereas replacement of serine residues in the putative activation

loop did not affect the ability of PKK to activate NF-κB. A catalytic inactive PKK mutant

inhibited NF-κB activation induced by phorphol ester and Ca2+-ionophore but it did not block

that mediated by tumor necrosis factor α, interleukin-1β or Nod1. Inhibition of NF-κB activation

by dominant negative PKK was reverted by co-expression of PKCβI, suggesting a functional

association between PKK and PKCβI. PKK-mediated NF-κB activation required IKKα and

IKKβ but not IKKγ, the regulatory subunit of the IKK complex. Moreover, NF-κB activation

induced by PKK was not inhibited by dominant negative Bimp1 and proceeded in the absence of

Bcl10, two components of a recently described PKC signaling pathway. These results suggest

that PKK is a member of the RICK/RIP family of kinases which is involved in a PKC-activated

NF-κB signaling pathway that is independent of Bcl10 and IKKγ.

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INTRODUCTION

NF-κB is a transcription factor that mediates the activation of a large array of target genes

which are involved in the regulation of diverse functions including inflammation, cell

proliferation and survival (1). During inflammatory responses, NF-κB is activated in response to

multiple stimuli including tumor necrosis factor (TNF), lipopolysaccharides (LPS) and

interleukin-1 (IL-1) (1). These trigger molecules interact with surface receptors or specific

intracellular sensors which lead to the activation of NF-κB through signal-specific mediators and

common downstream effectors such as IκBα and IκB kinase (IKK) (1,2). RICK and RIP are

highly related kinases which mediate NF-κB activation in the Nod1 (or Nod2) and TNFR1 (or

TRAIL) receptor signaling pathways, respectively (3-8). RICK and RIP contain N-terminal

kinase domains linked to intermediate (IM) regions but different C-terminal domains; a caspase-

recruitment domain (CARD) and a death domain (DD), respectively (9-13). These C-terminal

domains mediate the recruitment of RIP and RICK to upstream signaling components, whereas

the IM regions link these kinases to the common regulator IKK (9-13). The IM region of both

RIP and RICK is essential for NF-κB activation (9-13). Thus, RICK and RIP serve as bridging

molecules connecting signal-specific components to common mediators of NF-κB activation.

These observations suggest that proteins carrying kinase domains homologous to those of RIP

and RICK but different C-terminal domains might be involved in the activation of novel NF-κB

signaling pathways.

PKK , a mouse kinase composed of an N-terminal kinase domain, an IM region, and a C-terminal

domain containing 11 ankyrin repeats was recently identified for its ability to interact with

protein kinase C (PKC) isoform PKCβI while its human counterpart, named DIK, was shown to

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associate with PKCδ (14, 15). PKCs mediate intracellular signals triggered by stimulation of a

variety of extracellular ligands including those associated with G-coupled and antigen receptors

(16). Classical and novel PKCs are known to be activated by phorbol ester and intracellular Ca2+

and by phorbol ester only, respectively, and to induce the activation of multiple transcription

factors such as NF-κB and AP-1 (16). Recent studies have identified a PKC-dependent signaling

pathway of NF-κB activation which is mediated by Bcl10 (17-19). Bimps and MALT1 appear to

link PKC activation induced by surface receptors to Bcl10 and IKKs (17, 20).

It has been hypothesized that PKK and its human orthologue are somehow involved in a PKC-

associated signaling pathway (14, 15). However, the particular signaling pathway in which PKK

functions has not been previously addressed. We report here that PKK is highly homologous to

RIP and RICK. Expression of PKK induces the activation of NF-κB, and this activity requires the

kinase domain. We also provide evidence that PKK mediates the NF-κB activation induced by

phorbol ester and Ca2+-ionophore and specifically by PKCβI. These studies indicate that PKK is

a RICK/RIP-like molecule which is involved in an NF-κB signaling pathway mediated by

particular PKC isoforms.

