S-Y Liao1, C-W Chiang1,2, C-H Hsu3, Y-T Chen4, J Jen4, H-F Juan5, W-W Lai6and Y-C Wang1,4
Overexpression of Cys2His2 zinc-finger 322A (ZNF322A) oncogenic transcription factor is associated with lung tumorigenesis.
However, the mechanism of ZNF322A overexpression remains poorly understood. Here, we discover that protein stability of ZNF322A is regulated by coordinated phosphorylation and ubiquitination through the CK1δ/GSK3β/FBXW7α axis. CK1δ and GSK3β kinases sequentially phosphorylate ZNF322A at serine-396 and then serine-391. Moreover, the doubly phosphorylated ZNF322A protein creates a destruction motif for the ubiquitin ligase FBXW7α leading to ZNF322A protein destruction. Overexpression of FBXW7α induces ZNF322A protein degradation, thereby blocks ZNF322A transcription activity and suppresses ZNF322A-induced tumor growth and metastasis in vitro and in vivo. Clinically, overexpression of ZNF322A correlates with low FBXW7α or defective CK1δ/GSK3β-mediated phosphorylation in lung cancer patients. Multivariate Cox regression analysis indicates that patients with ZNF322A high/FBXW7 low expression profile can be used as an independent factor to predict the clinical outcome in lung cancer patients. Our results reveal a new mechanism of ZNF322A oncoprotein destruction regulated by the CK1δ/GSK3β/FBXW7α axis.
Deregulation of this signaling axis results in ZNF322A overexpression and promotes cancer progression.
Oncogene (2017)36, 5722–5733; doi:10.1038/onc.2017.168; published online 5 June 2017
INTRODUCTION
Cys2His2 zinc-finger (ZNF) proteins are the largest class of DNA-binding protein in eukaryotic transcription factors.1 Malfunction of ZNF transcription factors is involved in tumorigenesis and the regulatory mechanism varies among different ZNF proteins. For instance, ZNF24 represses transcription of vascular endothelial growth factor to inhibit angiogenesis and tumor growth of breast cancer.2 ZNF331 acts as a tumor suppressor and downregulates the gene expression associated with cell proliferation and invasion in gastric cancer.3In contrast, other ZNF proteins are defined as oncogenes, like ZNF217 and zinc-finger 322A (ZNF322A). Overexpression of ZNF217 promotes epithelial–mesenchymal transition (EMT) and chemoresistance in breast cancer.4,5Using lung cancer cell and xenograft models, we demonstrate that ZNF322A promotes tumor growth through positive transcriptional regulation of cyclin D1 and negative regulation of p53 expression, whereas upregulation of adducin 1 (ADD1) expression is required for ZNF322A-induced tumor metastases. Clinically, ZNF322A overexpression is found in Asian and Caucasian lung cancer patients and correlates with poor prognosis.6However, the mechanism of ZNF322A overexpression remains unclear.
It has been reported that the expression of many ZNF transcription factors are regulated by phosphorylation and ubiquitin–proteasome degradation. Glycogen synthase kinase 3 beta (GSK3β), the serine (Ser)/threonine (Thr) kinase, has been reported to regulate protein stability of many ZNF transcription factors like Slug, KLF2 and Snail through
phosphorylation-dependent proteasome degradation.7–9 Some protein substrates of GSK3β require priming phosphorylation on Ser/Thr amino residue located downstream of the GSK3β phosphorylation site. CK1 is known to primely phosphorylate substrates includingβ-catenin, Snail and pVHL, which is required for subsequent GSK3β-mediated phosphorylation.10–12 For example, phosphorylation of Snail at Ser-104 and Ser-107 by casein kinase 1ε (CK1ε) is required for subsequent GSK3β-mediated phosphorylation targeting it for proteasomal degradation.10 Of note, the F-box and WD40 repeat domain-containing 7 (FBXW7), a SCF (complex of SKP1, CUL1 and F-box protein) type E3 ubiquitin ligase, has been reported to involve in GSK3β-mediated protein degradation. The FBXW7 consensus-binding motif is called Cdc4 phosphodegron (CPD) (Ser/Thr)-PXX-(S/T/E), its Ser/Thr residues can be phosphorylated by GSK3β or other kinases and further recognized by FBXW7 E3 ubiquitin ligase.13,14FBXW7 regulates cell cycle progression and cell survival by promoting degradation of many oncogenic transcription factors such as c-Jun, MCL1, p100 and KLF5.15–18 These studies indicated that control of protein stability of transcription factors is associated with cancer progression.
