β-TrCP Restricts Lipopolysaccharide (LPS)-Induced Activation of TRAF6-IKK Pathway Upstream of IκBα Signaling

β transducin repeat-containing protein (β-TrCP) is a Skp1-Cul1-F-box ubiquitin ligase, which plays important roles in controlling numerous signaling pathways. Notably, β-TrCP induces ubiquitination and degradation of inhibitor of NF-κB (IκBα), thus triggering activation of NF-κB signaling. Here, we unexpectedly find that β-TrCP restricts TRAF6-IKK signaling upstream of IκBα induced by lipopolysaccharide (LPS). In LPS-Toll-like receptor 4 (TLR4) pathway, protein kinase D1 (PKD1) is essential for activation of TRAF6-IKK-IκBα signaling including TRAF6 ubiquitination, IKK phosphorylation and subsequent IκBα degradation. We found that LPS promotes binding of β-TrCP to PKD1, and results in downregulation of PKD1 and recovery of IκBα protein level. Knockdown of β-TrCP blocks LPS-induced downregulation of PKD1. Supplement of enough PKD1 in cells inhibits recovery of IκBα protein levels during LPS stimulation. Furthermore, we demonstrate that β-TrCP inhibits LPS-induced TRAF6 ubiquitination and IKK phosphorylation. Taken together, our findings identify β-TrCP as an important negative regulator for upstream signaling of IκBα in LPS pathway, and therefore renew the understanding of the roles of β-TrCP in regulating TLRs inflammatory signaling.


INTRODUCTION
The protein kinase D (PKD) family is a group of serine/threonine protein kinases that are comprised of three members (PKD1/PKCµ, PKD2, and PKD3/PKCν) (1). PKD family members have been shown to be essential for various cellular signaling pathways including activation of NF-κB and MAPKs, cell motility and adhesion, gene expression, and ROS (Reactive oxygen species) generation (2)(3)(4)(5). PKD family is also involved in regulation of immune and inflammatory responses. It has been reported that PKD2 promotes phosphorylation and degradation of interferon alpha receptor 1 (IFNAR1), thus restricting interferon-mediated antiviral immunity (6). In addition, increasing evidence demonstrates that PKD family plays important roles in regulating Toll-like receptor (TLR)-mediated inflammatory signaling (7,8). Recent studies found that a PKC/PKD inhibitor is able to inhibit lipopolysaccharide (LPS)-TLR4-mediated p38 activation and TNF-α secretion (9). PKD1, but not PKD2 and PKD3, is essential for MyD88-dependent ubiquitination of TNF-α receptor-associated factor 6 (TRAF6), phosphorylation of IKK, and subsequent activation of NF-κB and MAPKs in TLRs signaling (7). However, in spite of many important effects of PKD family on numerous signaling pathways, how cellular PKD protein levels are regulated remains largely unexplored.
In the present study, we unexpectedly found that β-TrCP negatively regulates TRAF6-IKK signaling upstream of IκBα induced by LPS. LPS is a kind of endotoxin secreted by Gramnegative bacteria. As an activation model of inflammation, LPS is widely used for studying the regulation mechanisms of inflammatory signaling pathways. LPS activates TLR4 signaling by recruiting MyD88 to form the TLR4/MyD88/IRAK receptor complex, which promotes PKD1 activation and subsequent TRAF6 ubiquitination (8). Ubiquitinated TRAF6 then leads to phosphorylation and activation of TAK1 and IKK. Activated IKK induces IκB phosphorylation, which recruits β-TrCP to target IκB for ubiquitination and degradation. Finally, NF-κB is released from the IκB-NF-κB complex and translocates into the nucleus in an activated status.
Here, we revealed that β-TrCP can also target PKD1, a signaling molecule upstream of IκBα in LPS signaling, and promotes PKD1 ubiquitination and degradation. We found that LPS promoted interaction between β-TrCP and PKD1, and results in downregulation of PKD1 and recovery of IκBα protein level. Knockdown of β-TrCP blocks LPS-induced downregulation of PKD1. Consistently, β-TrCP attenuates LPS-induced TRAF6 ubiquitination and IKK phosphorylation. Collectively, our findings uncover that β-TrCP acts as an important negative regulator for upstream signaling of IκBα in LPS signaling.