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MATERIALS AND METHODS

Cell Lines and Materials

Mouse embryonic fibroblasts (MEFs) lacking IKKα, IKKβ, both IKKα and IKKβ, Bcl10,

and IKKγ were described previously (18, 21, 22) and maintained in Dulbecco's MEM containing

10 % fetal bovine serum and antibiotics. IL-1β and TNFα were purchased from Collaborative

Biomedical Products (Bedford, MA). PMA, A23187 and other reagents were purchased from

Sigma Chemicals (St. Louis, MO). The partial nucleotide sequences of zebrafish cDNAs

encoding peptides with homology to RICK were found in expressed sequence tag (EST)

databases of GenBank using the TBLASTN program. The entire nucleotide sequences of EST

clones GenBank BF158596 (zebrafish PKK) and BG737635 (zebrafish RICK) were determined

by dideoxy sequencing.

Construction of Expression Plasmids

The open reading frame of mouse PKK was amplified by polymerase chain reaction (PCR) from

random-primed mouse embryo E15 cDNA and cloned into pcDNA3-Flag, pcDNA3-Myc, and

pcDNA3-HA (23). Deletion and site-directed mutants of PKK (residues 1-286 for KD, 1-439 for

∆ARD, 440-786 for ARD, 287-439 for IM, D143A, S171A/S173A/S177A for SSSAAA and

S171E/S173E/S177E for SSSEEE) were constructed by a PCR method and cloned into pcDNA3-

Myc. The authenticity of all constructs was confirmed by sequencing. pcDNA3-Nod1-Flag,

pcDNA3-Nod1-HA, pcDNA3-Bimp1-Flag, pcDNA3-Bimp1(117-1021)-Flag, pcDNA3-

Bcl10(CIPER)-Flag, pcDNA3-MALT1-(324-813)-Fpk3-Myc, pcDNA-IKKβ-Myc, pRK7-Flag-

IKKα-K44A, RSVMad-3MSS(Iκ-Bα-S32A/S36A), pRK7-Flag-IKKβ-K44A, pcDNA3-HA-

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IKKγ(134-419), pcDNA3-MyD88(1-109), pCEP4-HA-MEKK1, pcDNA3-Flag-IRF-1, pcDNA3-

p53, pTB701-HA-PKCβI, pTB701-HA-PKCβI(K371M), pTB701-HA-PKCε, pcDNA3-Flag-

DC-CIITA, pEF-BOS-β-gal, pBVIx-Luc, pGL3-(NF-AT)6-luc, MHC-II(Eα)-luc, and pGL3-

mdm2-luc have been described previously (8, 17, 19, 20, 23-34). pAP-1-luc was purchased from

Stratagene (La Jolla, CA).

Immunodetection of tagged proteins

HEK293T cells were co-transfected with pcDNA3-Myc-PKK and various expression

plasmids as described (8). Detection of expressed proteins was performed by immunoblotting as

described (8).

NF-κB activation assay

NF-κB activation assay was performed as described (8). Briefly, Rat1 fibroblasts, its

derivative 5R cell line, MEFs as well as HEK293T cells were co-transfected with 33 ng of the

reporter construct pBVIx-Luc, plus indicated amounts of each expression plasmid and 330 ng of

pEF-BOS-β-gal in triplicate as described (8). The total amount of transfected plasmid DNA was

adjusted with pcDNA3 vector such that it was constant within each individual experiment. 24 hr

post-transfection, cell extracts were prepared and luciferase activity was measured as described

(8). Results were normalized for transfection efficiency with values obtained with pEF-BOS-β-

gal.

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RESULTS AND DISCUSSION

PKK is highly related to RICK

To identify novel RICK-like molecules, public protein and nucleotide databases were

searched for homologous proteins using the entire human RICK sequence (9). As expected, we

identified RIP (E values; 4x 10-29 and 3x 10-29 for human and mouse RIP, respectively) and its

homologue RIP3 (E values; 1x10-31 and 5x 10-30 for human and mouse RIP3, respectively) as

molecules with significant homology to RICK (Fig. 1). In addition, the search identified PKK, a

kinase of unknown function, as the most homologous protein to RICK in available databases

(E=4x10-51 for mouse PKK and 4x10-50 for human PKK). We also identified zebrafish orthologues

of PKK and RICK. The domain structure of the fish PKK and RICK was identical to that of their

mammalian orthologues (Fig. 1A). Significantly, zebrafish PKK was more homologous to human