However, whether ZNF322A can be regulated by phosphorylation and ubiquitin–proteasome degradation remains elusive.
Here we demonstrate that ZNF322A is a short half-life protein regulated by coordinated phosphorylation and ubiquitination through CK1δ/GSK3β/FBXW7α signaling axis in normal physiological condition. Deregulation of this signaling axis results in ZNF322A protein overexpression and contributes to lung tumorigenesis.
1Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan, Taiwan;2Institute of Molecular Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan;3Department of Agricultural Chemistry, National Taiwan University, Taipei, Taiwan;4Department of Pharmacology, College of Medicine, National Cheng Kung University, Tainan, Taiwan;5Department of Life Science, Institute of Molecular and Cellular Biology, Graduate Institute of Biomedical Electronics and Bioinformatics, National Taiwan University, Taipei, Taiwan and6Department of Surgery, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan, Taiwan. Correspondence: Professor Y-C Wang, Department of Pharmacology and Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, No.1, University Road, Tainan 70101, Taiwan.
E-mail: [email protected]
Oncogene (2017)36, 5722–5733
© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved 0950-9232/17 www.nature.com/onc
RESULTS
ZNF322A is a short half-life protein regulated by ubiquitin– proteasome system
Many transcription factors are short-lived and regulated by the ubiquitin–proteasome system. Therefore, we used cycloheximide (CHX) chase assay to analyze the protein half-life of ZNF322A in human lung cell lines. Endogenous ZNF322A protein was found to have a short half-life of about 15 min (Supplementary Figure S1A) and ectopically expressed HA-tagged ZNF322A protein was similarly rapidly degraded with a half-life of 15 min in cancer cells and even as short as 6 min in BEAS-2B bronchial epithelial cells (Supplementary Figures S1B–D). ZNF322A protein level increased upon treatment with the proteasome inhibitor MG132 (Supplementary Figures S1E–I), suggesting that ZNF322A protein is regulated by the ubiquitin–proteasome system.
Phosphorylation of ZNF322A by CK1δ triggers the subsequent phosphorylation by GSK3β
Protein phosphorylation is often required for ubiquitination/protea-some-dependent degradation. Our liquid chromatography-tandem mass spectrometry (LC-MS/MS) experiment identified several ZNF322A phosphorylation sites in vivo including Ser-224 and Ser-391 with high LC-MS/MS experimental confidence, and Ser-83, Tyr-95, Ser-396 and Ser-399 with low LC-MS/MS experimental confidence. We focused on Ser-391 phosphorylation site with high LC-MS/MS experimental confidence (Supplementary Figure S1J) because Ser-391 was a putative GSK3β phosphorylation site predicted by several phosphorylation software. Priming phosphor-ylation of protein substrates by other kinases such as CK1 are required for subsequent GSK3β-mediated phosphorylation.10–12We observed that the putative CK1 phosphorylation sites Ser-396 and Ser-399 were close to Ser-391 site (Figure 1a, upper panel) and a highly conserved region between amino acids 390 and 402 was identified by sequence analysis across multiple species (Figure 1a, lower panel). Our pilot studies in cells showed that overexpression of CK1δ, but not CK1α or CK1ε, reduced the protein expression level of ZNF322A (Supplementary Figures S1K and L). Indeed, in vitro kinase assay showed that CK1δ phosphorylated bacterial expressed GST-tagged ZNF322A recombinant protein in a dose-dependent manner, and the phosphorylation signal was decreased upon treatment with CK1δ inhibitor IC261 (Figure 1b). In addition, the phosphorylation signal of phosphorylation-defective mutant of S396A (S396A-ZNF322A) or S399A (S399A-ZNF322A) was reduced compared with wild-type ZNF322A (WT-ZNF322A) (Figure 1c and Supplementary Figure S1M). However, CHX chase data showed that S396A-ZNF322A mutant maintained a longer protein half-life, whereas the half-life of S399A mutant was similar to that of WT-ZNF322A in cell-based experiments (Supplementary Figure S1N).
