Protein Kinase C-associated Kinase (PKK) Mediates Bcl10-independent NF-κB Activation Induced by Phorbol Ester*

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 phorbol 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.

NF-B 1 is a transcription factor that mediates the activation of a large array of target genes that are involved in the regulation of diverse functions including inflammation, cell proliferation, and survival (1). During inflammatory responses NF-B 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 that lead to the activation of NF-B through signal-specific mediators and common downstream effectors such as IB␣ and IB kinase (IKK) (1,2). RICK and RIP are highly related kinases that mediate NF-B activation in the Nod1 (or Nod2) and TNFR1 (or TRAIL) receptor signaling pathways, respectively (3)(4)(5)(6)(7)(8). RICK and RIP contain N-terminal kinase domains linked to intermediate (IM) regions but the following different C-terminal domains: a caspase-recruitment domain (CARD) and a death domain (DD), respectively (9 -13). These C-terminal domains mediate 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-B activation (9 -13). Thus, RICK and RIP serve as bridging molecules connecting signal-specific components to common mediators of NF-B 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-B 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 PKC␤I, whereas its human counterpart named DIK was shown to associate with PKC␦ (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 Ca 2ϩ and by phorbol ester only, respectively, and to induce the activation of multiple transcription factors such as NF-B and AP-1 (16). Recent studies have identified a PKCdependent signaling pathway of NF-B activation that is mediated by Bcl10 (17)(18)(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-B, and this activity requires the kinase domain. We also provide evidence that PKK mediates the NF-B activation induced by phorbol ester and Ca 2ϩ -ionophore and specifically by PKC␤I. These studies indicate that PKK is a RICK/RIP-like molecule that is involved in an NF-B signaling pathway mediated by particular PKC isoforms.

MATERIALS AND METHODS
Cell Lines and Materials-Mouse embryonic fibroblasts (MEFs) lacking IKK␣, IKK␤, both IKK␣ and IKK␤, Bcl10, and IKK␥ were described previously (18,21,22) and were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and antibiotics. IL-1␤ and TNF␣ were purchased from Collaborative Biomedical Products (Bedford, MA). PMA, A23187, and other reagents were purchased from Sigma. The partial nucleotide sequences of zebrafish cDNAs encoding peptides with homology to RICK were found in expressed sequence tag (EST) databases of GenBank TM using the TBLASTN program. The entire nucleotide sequences of EST clones, GenBank TM accession numbers BF158596 (zebrafish PKK) and BG737635 (zebrafish RICK), were determined by dideoxy sequencing.
NF-B Activation Assay-An NF-B 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-␤-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 h 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-␤-gal.

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: 4 ϫ 10 Ϫ29 and 3 ϫ 10 Ϫ29 for human and mouse RIP, respectively) and its homologue RIP3 (E values: 1 ϫ 10 Ϫ31 and 5 ϫ 10 Ϫ30 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 ϭ 4 ϫ 10 Ϫ51 for mouse PKK and 4 ϫ 10 Ϫ50 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 ϭ 5 ϫ 10 Ϫ50 ) 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 ϭ 4 ϫ 10 Ϫ31 ) and RIP3 (E ϭ 5 ϫ 10 Ϫ32 and 3 ϫ 10 Ϫ30 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 IKK␥ and NF-B-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.
PKK Activates NF-B 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-B. Transfection of the wild-type (WT) PKK cDNA into HEK293T cells induced activation of NF-B in a dose-dependent manner as measured with a reporter luciferase construct ( Fig. 2A). The induction of NF-B 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-B Activation-To identify the domains of PKK that are required for NF-B 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-B activation, whereas 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-B 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).
Human and mouse PKK contain a SXXXS 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-B when compared with the wild-type kinase (Fig. 3C). Similarly, replacement of S171, S173, and S177 for glutamic acid residues, which are associated with constitutive activation of serine-threonine kinases, did not enhance the ability of PKK to induce NF-B (Fig. 3C). Close inspection of zebrafish PKK revealed that the fish kinase lacks serines at positions 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-B is not regulated by phosphorylation of its putative activation loop.
PKK Is Involved in PMA/Ca 2ϩ -ionophore-induced NF-B Activation-PKK is known to interact with PKC␤I, 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 that links PKC activation to NF-B induction (17,18). Therefore, we next tested whether PKK regulates an NF-B signaling pathway mediated by Bimp1, Bcl10, and MALT1 in HEK293T cells that are known to express endogenous PKK (15). Treatment of HEK293T cells with PMA/Ca 2ϩ -ionophore induced NF-B 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 D143A did not block NF-B activation induced by Bimp1, Bcl10, oligomerized MALT1, TNF␣ (Fig. 4A), IL-1␤, or Nod1. 2 Additional control experiments shown in Fig. 4A revealed that activation of NF-B induced by PKK, Bimp1, Bcl10, activated MALT1, PMA/Ca 2ϩionophore, or TNF␣ could be inhibited by a dominant interfering form of IKK␤ but not by that of MyD88, an essential mediator of IL-1/Toll receptor signaling (32). Because PKK associates with PKC␤I (14), we tested if the PKK D143A mutant inhibits PMA-induced NF-B activation through a functional interaction with PKC␤I. Expression of PKC␤I reverted the effect of the PKK D143A mutant, whereas a kinase negative mutant of PKC␤I (K371M) and PKC⑀ did not (Fig. 4B). The mechanism by which PKC␤I reverts the dominant negative effect of the PKK mutant is unclear. A possible explanation is that overexpressed catalytically active PKC␤I competes out dominant negative PKK for cellular factor(s) necessary for function. The selective effect of PKC␤I is consistent with the observation that PKK interacts with PKC␤I (14) but not with 2 A. Muto, N. Inohara, and G. Nú ñ ez, unpublished results. PKC⑀. 2 In addition, activation of AP-1 induced by PMA/Ca 2ϩionophore was specifically inhibited by PKK dominant negative (Fig. 4C), suggesting that PKK also acts in a PMA-induced AP-1 signaling pathway activated by PKC␤I.