RICK (E=5 x10-50) than human RICK to human RIP or RIP3 (Fig. 1B). As expected from the

homology between RICK and RIP, PKK also exhibited significant similarity to RIP (E=4x10-31)

and RIP3 (E=5x10-32and E=3x10-30 for human and mouse, respectively) (Fig. 1B). These results

indicate that PKK is a novel member of the RICK/RIP family of kinases. Further analysis of

protein sequences revealed that the homology between PKK and RICK-related kinases was

restricted to their kinase domains in that no significant similarity was identified in the IM and C-

terminal domains. Consistent with these findings, RICK and RIP have C-terminal CARD and

DD, respectively, whereas PKK contains 11 ankyrin repeats in its C-terminus (Fig. 1A). The IM

region of RICK and RIP is serine/threonine-rich and essential for the interaction with IKKγ and

NF-κB inducing activity (8, 35). Interestingly, the IM region of PKK was also serine-threonine-

rich, but it did not exhibit any significant amino acid homology to that of RICK and RIP.

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PKK activates NF-κB and AP-1

Given the amino acid and structural homology between PKK and RICK-related kinases, we

first tested whether expression of PKK activates NF-κB. Transfection of the wild-type (wt) PKK

cDNA into HEK293T cells induced activation of NF-κB in a dose-dependent manner, as

measured with a reporter luciferase construct (Fig. 2A). The induction of NF-κB by PKK was

specific in that transfection of the PKK cDNA did not induce transactivation of NF-AT, NF-IL6,

p53, IRF-1 and class II MHC-dependent promoters (Fig. 2B). In control experiments, the

transcriptional activity of the reporter constructs was stimulated by expression of proteins known

to induce their activation (Fig. 2B). We also found that expression of PKK induced significant

activation of AP-1 (Fig. 2B) as did expression of MEKK1, a known activator of AP-1 (26).

The kinase domain of PKK is essential for NF-κB activation

To identify the domains of PKK that are required for NF-κB activation, a series of deletion

mutants carrying each domain alone or in combination were constructed (Fig. 3A). Expression of

PKK mutants containing the kinase domain resulted in NF-κB activation, while mutants

containing the IM region and/or ankyrin repeats-containing domain (ARD) alone were inactive

(Fig. 3C). Immunoblotting analysis showed that the lack of activity of the mutants could not be

explained by different expression levels of the mutant proteins (Fig. 3C, inset). Thus, the kinase

domain of PKK is necessary and sufficient for NF-κB activation, suggesting that the catalytic

region acts as an effector domain in PKK signaling. Consistent with this hypothesis, replacement

of the conserved aspartate residue (D143) in the catalytic site for alanine rendered PKK inactive

(Fig. 3C).

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Human and mouse PKK contain a Ser-X-X-X-Ser motif (SHDLS) at positions 173-177 in their

putative activation loops (Fig. 3B). The corresponding serine residues of mitogen-activated

protein (MAP) kinase kinases and IKKs are often phosphorylated by other serine protein kinases

resulting in kinase transactivation (30, 31). Substitution of the conserved serine residues, S173

and S177 as well as S171 for alanine, did not alter the ability of PKK to induce NF-κB when

compared to the wild-type kinase (Fig. 3C). Similarly, replacement of S171, S173 and S177 for

glutamic acid residues which is associated with constitutive activation of serine/threonine kinases

did not enhance the ability of PKK to induce NF-κB (Fig. 3C). Close inspection of zebrafish

PKK revealed that the fish kinase lacks serines at position 171 and 173 and tyrosine residues in

its putative activation loop (Fig. 3B) This finding indicates that the canonical motif in the

activation loop of kinases is not evolutionarily conserved in PKK. Together, these observations

suggest that the ability of PKK to activate NF-κB is not regulated by phosphorylation of its

putative activation loop.

