Human embryonic kidney cells (HEK293T) and RAW264.7 cell lines were obtained from ATCC. All cells were cultured at 37°C under 5% CO₂ in Dulbecco's modified Eagle's medium (DMEM; Hyclone) supplemented with 10% FBS (GIBCO, Life Technologies), 100 units/mL penicillin, and 100 μg/mL streptomycin. According to the manufacturer's recommendation, plasmids were transfected into cells by using Longtrans (Ucallm).

Plasmids encoding β-TrCP and HA-β-TrCP were gifts from Dr. Serge Y. Fuchs (University of Pennsylvania). Expression plasmids HA- or GST-tagged PKD1, PKD2 and PKD3 were generated using PCR amplified from a HEK293T cDNA library. HA-Ub, HA-Ub-R48K, and HA-Ub-R63K were described previously (17). Myc-His-PKD1(1–444) and Myc-His-PKD1(445–912) were generated using PCR amplified from HA-PKD1. Myc-PKD1-Δ(172–312) and Myc-PKD1-Δ(311–444) were generated using PCR amplified from Myc-His-PKD1(1–444). Shβ-TrCP were purchased from GENECHEM (Shanghai, China). All mutations were generated by QuickChange site-Directed Mutagenesis Kit (Stratagene). All the plasmids were confirmed by DNA sequencing. Cycloheximide, MG132 and LPS were purchased from Sigma.

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 5′-CTCCAATAGTGCCGTTTCCG-3′; β-actin, 5′-ACCAACTGGGACGACATGGAGAAA-3′ and 5′-ATAGCACAGCCTGGATAGCAACG-3′. The relative expression of the target genes mRNA was normalized to β-actin mRNA. The results were analyzed from three independent experiments and are shown as the average mean ± SD.

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). The following antibodies specific for GST (1:2000, HaiGene, M0301), PKD1 (1:500, Santa Cruz, sc-639), β-TrCP (1:1000, Cell signaling, #4394), HA (1:2000, abcam, ab9110), K63Ub (1:500, Cell signaling, D7A11), tubulin (1:5000, Proteintech, 66031-1-Ig), β-actin (1:5000, Proteintech, 66009-1-Ig), Myc (1:1000, abmart, #284566), IκBα (1:2000, Cell signaling, #4814), TRAF6 (1:500, Santa Cruz, sc-8409), Ubiquitin (1:1000, Santa Cruz, sc-8017), IKK (1:1000, Cell signaling, #2682), and pS176/18-IKK (1:1000, Cell signaling, #2697) have been used.

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.

RAW264.7 cells were treated with LPS (2.5 μg/mL) for different times. Cells were then harvested on ice using lysis buffer containing 150 mM NaCl, 20 mM Tris-HCl (pH 7.4), 1% Nonidet P-40, 0.5 mM EDTA, PMSF (50 μg/ml), and protease inhibitor mixtures (Sigma) and subjected to analysis by immunoblotting or immunoprecipitation.

Small guide RNAs targeting mus PKD1: 1^# (5′-GCCCAGTCCGCTGCTGCCCG-3') and 2^# (5′-GCCGCACTGGTCCCAGGGTC-3′) were cloned into the lentiCRISPRv2 vector and were transfected into HEK293T cells. Forty eight hours after transfection, the supernatant was used to infect RAW264.7 cells. Then the RAW264.7 cells were cultured under puromycin selection until further experiments.

To analyze ubiquitination of TRAF6 in LPS signaling, RAW264.7 cells were treated with LPS (2.5 μg/mL) for 0, 15, 30 min. Cells were harvested in lysis buffer containing 150 mM NaCl, 20 mM Tris-HCl (PH 7.4), 1% NonidetP-40, 0.5 mM EDTA, PMSF (50 μg/ml), N-ethylmaleimide (10 mM) and protease inhibitors mixtures (Sigma). Endogenous TRAF6 proteins were immunoprecipitated using a specific TRAF6 antibody and then subjected to ubiquitination analysis using a specific ubiquitin (Ub) antibody by western blotting.

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.

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+nS 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.