These data suggested that Ser-396 was the main phosphorylation site of CK1δ in regulating ZNF322A protein stability.
Next, we examined whether ZNF322A phosphorylation by CK1δ may create the priming phosphorylation site, which could facilitate GSK3β-mediated phosphorylation. Indeed, the results of the in vitro kinase assay showed that CK1δ-mediated phosphor-ylation further enhanced ZNF322A phosphorphosphor-ylation by GSK3β (lanes 5 and 6 vs lanes 2 and 3, Figure 1d), and the phosphorylation signal was attenuated upon treatment of GSK3β inhibitor SB216763 (lanes 8 and 9 vs lanes 5 and 6, Figure 1d).
In addition, S391A phosphorylation-defective mutant (391A-ZNF322A) obviously blocked ZNF322A phosphorylation by GSK3β compared with WT-ZNF322A (lanes 4 vs 3; lanes 8 vs 7, Figure 1e), indicating that Ser-391 is the phosphorylation site of GSK3β. Notably, CK1δ phosphorylation-defective mutant S396A-ZNF322A protein greatly decreased GSK3β-mediated phosphorylation level (lanes 4 vs 3, Figure 1f) and attenuated CK1δ/GSK3β-mediated phosphorylation level (lanes 8 vs 7, Figure 1f). Together, these data suggested that CK1δ-mediated
ZNF322A phosphorylation at Ser-396 is a prerequisite for effective GSK3β-mediated phosphorylation at Ser-391 of ZNF322A.
CK1δ and GSK3β promote ZNF322A protein degradation Given that ZNF322A is phosphorylated in vitro by CK1 and GSK3β, both of which have been reported to regulate protein stability through phosphorylation-dependent proteolysis, we next determined if ZNF322A could interact with CK1δ and GSK3β in cells. The immunoprecipitation (IP) results showed that ZNF322A interacted with CK1δ and GSK3β (Supplementary Figures S2A and B). Of note, co-IP data showed that CK1δ, GSK3β and ZNF322A existed in the same protein complex (Figure 2a). As expected, overexpression of CK1δ or GSK3β reduced ZNF322A protein level in both H1299 and H460 cells, this decrease in ZNF322A protein was blocked by MG132 treatment (Figures 2b and c;
Supplementary Figures S2C and D). Similarly, constitutively active GSK3β (GSK3β-CA), but not the kinase-dead GSK3β (GSK3β-KD), reduced the ZNF322A protein level (Supplementary Figure S2E).
Conversely, ZNF322A protein expression level was increased in cells treated with CK1 inhibitors IC261 and D4476 or GSK3β inhibitor GSK3β XI (Figures 2d and e; Supplementary Figures S2F and G). In addition, depletion of CK1δ and GSK3β by short hairpin RNAs increased ZNF322A protein level (Supplementary Figure S2H), but did not affect ZNF322A mRNA levels (Supplementary Figures S2I and J), indicating that CK1δ and GSK3β regulated ZNF322A protein stability via post-translational modification.
We further analyzed the protein half-life of ZNF322A by CHX chase assay upon depletion of CK1δ or GSK3β. Our results confirmed that the half-life of ZNF322A protein was extended upon depletion of CK1δ or GSK3β (Figures 2f and g;
Supplementary Figures S2K and L). Importantly, our cell-based ubiquitination assay revealed that knockdown of CK1δ or GSK3β reduced ZNF322A protein ubiquitination (Figures 2h and i), whereas ectopic expression of CK1δ or GSK3β-CA increased ZNF322A protein ubiquitination (Figure 2j; Supplementary Figures S2M and N). Importantly, the ubiquitination level of the S391A/S396A-ZNF322A mutant protein was decreased upon GSK3β overexpression as compared with WT-ZNF322A (Figure 2k). Collectively, these results indicated that both CK1δ and GSK3β promoted ZNF322A protein degradation via phosphorylation at Ser-391 and Ser-396.