PKK is involved in PMA/ Ca2+-ionophore-induced NF-κB activation

PKK is known to interact with PKCβI, suggesting that these proteins may function in a

common signaling pathway (14). Recent studies have revealed that Bimp1, Bcl10 and MALT1

are components of a receptor-mediated signaling pathway which links PKC activation to NF-κB

induction (17, 18). Therefore, we next tested whether PKK regulates an NF-κB signaling

pathway mediated by Bimp1, Bcl10 and MALT1 in HEK293T cells which are known to express

endogenous PKK (15). Treatment of HEK293T cells with PMA/ Ca2+-ionophore induced NF-κB

activation which was inhibited by the PKK mutant carrying an alanine substitution at the catalytic

aspartate residue (D143A) (Fig. 4A). The inhibitory effect was specific in that expression of PKK

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D143A did not block NF-κB activation induced by Bimp1, Bcl10, oligomerized MALT1, TNFα

(Fig. 4A), IL-1β or Nod12. Additional control experiments shown in Fig. 4A revealed that

activation of NF-κB induced by PKK, Bimp1, Bcl10, activated MALT1, PMA/Ca2+-ionophore or

TNFα could be inhibited by a dominant interfering form of IKKβ but not by that of MyD88, an

essential mediator of IL-1/Toll receptor signaling (32). Because PKK associates with PKCβI

(14), we tested if the PKK D143A mutant inhibits PMA-induced NF-κB activation through a

functional interaction with PKCβI. Expression of PKCβI reverted the effect of the PKK D143A

mutant, whereas a kinase negative mutant of PKCβI (K371M) and PKCε did not (Fig. 4B). The

mechanism by which PKCβI reverts the dominant negative effect of the PKK mutant is unclear.

A possible explanation is that overexpressed catalytically active PKCβI competes out PKK-DN

for cellular factor(s) necessary for function. The selective effect of PKCβI is consistent with the

observation that PKK interacts with PKCβI (14) but not with PKCε2. In addition, activation of

AP-1 induced by PMA/Ca2+-ionophore was specifically inhibited by PKK dominant negative

(Fig. 4C), suggesting that PKK also acts in a PMA-induced AP-1 signaling pathway activated by

PKCβI.

NF-κB activation induced by PKK requires IKKα and IKKβ but not IKKγ

NF-κB activation by RICK and RIP is mediated by the IKK complex, a universal regulator which

phosphorylates IκBα resulting in degradation of IκBα and nuclear translocation of NF-κB (2, 8).

To determine whether NF-κB activation by PKK is also dependent on IKKs, PKK was co-

expressed with the catalytic inactive forms of IKKα and IKKβ. NF-κB activation induced by

PKK as well as that induced by PMA/Ca2+-ionophore, IL-1β and TNFα, was inhibited by

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catalytic inactive IKKα and IKKβ (Fig. 5A). In control experiments, PKK-mediated NF-κB

activation was not affected by dominant negative forms of Bimp1 or MyD88 (Fig. 5A). The

ability of PKK to activate NF-κB was also determined in MEFs lacking IKKα and IKKβ.

Whereas PKK activated NF-κB in wt fibroblasts, it was unable to induce NF-κB in cells lacking

IKKβ or in cells lacking both the IKKα and IKKβ proteins (Fig. 5B). These results suggest that

NF-κB activation induced by PKK requires catalytic IKKs. However, we found that purified

PKK did not phosphorylate IKKα or IKKβ in vitro2 suggesting that PKK does not function

through direct phosphorylation and activation of the IKK complex.

Next we tested if NF-κB activation by PKK requires IKKγ, a regulatory component of the IKK complex

(18, 33-35). PKK was co-expressed with a truncated mutant of IKKγ (residues 134-419) which inhibits

NF-κB activation induced by RIP and RICK (8). Surprisingly, co-expression of the IKKγ mutant did not

inhibit PKK-mediated NF-κB activation (Fig. 5A). To verify the latter result, we tested the ability of

PKK to activate NF-κB in parental Rat1 fibroblasts and IKKγ-deficient 5R cells, a Rat1 derivative cell

line that is defective in IKKγ (22). Expression of PKK induced NF-κB activity not only in parental Rat1

cells but also in 5R cells (Fig. 5C). As a control, stimulation with TNFα, IL-1β, LPS, or expression of

Nod1, which require IKKγ, induced NF-κB activation in parental Rat1 but not in 5R cells (Fig. 5C). It

was shown in Fig. 3 that the IM region of PKK is not essential for NF-κB activation. In contrast, the

same region of RIP and RICK is essential for NF-κB activation and mediates the interaction with

IKKγ (8, 35). Thus, unlike in RICK and RIP, the IM region of PKK and IKKγ are dispensable for NF-κB

activation.