NF-B Activation Induced by PKK Requires IKK␣ and IKK␤
but Not IKK␥-NF-B activation by RICK and RIP is mediated by the IKK complex, a universal regulator that phosphorylates IB␣ resulting in degradation of IB␣ and nuclear transloca- tion of NF-B (2,8). To determine whether NF-B activation by PKK is also dependent on IKKs, PKK was co-expressed with the catalytic inactive forms of IKK␣ and IKK␤. NF-B activation induced by PKK as well as that induced by PMA/Ca 2ϩionophore, IL-1␤ and TNF␣, was inhibited by catalytic inactive IKK␣ and IKK␤ (Fig. 5A). In control experiments, PKK-mediated NF-B activation was not affected by dominant negative forms of Bimp1 or MyD88 (Fig. 5A). The ability of PKK to activate NF-B was also determined in MEFs lacking IKK␣ and IKK␤. Whereas PKK activated NF-B in WT fibroblasts, it was unable to induce NF-B in cells lacking IKK␤ or in cells lacking both the IKK␣ and IKK␤ proteins (Fig. 5B). These results suggest that NF-B activation induced by PKK requires catalytic IKKs. However, we found that purified PKK did not phosphorylate IKK␣ or IKK␤ in vitro, 2 suggesting that PKK does not function through direct phosphorylation and activation of the IKK complex.
Next we tested if NF-B activation by PKK requires IKK␥, a regulatory component of the IKK complex (18,(33)(34)(35). PKK was co-expressed with a truncated mutant of IKK␥ (residues 134 -419) that inhibits NF-B activation induced by RIP and RICK (8). Surprisingly, co-expression of the IKK␥ mutant did not inhibit PKK-mediated NF-B activation (Fig. 5A). To verify the latter result, we tested the ability of PKK to activate NF-B in parental Rat1 fibroblasts and IKK␥-deficient 5R cells, a Rat1 derivative cell line that is defective in IKK␥ (22). Expression of PKK induced NF-B activity not only in parental Rat1 cells but also in 5R cells (Fig. 5C). As controls, stimulation with TNF␣, IL-1␤, or LPS, or expression of Nod1 (all of which require IKK␥) induced NF-B 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-B activation. In contrast, the same region of RIP and RICK is essential for NF-B activation and mediates the interaction with IKK␥ (8,35). Thus, unlike in RICK and RIP, the IM region of PKK and IKK␥ are dispensable for NF-B activation.
Conversely, Fig. 5A demonstrated that a dominant negative form of Bimp1 had no effect on PKK-mediated NF-B activation. To determine whether PKK could act upstream of Bcl10, we tested the ability of PKK to induce NF-B in MEFs deficient in Bcl10 (18). Both PKK and Nod1 induced NF-B activation in both Bcl10 ϩ/and Bcl10 Ϫ/Ϫ MEFs (Fig. 5D). In control experiments shown in Fig. 5D, Bcl10 was required for NF-B activation induced by Bimp1, a protein that acts upstream of Bcl10 to activate NF-B (17). Together with the results shown in Fig.  4A, these results suggest that PKK functions in a PKC signaling pathway of NF-B activation that is independent from Bcl10.
We provide evidence that PKK is an NF-B-activating kinase. The activity of PKK is consistent with its homology to RICK and RIP, two serine-threonine kinases that activate NF-B. Another member of the family, RIP3, has been shown to activate or inhibit NF-B activation, probably depending on the cellular context (36 -38). Thus, PKK appears to represent the fourth member of the RIP/RICK family of NF-B activating kinases. Unlike RIP and RICK (8), the catalytic activity of PKK was required for NF-B activation. These results indicate that PKK is unique among the RICK-related kinases and suggest that the mechanism by which PKK activates NF-B is distinct from that utilized by RIP and RICK. We hypothesize that PKK activates NF-B through the phosphorylation of protein target(s).
PKK was originally identified as a binding partner of PKC␤I, and it was suggested to function in a PKC signaling pathway (14). Consistent with this proposed model, we show that a dominant negative mutant of PKK inhibits PMA/Ca 2ϩ -ionophore-mediated NF-B activation, an effect that was reverted by expression of PKC␤I. Several studies have implicated PKC␤I in the activation of NF-B in cells derived from several tissues including the heart and kidney (39 -41), which reportedly exhibit high expression of PKK (14,15). We hypothesize that PKK functions in these tissues to regulate a Bcl10-independent PKC␤I-mediated signaling pathway of NF-B activation. Bcl10 appears to regulate PKC signaling pathways involved in antigen receptor stimulation and neurogenesis (18). The physiological upstream signals that activate PKK through PKC␤I remain to be elucidated. Additional studies are necessary to identify protein substrate(s) of PKK that may reveal the mechanism by which this RICK-related kinase activates NF-B.