The CK1δ–GSK3β axis phosphorylates ZNF322A at Ser-396 and Ser-391, and thus promotes ZNF322A turnover
We have shown that phosphorylation of ZNF322A by CK1δ at Ser-396 triggered the subsequent phosphorylation of Ser-391 by GSK3β in vitro. In addition, both CK1δ and GSK3β promoted ZNF322A protein degradation in cells. It is conceivable that Ser-391 and Ser-396 double phosphorylation mediated by GSK3β and CK1δ has a role in the regulation of ZNF322A protein degradation. As expected, phosphorylation-defective S391A-, S396A- and S391A/396A-ZNF322A proteins all exhibited longer protein half-life than WT-ZNF322A protein (Figures 3a–c).
To further characterize the interplay between GSK3β and CK1δ in promoting ZNF322A protein destruction in the context of phosphorylation at Ser-391 and Ser-396, we generated anti-phospho-ZNF322A-specific antibodies, which could recognize phosphorylation of ZNF322A at Ser-391 or at Ser-396 (Supplementary Figures S3A-D). Using these anti-phospho-ZNF322A antibodies, we found that overexpression of GSK3β or CK1δ promoted endogenous ZNF322A phosphorylation level at Ser-391 and Ser-396, respectively (Figures 3d and e). Conversely, phosphorylation levels of Ser-391 and Ser-396 were attenuated by GSK3β inhibitor and CK1δ inhibitor treatments, respectively, accompanied with an increase of total ZNF322A protein level in WT-ZNF322A-expressing cells (Figures 3f and g; Supplementary Accumulated ZNF322A oncoprotein in lung cancer
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Figures S3E–G). Importantly, overexpression of GSK3β could not promote degradation of phosphorylation-defective S391A-ZNF322A mutant protein compared with WT-ZNF322A protein. Similarly, S396A-ZNF322A mutant was not degraded by overexpression of CK1δ (Figures 3h and i). These data suggested that GSK3β and CK1δ phosphorylate ZNF322A at Ser-391 and Ser-396, respectively, thereby resulting in ZNF322A protein turnover.
Next, we verified that CK1δ-mediated ZNF322A phosphorylation at Ser-396 may be critical for ZNF322A protein degradation targeted by GSK3β. To this end, we first examined the protein interaction between S396A-ZNF322A mutant and GSK3β.
The IP assay results showed that the protein interaction between S396A-ZNF322A and GSK3β was attenuated compared with WT-ZNF322A (Supplementary Figure S4A). Moreover, knocking down of CK1δ indeed abolished the protein interaction between WT-ZNF322A and GSK3β (Supplementary Figure S4B). These data suggested that CK1δ-mediated ZNF322A phosphorylation at Ser-396 was required for targeting of ZNF322A protein by GSK3β.
Moreover, we ectopically expressed WT- or S396A-ZNF322A in GSK3β-overexpressing cells. As expected, high level of S396A-ZNF322A was expressed in cells even when GSK3β was overexpressed (Figure 3j), supporting the notion that GSK3β phosphorylates ZNF322A in a CK1δ phosphorylation-dependent manner to regulate ZNF322A protein level. In addition, phosphorylation-mimetic mutant S396E-ZNF322A showed lower expression level than WT-ZNF322A. The expression level of phosphorylation-mimetic mutant S396E-ZNF322A could be restored to the level similar to that of WT-ZNF322A upon GSK3β depletion, suggesting the importance of subsequent S391 phosphorylation by GSK3β in regulation of ZNF322A protein expression (Figure 3k). In addition, GSK3β-induced WT-ZNF322A
protein degradation could be blocked by depletion of CK1δ (Figure 3l; Supplementary Figure S4C). Consistently, overexpres-sion of CK1δ promoted WT-ZNF322A protein degradation, whereas GSK3β depletion blocked CK1δ-mediated WT-ZNF322A protein turnover (Figure 3m; Supplementary Figure S4D).
Collectively, these data suggested that the sequential order of phosphorylation of ZNF322A at Ser-396 by CK1δ and then Ser-391 by GSK3β is crucial for ZNF322A protein degradation.
FBXW7α is an E3 ligase targeting ZNF322A protein for ubiquitination and degradation
We next addressed the question of which E3 ubiquitin ligase is required for CK1δ/GSK3β-mediated ZNF322A protein degradation.