Bcl10 is not required for PKK-mediated NF-κB activation

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Bimp1 and its interacting partners Bcl10 and MALT1 have been shown to act downstream of

PKC in a signaling pathway leading to NF-κB activation (17, 18). In Fig. 4A, we showed that

NF-κB activation induced by expression of Bimp1, Bcl10 and activated MALT1 is unaffected by

dominant negative PKK. Conversely, Fig. 5A demonstrated that a dominant negative form of

Bimp1 had no effect on PKK mediated NF-κB activation. To determine whether PKK could act

upstream of Bcl10, we tested the ability of PKK to induce NF-κB in MEFs deficient in Bcl10

(18). Both PKK and Nod1 induced NF-κB activation in both Bcl10+/- and Bcl10-/- MEFs (Fig.

5D). In control experiments shown in Fig. 5D, Bcl10 was required for NF-κB activation induced

by Bimp1, a protein that acts upstream of Bcl10 to activate NF-κB (17). Together with the results

shown in Fig. 4A, these results suggest that PKK functions in a PKC signaling pathway of NF-κB

activation that is independent from Bcl10.

We provide evidence that PKK is an NF-κB-activating kinase. The activity of PKK is consistent

with its homology to RICK and RIP, two serine-threonine kinases that activate NF-κB. Another

member of the family, RIP3 has been shown to activate or inhibit NF-κB activation, probably

depending on the cellular context (36-38). Thus, PKK appears to represent the fourth member of

the RIP/RICK family of NF-κB activating kinases. Unlike RIP and RICK (8), the catalytic

activity of PKK was required for NF-κB activation. These results indicate that PKK is unique

among the RICK-related kinases and suggest that the mechanism by which PKK activates NF-κB

is distinct from that utilized by RIP and RICK. We hypothesize that PKK activates NF-κB

through the phosphorylation of protein target(s).

PKK was originally identified for its interaction with PKCβI and suggested to function in a PKC

signaling pathway (14). Consistent with this proposed model, we show that a dominant negative

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mutant of PKK inhibits PMA/Ca2+-ionophore-mediated NF-κB activation, an effect which was

reverted by expression of PKCβI. Several studies have implicated PKCβI in the activation of NF-

κB in cells derived from several tissues including the heart and kidney (39-41) which have been

reported to exhibit high expression of PKK (14, 15). We hypothesize that PKK functions in these

tissues to regulate a Bcl10-independent PKCβI-mediated signaling pathway of NF-κB activation.

Bcl10 appears to regulate PKC signaling pathways involved in antigen-receptor stimulation and

neurogenesis (18). The physiological upstream signals that activate PKK through PKCβI remain

to be elucidated. Likewise, additional studies are necessary to identify protein substrate(s) of

PKK which may reveal the mechanism by this RICK-related kinase activates NF-κB.

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ACKNOWLEDGMENTS

We thank C.H. Chang, G. Grabtree, A. Levine, M.H. Cobb and S. Kuroda for providing

expression plasmids, V. Rivera (Ariad Pharmaceuticals) for providing dimerization agent

AP1510, Q. Li and I.M. Verma for providing MEFs deficient in IKKs and S. Chen for technical

support.

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FOOTNOTES

*The work was supported in part by Grant GM60421 from the National Institutes of Health (to

N.I.) and Grant CA84064 from the National Institutes of Health and Grant 1506 from the

Michigan Life Sciences Corridor Fund (to G.N.). A.M. was supported in part by a research

fellowship from Uehara Memorial Foundation, Japan. L.M.M-L. was supported by University of

Michigan Pediatric Research Training Grant T32-HL07622 from the National Institutes of

Health. P.C.L. was supported by an individual National Research Service Award Grant F23-

CA88470-01 from the National Institutes of Health.

1 Abbreviations used are: PKC, protein kinase C; NF-κB, nuclear factor κB; IκB, inhibitor of NF-

κB; IKK, IκB kinase; AP-1, activator protein-1; PMA, phorbol myristylacetate; TNFα, tumor

necrosis factor α; LPS, lipopolysaccharides; IL-1, interleukin-1; CARD, caspase-recruitment

domain; DD, death domain; LRRs; leucine-rich repeats; IM, intermediate; ARD, ankyrin

repeats-containing domain; EST, expressed sequence tag; HA, hemagglutinin; wt, wild-type;

MEF, mouse embryonic fibroblast; NF-AT, nuclear factors of activated T cell; NF-IL6,

nuclear factor-interleukin-6; MHC, major histocompatibility complex; CIITA, MHC class II

transcriptional activator; IRF-1, interferon regulatory factor-1; MAP, mitogen-activated

protein.