RNA Isolation and Real-Time PCR
Total RNAs were extracted from HEK293T cells using TRIzol reagent (Invitrogen). The cDNA was produced by reverse transcription using oligo (dT) and analyzed by quantitative real-time PCR (qPCR) with PKD1, β-actin primers using SYBR Green Supermix (Bio-Rad Laboratories). The primer sequences were as follows: PKD1, 5 ′ -GCCAACAGAACCATCAGTCC-3' and The relative expression of the target genes mRNA was normalized to βactin mRNA. The results were analyzed from three independent experiments and are shown as the average mean ± SD.

Immunoblotting and Immunoprecipitation
Immunoblotting and immunoprecipitation were performed as described previously (17,18). Briefly, all cells were harvested on ice using lysis buffer. N-ethylmaleimide (10 mM) was added to the lysis buffer when protein ubiquitination was detected. Immunoprecipitation was performed using specific antibodies overnight on a rotor at 4 • C. Equivalent quantity of proteins were subjected to SDS-PAGE followed by transferring to PVDF membranes (Millipore). Then membranes were blocked with 5% non-fat milk or 5% BSA, and incubated with the corresponding primary antibodies, followed by the respective HRP-conjugated Goat anti-mouse or Goat anti-rabbit (Bioworld) secondary antibodies. Immunoreactive bands were depicted using SuperSignal West Dura Extended kits (Thermo Scientific).

Cycloheximide Chase Assay
The half-life of proteins was determined by cycloheximide (CHX) chase assay. HEK293T cells were transfected with GST-PKD1, with or without shβ-TrCP in 6-well culture plates. Seventy two hours after transfection, cells were treated with DMSO or CHX (50 mg/mL) for 0, 6, 12 h. Furthermore, cells were harvested on ice and subjected to analysis by western blotting.

Statistical Analysis
Comparison between different groups was analyzed by using a two-tailed Student's T-test. All differences were considered that p < 0.05 represent statistically significant.

β-TrCP Negatively Regulates Protein Levels of PKD1, but Not PKD2 and PKD3
To explore the ubiquitin-mediated regulation of PKD family, we firstly analyzed the amino acid sequences of PKD family. We noted that PKD family members possess a putative DSG(X) 2+n S motif, which has been demonstrated to be a characteristic of substrates for ubiquitin E3 ligase β-TrCP. Thus, we hypothesized that PKD family members could be potential substrates of β-TrCP. To determine the possible effect of β-TrCP on PKD family, we firstly analyzed the effects of β-TrCP overexpression on exogenous PKD family members. The results showed that overexpression of β-TrCP significantly downregulated protein levels of exogenous GST-PKD1 ( Figure 1A), but not GST-PKD2 ( Figure 1B) and GST-PKD3 ( Figure 1C). To confirm the effect of β-TrCP on endogenous PKD1, we transfected cells with increasing amount of β-TrCP. We found that overexpression of β-TrCP gradually lowered protein levels of endogenous PKD1 ( Figure 1D). Furthermore, endogenous β-TrCP was knocked down by shRNAs against β-TrCP (shβ-TrCP). We found that knockdown of β-TrCP upregulated protein levels of endogenous PKD1 in cells ( Figure 1E). In addition, we found that overexpression of β-TrCP did not decrease PKD1 mRNA levels ( Figure 1F), suggesting that β-TrCP regulates PKD1 at protein level. Taken together, our results suggest that β-TrCP is a negative regulator of cellular PKD1 protein.
Given that β-TrCP induces K48-linked ubiquitination of PKD1, we speculated that the protein stability of PKD1 could be regulated by β-TrCP. To address this hypothesis, we carried out cycloheximide (CHX) pulse chase analysis of PKD1 protein.