FBXW7, a SCF type E3 ubiquitin ligase, binds to the CPD motif in its protein substrates and targets them for degradation. Sequence analysis revealed that ZNF322A protein contains CPD-like sequences from amino acids 391 to 396, which were highly conserved in ZNF322A orthologs (Supplementary Figure S5A).
Therefore, we investigated whether ZNF322A was the target of FBXW7 E3 ligase. The IP results showed that ZNF322A interacted with FBXW7α in both H460 and H1299 cells (Figures 4a and b; Supplementary Figure S5B). In addition, overexpression of FBXW7α decreased endogenous or ectopically expressed ZNF322A protein level (Figures 4c and d;
Supplementary Figure S5C). Furthermore, knockdown of FBXW7α increased ZNF322A protein half-life (Figure 4e), but did not affect ZNF322A mRNA level (Figure 4f), suggesting that FBXW7α regulates ZNF322A protein expression at the post-translational modification level.
As MG132 treatment blocked FBXW7α-induced ZNF322A protein degradation (Supplementary Figure S5D), we therefore Figure 1. CK1δ-primed phosphorylation of ZNF322A at Ser-396 is required for GSK3β-mediated phosphorylation of ZNF322A at Ser-391 in vitro. (a) Sequence alignment of GSK3β and CK1 consensus motifs in ZNF322A (upper) and across multiple species (lower). The targeted phosphorylation residue of GSK3β is shown in red and CK1 in blue. (b) In vitro kinase assay using recombinant CK1δ and GST-ZNF322A upon treatment with or without CK1 inhibitor IC261 in the presence ofγ-[ATP]-32P. (c) In vitro kinase assay in the presence of recombinant CK1δ, wild-type (WT) or S396A mutant (S396A) GST-ZNF322A recombinant protein. (d) In vitro kinase assay using GST-ZNF322A pre-incubated with or without recombinant CK1δ before incubation with recombinant GSK3β upon treatment with or without GSK3β inhibitor (GSK3βI, SB216763). (e) In vitro kinase assay using CK1δ-primed GST-ZNF322A (WT or S391A) and recombinant GSK3β upon treatment with or without GSK3βi. (f) In vitro kinase assay using CK1δ-primed GST-ZNF322A (WT or S396A) and recombinant GSK3β upon treatment with or without GSK3βi.
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determined the ZNF322A protein ubiquitination level upon FBXW7α manipulation. Cell-based ubiquitination assay showed that overexpression of FBXW7α increased ZNF322A protein ubiquitination level (Figure 4g; Supplementary Figure S5E), whereas depletion of FBXW7α decreased ZNF322A protein ubiquitination (Figure 4h). Moreover, FBXW7α-promoted ubiquitination of ZNF322A was blocked by lysine (K) 48R mutant, but not by K63R mutant, indicating the formation of K48-linked ubiquitination by FBXW7α (Supplementary Figure S5F).
Collectively, these results supported a novelfinding that FBXW7α acted as an E3 ligase for ZNF322A protein degradation.
Phosphorylation of Ser-391 and Ser-396 facilitates the recognition and degradation of ZNF322A by FBXW7α
Our data so far lead us to speculate that CK1δ/GSK3β-mediated ZNF322A phosphorylation is required for FBXW7α-promoted ZNF322A protein destruction. Indeed, CK1δ or GSK3β depletion blocked FBXW7α-mediated ZNF322A protein degradation (Supplementary Figures S5G and H). Notably, FBXW7α degraded WT-ZNF322A protein but not phosphorylation-defective S391A/396A-ZNF322A mutant (Figure 4i). Cell-based ubiquitination assay showed decrease in S391A/396A-ZNF322A mutant protein ubiquitination by FBXW7α compared with WT-ZNF322A (Figure 4j).