2 Muto, A, Inohara, N and Núñez, G., unpublished results.

3The nucleotide sequences of the zebrafish PKK, zebrafish RICK and mouse RICK cDNAs are

available in GenBank as accession number AF487541, AF487540 and AF487539,

respectively.

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FIGURE LEGENDS

Figure 1. Homology between PKK and RICK-related Proteins

(A) Schematic representation of human, mouse, zebrafish PKK and related kinases. Kinase

domain, KD; intermediate region, IM; ankyrin repeats-containing domain, ARD; caspase-

recruitment domain, CARD; death domain, DD.

(B) Homology among PKK and related kinases. The homology between PKK (GenBank

accession numbers human, AJ278016; mouse, AF302127; zebrafish, AF487541), RICK (human,

AC004003; mouse, AF487539; zebrafish, AF487540), RIP (human, NM003804; mouse,

NM009068) and RIP3 (human, AF156884; mouse, AF178953) was calculated by BLASTP and is

given as an E value. 0 indicates E value is less than 10-152.

Figure 2. Expression of PKK Activates NF-κB and AP-1

(A) PKK activates NF-κB in a dose-dependent manner. HEK293T cells were transfected with

control plasmid (-) or indicated amount of pcDNA3-Myc-PKK. Induction of NF-κB activation

was determined from triplicate culture of HEK293T cells co-transfected with the indicated

amount of wt or mutant PKK expression plasmids in the presence of pBVIx-Luc and pEF-BOS-

β-gal as described in Materials and Methods. Values represent mean of normalized values ± SD

of triplicate cultures. Expression of Myc-tagged PKK protein was determined in cell extracts by

immunoblotting (inset). Arrowhead indicates PKK protein.

(B) Specific activation of NF-κB and AP-1 by PKK. HEK293T cells were co-transfected with

control plasmid (-), 3.3 ng of pcDNA3-Myc-PKK, 33 ng of pcDNA3-p53, 0.33 ng of pcDNA3-

Flag-DC-CIITA, 17 ng of pCEP4-HA-MEKK1 and 17 ng of pcDNA3-Flag-IRF-1 plasmid DNA.

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Specific transactivation by NF-κB, AP-1, NF-AT, NF-IL6, p53, CIITA, and IRF-1 activation was

determined using 3.3 ng of the corresponding luciferase reporter constructs and pEF-BOS-β-gal

as described in Materials and Methods. Values represent mean of normalized values ± SD of

triplicate cultures.

Figure 3. Mutational Analysis of PKK

(A) wild type (WT) and mutant PKK proteins. KD, IM and ARD are indicated by black, open,

and hatched boxes, respectively. Numbers represent the position of amino acid residues in PKK

protein.

(B) Amino acid alignment of the putative loop region of PKKs. Amino acid sequences (163 to

183) from human (Hs), mouse (Ms) and zebrafish (Dr) PKK proteins are shown. Serine residues

in the putative activation loop region are indicated by asterisks.

(C) Functional and expression analysis of wt and mutant PKK proteins. HEK293T cells were

transfected with control plasmid (-) or indicated amount of Myc-tagged PKK plasmid DNA.

Induction of NF-κB activation was determined from triplicate culture of HEK293T cells co-

transfected with the indicated amount of wt or mutant PKK expression plasmids in the presence

of pBVIx-Luc and pEF-BOS-β-gal as described in Materials and Methods. Values represent

mean of normalized values ± SD of triplicate cultures. Immunobloting analysis of the expressed

Myc-tagged PKK proteins is shown on top panel. Molecular weight markers are indicated on the

left.

Figure 4. PKK Mediates Phorbol Esther-induced NF-κB Activation.

(A) Inhibition of PMA/Ca2+-ionophore-induced NF-κB activation by dominant negative PKK.