NS 354 G Motif of PKD1 Is Required for β-TrCP Binding and Degradation of PKD1
Previous studies have demonstrated that the conserved DSG(X) 2+n S motif is an important characteristic of protein substrates for β-TrCP binding and subsequent protein substrates degradation. And mutation of the first serine (S) in this DSG(X) 2+n S motif of a protein substrate will result in its inability to bind with β-TrCP. By analyzing the amino acid sequences of PKD1, we found that PKD1 does possess this DSG(X) 2+n S motif. Therefore, we firstly determined the interaction between β-TrCP and PKD1. By co-immunoprecipitation assays, we found that β-TrCP is able to interact with PKD1 ( Figure 3A). To our surprise, mutation of this serine 380 in DSG(X) 2+n S motif of PKD1 did not obviously affect the binding of β-TrCP to PKD1-S 380 A (Figure 3B), indicating that β-TrCP interacts with PKD1 in an non-canonical manner.

LPS Promotes Binding of β-TrCP to PKD1 and Downregulates PKD1 Protein Levels
Our above findings reveal that β-TrCP interacts with PKD1, and promotes PKD1 ubiquitination and degradation. Interestingly, we noticed that both β-TrCP and PKD1 are involved in LPS induced inflammatory signaling pathway ( Figure 4A). Thus, we sought to determine whether the regulation of β-TrCP on PKD1 occurs in LPS inflammatory signaling. To this end, the interaction between β-TrCP and PKD1 in LPS signaling was analyzed. We found that LPS significantly promoted binding of β-TrCP to PKD1 (Figure 4B), and simultaneously upregulated PKD1 ubiquitination (Figure 4B). These data suggest that β-TrCP could interact with and regulate PKD1 during LPS stimulation.
To further study the possible regulation of LPS stimulation on PKD1, we analyzed protein levels of PKD1 in RAW264.7 cells under the conditions of LPS treatment for various times. Using a specific antibody against PKD1, we found that PKD1 protein levels were significantly reduced at 90 min and 180 min stimulation by LPS, whereas IκBα levels gradually increased simultaneously ( Figure 4C). Importantly, when β-TrCP was knocked down, LPS-induced downregulation of PKD1 was markedly inhibited (Figure 4D), suggesting that β-TrCP is responsible for PKD1 downregulation in LPS signaling. Collectively, we demonstrate that LPS stimulation can promote binding of β-TrCP to PKD1, and results in PKD1 downregulation.

Supplement of PKD1 in Cells Inhibits Recovery of IκBα Protein Levels in LPS Signaling
Interestingly, from the above results we observed a negative correlation between PKD1 and IκBα protein levels in LPS signaling ( Figure 4C). It is actually not difficult to understand. It has been clearly demonstrated that LPS stimulation can activate TLR4 signaling and lead to IκBα protein degradation very rapidly. In conjunction with our findings here showing that the upstream signaling molecule PKD1 can be downregulated after LPS stimulation, we speculate that the LPS-TLR4 signaling could be inhibited during the PKD1 downregulation stage, which could result in decrease of IκBα phosphorylation and recovery of IκBα protein levels. That is, PKD1 downregulation during LPS stimulation could contribute to recovery of IκBα protein levels. Next, we try to provide more evidence for this speculation. Given that manipulation of β-TrCP could directly affect IκBα, which impedes the observation of the dynamic changes of IκBα protein levels during LPS stimulation, we took advantage of PKD1 overexpression to supplement PKD1 levels in cells. Firstly, RAW264.7 cells were transfected with increasing dose of PKD1. Cells were then treated with LPS for 90 min. Our data showed that PKD1 overexpression continuously lowered IκBα protein levels in a dose-dependent manner ( Figure 5A). Conversely, knockout of PKD1 in RAW264.7 cells by CRISPR-Cas9 genome editing substantially upregulated IκBα protein levels in LPS signaling ( Figure 5B). Furthermore, we analyzed  the dynamic changes of IκBα protein levels in LPS signaling. The result showed that during LPS stimulation, IκBα protein levels significantly decreased at 30 min time point. After that, IκBα protein levels were gradually recovered ( Figure 5C, left panel). When cells were supplemented with PKD1, the recovery of IκBα protein levels was dramatically delayed (Figure 5C, right panel). Collectively, we believe that PKD1 downregulation could contribute to recovery of IκBα protein levels in LPS signaling.