To explore the mechanisms of PKD1 downregulation mediated by β-TrCP, we firstly took advantage of a proteasomal inhibitor MG132. Our results showed that MG132 inhibited β-TrCP-mediated PKD1 downregulation (Figure 2A), suggesting that β-TrCP promotes PKD1 downregulation via the proteasome pathway. Thus, we next analyzed whether β-TrCP is capable of regulating ubiquitination levels of PKD1. We found that overexpression of β-TrCP obviously promoted ubiquitination of GST-PKD1, but not GST-PKD2 and GST-PKD3 (Figure 2B), suggesting that β-TrCP specifically targets PKD1 member of PKD family. Furthermore, knockdown of β-TrCP upregulated PKD1 protein levels (Figure 2C, left panel) and significantly downregulated ubiquitination levels of PKD1 (Figure 2C, right panel). Next, we tried to determine whether β-TrCP induces K48-linked or K63-linked ubiquitination of PKD1. To this end, Ub-R48K (all lysines in Ub are mutated to arginines except lysine 48 residue) and Ub-R63K (all lysines in Ub are mutated to arginines except lysine 63 residue) were used to analyze the types of PKD1 ubiquitination. Our data showed that overexpression of β-TrCP significantly increased K48-linked polyubiquitination of PKD1 (Figure 2D). However, β-TrCP did not obviously affect K63-linked ubiquitination of PKD1 (Figure 2E).

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. Cells transfected with control vector (shCON) or shβ-TrCP were treated with protein synthesis inhibitor CHX. We found that knockdown of β-TrCP in cells substantially inhibited PKD1 degradation (Figure 2F), suggesting that β-TrCP knockdown enhances PKD1 protein stability. Furthermore, overexpression of β-TrCP accelerated the degradation of endogenous PKD1 protein (Figure 2G). In conjunction with the above observation showing that β-TrCP induces PKD1 K48-linked polyubiquitination, we think that β-TrCP can specifically promote PKD1 ubiquitination and degradation.

Previous studies have demonstrated that the conserved DSG(X)_2+nS 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+nS 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+nS 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+nS motif of PKD1 did not obviously affect the binding of β-TrCP to PKD1-S₃₈₀A (Figure 3B), indicating that β-TrCP interacts with PKD1 in an non-canonical manner.

We noticed that recent studies have reported several “atypical” substrates of β-TrCP, including STAT1 (19), Weel (20), GHR (21), ELAVL1/huR, CDC25B, and TP53 (22), which do not contain the classic DSG(X)_2+nS motif. Instead, most of these atypical substrates have the XSG(X)_2+nS motif. Therefore, we analyzed the potential XSG(X)_2+nS motif in various species of PKD1 including Homo Sapiens, Mus Musculus, Rattus norvegicus, Desmodus rotundus, Pongoabelii, Chrysemys picta bellii (Figure 3C). To determine which XSG(X)_2+nS motif of PKD1 is pivotal for binding with β-TrCP, we firstly constructed two PKD1 deletion mutants, PKD1 (1–444) and PKD1 (445–912) (Figure 3D). The ability of β-TrCP to downregulate two PKD1 mutants was analyzed. Our results showed that PKD1 (1–444), but not PKD1 (445–912), can be downregulated by β-TrCP overexpression (Figure 3E), indicating that the potential XSG(X)_2+nS motif locates in PKD1 (1–444). PKD1 (1–444) possesses three conserved XSG(X)_2+nS motifs (Figure 3D). Thus, we constructed another two PKD1 mutants, PKD1-Δ(172–312) and PKD1-Δ(311–444) (Figure 3D). We found that deletion of the 311–444 motif, but not the 172–312 motif, abolished β-TrCP-mediated downregulation of PKD1 (Figure 3F). These results suggested that the key XSG(X)_2+nS motif locates in PKD1-Δ(311–444), which has two XSG(X)_2+nS motifs, NS₃₅₄G and DS₃₈₀G. Our previous data have demonstrated that the DS₃₈₀G motif of PKD1 is not essential for interaction between β-TrCP and PKD1 (Figure 3B). Therefore, we made a new PKD mutant, PKD1-S₃₅₄A. Our data showed that mutation of serine 354 of PKD1 abolished the binding of β-TrCP to PKD1 (Figure 3G). Consistently, β-TrCP cannot downregulate PKD1-S₃₅₄A protein, as compared with PKD1-wild type (WT) (Figure 3H). Taken together, we demonstrate that the NS₃₅₄G motif of PKD1 is required for β-TrCP binding and PKD1degradation.

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.

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.

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.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.