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Induction of NF-κB activation was determined in triplicate cultures of HEK293T cells

cotransfected with 3 ng of pcDNA3-Bimp1-Flag, 30 ng of pcDNA3-Bcl10-Flag, 67 ng of

pcDNA3-MALT1(324-813)-Fpk3-Myc, or stimulated with 50 ng/ml PMA and 0.7 µg/ml Ca2+-

ionophore A23187 for 6 hr or 10 ng/ml TNFα for 2 hrs in the presence of 167 ng of pcDNA3-

HA-PKK D143A, pRK7-Flag-IKKβ-K44A or control plasmid in the presence of pBVIx-Luc and

pEF-BOS-β-gal. Results are presented as a percent of values obtained with Bimp1 and control

plasmid. MALT1(324-813)-Fpk3 was activated by oligomerization with 100 nM AP1510 for 6

hrs. In the experiment shown, Bimp1, Bcl10, oligomerized MALT1, PMA and Ca2+ ionophore,

and TNFα induced 141 ± 12, 96 ± 6, 13 ± 1, 9 ± 1 and 138 ± 26 fold activation of NF-κB,

respectively. Values represent mean of normalized values ± SD of triplicate cultures.

(B) Inhibition of PMA/Ca2+-ionophore-induced NF-κB activation is reverted by wt PKCβI but

not a kinase inactive mutant of PKCβI or PKCε. Induction of NF-κB activation was determined

from triplicate culture of HEK293T cells co-transfected with 167 ng of pTB701-HA-PKCβI,

pTB701-HA-PKCβI (K371M), pTB701-HA-PKCε, or control plasmid and 34 ng of pcDNA3-

HA-PKK D143A in the presence of pBVIx-Luc and pEF-BOS-β-gal and stimulated with or

without 5 ng/ml PMA and 70 ng/ml A23187 for 6 hrs.

(C) Inhibition of PMA/Ca2+-ionophore-mediated AP-1 activation by dominant negative PKK.

Induction of NF-κB activation was determined in triplicate cultures of HEK293T cells transfected

with 167 ng of pcDNA3-HA-PKK D143A and stimulated with 50 ng/ml PMA and 0.7 µg/ml of

A23187 for 6 hrs or left alone in the presence of pAP-1-luc and pEF-BOS-β-gal. Results are

presented as a percent of values obtained with control plasmid.

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Figure 5. PKK Acts through the IKK complex and independently of Bcl10 to activate NF-

κB.

(A) PKK-induced NF-κB activation is inhibited by dominant negative forms of IKK α and IKKβ

but not by those of IKKγ, Bimp1, nor MyD88. Induction of NF-κB activation was determined in

triplicate cultures of HEK293T cells transfected with 1.6 ng of pcDNA3-Myc-PKK, or stimulated

with 50 ng/ml PMA and 0.7 µg/ml of A23187, 10 ng/ml IL-1β or 10 ng/ml TNFα for 4 hrs in the

presence of pBVIx-Luc and pEF-BOS-β-gal. Results are presented as a percent of values

obtained with PKK and control plasmid. In the experiment shown, PKK, PMA/Ca2+-ionophore,

IL-1β and TNFα induced 55 ± 3, 196 ± 15, 423 ± 22 and 183 ± 55 fold activation of NF-κB,

respectively. Values represent mean of normalized values ± SD of triplicate cultures. (B) PKK-

mediated NF-κB activation requires IKKα and IKKβ. Induction of NF-κB activation was

determined in wt, IKKα-/-, IKKβ -/- and IKKα-/-/IKKβ -/- MEFs transfected with 100 ng of

pcDNA3-Flag-PKK, pcDNA3-Nod1-Flag and pcDNA-IKKβ-Myc in the presence of pBVIx-Luc

and pEF-BOS-β-gal. Results were normalized according to the value obtained with cells

transfected with vector alone which was considered as 1. In the experiment, relative κB-

dependent activity of wt, IKKα-/-, IKKβ-/- and IKKα-/-/IKKβ-/- MEFs with control vector was 1,

0.08, 0.11 and 0.006, respectively. (C) PKK induced NF-κB activation in both parental Rat-1 and

IKKγ-deficient 5R cells. Induction of NF-κB activation was determined in Rat-1 and IKKγ-

deficient 5R MEFs transfected with 330 ng of pcDNA3-Flag-PKK, pcDNA-IKKβ-Myc,

pcDNA3-Nod1-Flag, or stimulated with 10 ng/ml TNFα, 10 ng/ml IL-1β or 1 µg/ml LPS in the

presence of pBVIx-Luc and pEF-BOS-β-gal. (D) PKK-mediated activation of NF-κB in the

absence of Bcl10. Bcl10+/- and Bcl10-/- MEFs were transfected with 900 ng of the indicated

expression plasmid: pcDNA3-Flag-PKK, pcDNA3-Nod1-HA or pcDNA3-Bimp1-Flag.