β-TrCP Negatively Regulates LPS-Induced TRAF6 Ubiquitination and IKK Activation
Given that we have demonstrated that β-TrCP promotes PKD1 downregulation in LPS signaling, we further study whether β-TrCP can regulate the signaling pathway downstream of PKD1 and upstream of IκBα. In LPS signaling, PKD1 activation leads to TRAF6 polyubiquitination and subsequent IKK phosphorylation and activation. Therefore, we analyzed the roles of β-TrCP in regulating TRAF6 ubiquitination and IKK phosphorylation in LPS signaling. Our data showed that β-TrCP overexpression significantly inhibited LPS-stimulated ubiquitination of TRAF6 ( Figure 6A). Conversely, knockdown of β-TrCP remarkably promoted TRAF6 ubiquitination induced by LPS (Figure 6B). Furthermore, we found that overexpression of β-TrCP reduced K63-linked polyubiquitination of TRAF6 ( Figure 6C), but had no obvious effect on TRAF6 protein levels ( Figure 6D), suggesting that β-TrCP inhibits TRAF6 signaling activation. Next, we studied whether β-TrCP could regulate IKK phosphorylation. Using a specific antibody against activated IKK at Ser176/180 site, we found that β-TrCP obviously inhibited LPS-stimulated IKK phosphorylation ( Figure 6E). Taken together, our findings demonstrate that β-TrCP negatively regulates LPS-induced TRAF6 K63-linked ubiquitination and IKK activation.

DISCUSSION
PKD1, as a ubiquitous serine-threonine protein kinase, plays crucial roles in multiple biological processes, including cell growth, adhesion, motility, and angiogenesis (2)(3)(4)(5). Stable PKD1 levels in cells are extremely important for normal physiology functions of cells. Dysregulated PKD1 expression could result in the pathogenesis of some cancers. PKD1 could be activated by various stimuli including growth factor (23), oxidative stress (24), and apoptotic signaling (25). Thus far, the regulation of PKD1 expression remains largely unexplored. It has been reported that PKD1 expression is gradually reduced during breast cancer progression, which could be directly associated with hypermethylation of PKD1 promoter (26). Recent studies demonstrated that there is a correlation between the expression of PKD1 and ERα in breast cancer Forty eight hours after transfection, cells were stimulated with LPS (2.5 µg/mL) for 0, 15, 30 min. Endogenous TRAF6 were immunoprecipitated, and K63-linked polyubiquitination and protein levels of TRAF6 were analyzed using indicated antibody. (D) Total protein levels of TRAF6 were analyzed using indicated antibodies. (E) RAW264.7 cells were transfected with or without β-TrCP. Forty eight hours after transfection, cells were stimulated with LPS (2.5 µg/mL) for 0, 15, 30 min. The levels of p-IKK, IKK, β-TrCP, and Actin were analyzed by immunoblotting as indicated. (F) Hypothetical model for β-TrCP-mediated regulation of LPS inflammatory signaling pathway. LPS stimulation activates TLR4-PKD1-TRAF6-IKK signaling, which rapidly induces IκBα phosphorylation and subsequent IκBα ubiquitination and degradation mediated by β-TrCP. Finally, NF-κB inflammatory signaling was activated. During LPS stimulation, β-TrCP also interacts with PKD1, which results in PKD1 ubiquitination and degradation, and leads to inhibition of LPS-TLR4-PKD1-TRAF6-IKK signaling. Together, β-TrCP plays both promotion and brake roles in LPS-induced inflammatory signaling.
cells (27). However, ubiquitination-mediated regulation of PKD1 remains unknown so far. In this study, we for the first time reveal that ubiquitin E3 ligase β-TrCP regulates PKD1 protein levels in cells. We clearly demonstrated that in LPS signaling β-TrCP can interact with PKD1, which leads to PKD1 downregulation. Furthermore, β-TrCP-mediated downregulation of PKD1 restricts LPS-induced TRAF6 K63linked polyubiquitination and IKK phosphorylation. Thus, our findings uncover the novel regulation mediated by the β-TrCP-PKD1 axis in LPS inflammatory signaling. In addition, it has been reported that PKD1 can be activated by TLRmediated MyD88 signaling pathway. Thus, it could be interesting to observe the effects of β-TrCP on PKD1 levels in different TLR ligands-activated signaling pathways in the future.
It has been well-characterized that β-TrCP's substrates contain a consensus DSG(X) 2+n S degron. The serine residues in this degron could be phosphorylated by certain protein kinases, which results in β-TrCP recognition and subsequent ubiquitination of the substrates (28)(29)(30). In this study, we found that PKD1 does possess a potential DSG(X) 2+n S motif. However, this DSG(X) 2+n S motif of PKD1 is not important for β-TrCP binding and PKD1 degradation. Through analyzing all conserved XSG(X) 2+n S motif in various species of PKD1, we finally found the NSG(X) 2+n S motif of PKD1 is essential for β-TrCP binding and subsequent degradation of PKD1. As a matter of fact, some studies have reported several "atypical" substrates of β-TrCP, including STAT1 (19), Weel (20), and GHR (21), which do not have the classic DSG(X) 2+n S motif. Here, our study uncovers a new "atypical" degron in PKD1 for β-TrCP-mediated degradation.
Prevailing thought holds that β-TrCP is a positive regulator for TLRs-NF-κB inflammatory signaling, because β-TrCP induces IκBα degradation and NF-κB release. In this study, we demonstrate that β-TrCP could be a negative regulator for upstream signaling of TLRs-NF-κB pathway. LPS stimulation activates TLR4-PKD1-TRAF6-IKK-IκBα signaling, which leads to β-TrCP-mediated ubiquitination and degradation of IκBα rapidly. Unexpectedly, we find that during LPS stimulation β-TrCP also binds to PKD1, which results in PKD1 ubiquitination and degradation. PKD1 downregulation inhibits further activation of LPS-PKD1-TRAF6-IKK signaling ( Figure 6F). As observed in this study, β-TrCP-mediated PKD1 downregulation inhibits TRAF6 K63-linked polyubiquitination and subsequent IKK phosphorylation. Also, we observed that PKD1 protein levels are negatively correlated with IκBα levels in LPS signaling, and supplement of PKD1 in cells significantly delays recovery of IκBα protein levels. Taken together, we think that β-TrCP plays both promotion and brake roles in LPS-induced inflammatory signaling. The dual effects of β-TrCP may provide the balance for LPS inflammatory signaling. Based on the dual effects of β-TrCP, we focus on studying LPS-IKK upstream signaling, rather than analyzing cytokines production downstream of IκBα signaling.
In summary, we demonstrate that β-TrCP specifically targets PKD1 for K48 ubiquitination and degradation in an "atypical" binding manner. Furthermore, we uncover that during LPS stimulation β-TrCP can interact with PKD1 and promotes PKD1 downregulation, which in turn restricts TRAF6-IKK signaling upstream of IκBα in LPS inflammatory pathway. Our findings reveal that β-TrCP is an important negative regulator for upstream signaling of IκBα in LPS signaling, and therefore renew our understanding of the roles of β-TrCP in TLRs inflammatory signaling.