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BAKD IM ARD

KD IM CARD

KD IM DD

PKK

RICK

RIP

Figure. 1 Muto et al.

SUBJECTPKK RICK RIP RIP3

QUERYhuman mouse zebrafish human mouse zebrafish human mouse human mouse

PKKhuman

mouse

zebrafish

RICKhuman

mouse

zebrafish 1X10-144

RIPhuman

mouse

RIP3 humanmouse

1X10-51

4X10-53

2X10-50

1X10-147

1X10-152

0

9X10-30

3X10-27

8X10-36

6X10-36

4X10-53

0

0

0

8X10-52

8X10-52

7X10-32

7X10-30

1X10-35

4X10-34

7X10-54

0

0

0

4X10-53

1X10-52

5X10-34

5X10-29

7X10-34

9X10-34

2X10-50

0

0

0

6X10-51

9X10-30

2X10-31

3X10-29

4X10-38

6X10-36

1X10-152

1X10-31

4X10-31

5X10-33

1X10-32

0

0

1X10-50

3X10-53

1X10-51

4X10-29

3X10-29

1X10-31

5X10-30

4X10-50

4X10-51

5X10-50

0

0

2X10-28

4X10-31

3X10-33

4X10-31

2X10-29

2X10-30

00

1X10-32

1X10-29

4X10-26

3X10-29

1X10-28

8X10-30

4X10-30

6X10-30

00

3X10-34

5X10-30

7X10-34

5X10-32

5X10-31

9X10-38

7X10-29

3X10-29

4X10-30

1X10-30

01X10-135

2X10-31

3X10-30

1X10-30

7X10-33

5X10-28

2X10-26

2X10-31

2X10-26

1X10-137

0

E value

25

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Figure. 2 Muto et al.

NF

-κB

act

ivat

ion

(fo

ld)

A

0

200

400

600

800

0 10 20 50 100 ng

B

0NF-κB AP-1 NF-AT NF-IL6 p53 CIITA IRF-1

Tra

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rip

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50

100(-)PKKp53CIITAMEKK1IRF-1

26

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Figure. 3 Muto et al.

A

B Hs�163� GLAKCNGLSHSHDLSMDGLFG� 183Ms�163� GLAKCNGMSHSHDLSMDGLFG� 183Dr�163� GLARWNGFARDDDISRDGFCG� 183

* * *

WT

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KD ARDIM

D143A D

A

SSSAAA

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1

1

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1 286

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287 439

440 786

786

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171 177

171 177

C

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0

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400

600

800

1000

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1500

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350

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27

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Figure. 4 Muto et al.

A

0

50

100

150

(-) PKK IKKβ MyD88

NF

-κB

act

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(%

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Bimp1Bcl10oligo-MALT1PMA/IonoTNFα

Dominant Negative

B

Dominant Negative

0

10

20

(-) (-) PKK MyD88A

P-1

act

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(fo

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0

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Figure. 5 Muto et al.

(-) PKK Nod1 Bimp1

NF

-κB

act

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(fo

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0

5

10

15

20

25 Bcl10+/-Bcl10-/- #1Bcl10-/- #2

DC

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0

5

10

15

20

25

(-) PKK Nod1 IKKβ

NF

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PKKPMA/IonoIL-1βTNFα

29

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Felicia F. Chen, Amy Lin, Tak W. Mak, Gabriel Nunez and Naohiro InoharaAkihiro Muto, Jurgen Ruland, Linda M. McAllister-Lucas, Peter C. Lucas, Shoji Yamaoka,

B activation induced by phorbol esterκPKK mediates Bcl10-independent NF-

published online June 28, 2002J. Biol. Chem. 

  10.1074/jbc.M202222200Access the most updated version of this article at doi:

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