Figure 2. CK1δ and GSK3β promote ZNF322A protein degradation. (a) H1299 cells were co-transfected with wild-type GFP-tagged ZNF322A (GFP-WT-ZNF322A), Flag-tagged CK1δ (Flag-CK1δ) and Myc-tagged constitutively active GSK3β (Myc-GSK3β) for 24 h. Cells were treated with the proteasome inhibitor MG132 before being harvested. Co-IP experiment was performed using anti-GFP to pull down GFP-ZNF322A interacting proteins. (b, c) IB analysis of endogenous ZNF322A protein level in H1299 (upper) and H460 (lower) cells expressing with or without Flag-CK1δ (b) or in cells expressing with or without Myc-GSK3β (c). (d, e) IB analysis of endogenous ZNF322A protein level in H1299 (upper) and H460 (lower) cells treated with or without CK1 inhibitor IC261 (d) or GSK3β inhibitor GSK3β XI (e). (f, g) IB analysis of cell lysates from H460 sh-Ctrl and sh-CK1δ
#1 (f) or from H460 sh-Ctrl and sh-GSK3β #1 (g) cells expressing HA-ZNF322A for 24 h before CHX (20 μg/ml) treatment at the indicated times.
Quantification of ZNF322A band intensities was normalized to GAPDH, and then normalized to the time-point: 0 min. Data are presented as mean± s.e.m. (h, i) IB analysis of the anti-GFP IP products from H460 Ctrl and CK1δ cells (#1 and #2 clones) (h) or from H460 Ctrl and sh-GSK3β cells (#1 and #2 clones) (i). Where indicated, HA-Ub or GFP-WT-ZNF322A was included in the transfection. Cells were treated with proteasome inhibitor MG132 (10μM) for 6 h before being harvested. (j, k) IB analysis of the anti-GFP IP products from H460 cells expressing Flag-CK1δ (j) or cells expressing Myc-GSK3β (k). Where indicated, HA-Ub, GFP-WT-ZNF322A or GFP-S391A/396A-ZNF322A was included in the transfection. Cells were treated with the proteasome inhibitor MG132 before being harvested. IB, Immunoblotting.
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Consistently, overexpression of FBXW7α increased WT-ZNF322A protein ubiquitination level, whereas treatment with CK1δ or GSK3β inhibitor significantly attenuated FBXW7α-mediated WT-ZNF322A protein ubiquitination (Supplementary Figures S5I and J).
Our IP data showed that GFP-tagged WT-ZNF322A interacted with FBXW7α, whereas such protein interaction was attenuated
in cells expressing GFP-tagged S391A/396A-ZNF322A mutant (lanes 4 vs 3, HA-blot, Figure 4k). Molecular modeling of FBXW7 in complex with ZNF322A doubly S391/S396-phosphorylated peptide was performed based on cyclin E-FBXW7 complex.19 The model revealed that the doubly phosphorylated peptide can potentially bind to the narrow Figure 3. Sequential phosphorylation of ZNF322A at Ser-396 by CK1δ and Ser-391 by GSK3β is crucial for ZNF322A protein degradation.
(a–c) IB analysis of cell lysates from H460 cells expressing with S391A (a), S396A (b) and S391A/396A (c) mutant HA-ZNF322A for 24 h before CHX treatment. Quantification of ZNF322A band intensities was performed as described in Figure 2f. Data of WT-ZNF322A are included for comparison. Data are presented as mean± s.e.m. (d, e) IB analysis of endogenous total ZNF322A protein and p-S391 (d) or p-S396 (e) expression in H1299 cells expressing Myc-GSK3β-CA (d) or Flag-CK1δ (e). Cells were treated with MG132 for 6 h before being harvested.
(f, g) IB analysis of total ZNF322A protein and p-S391 (f) or p-S396 (g) expression in H1299 cells expressing HA-ZNF322A treated with or without GSK3β inhibitor GSK3β XI (f) or CK1δ inhibitor IC261 (g) for 6 h. (h) IB analysis of the cell lysates from H460 cells expressing WT- or S391A-ZNF322A together with or without Myc-GSK3β. (i, j) IB analysis of cell lysates from H460 cells expressing Flag-CK1δ (i) or Myc-GSK3β (j).
Where indicated, WT- or S396A-ZNF322A construct was included in the transfection. (k) IB analysis of the cell lysates from H460 cells expressing WT- or S396E-ZNF322A together with or without the sh-GSK3β. (l, m) IB analysis of WT-ZNF322A level from H1299 sh-Ctrl or sh-CK1δ cells reconstituted GSK3β (l) or from H1299 sh-Ctrl or sh-GSK3β cells reconstituted CK1δ (m). IB, Immunoblotting.
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face of the FBXW7-WD40 domain (Figure 4l). Notably, the two phosphoserine residues Ser-391 and Ser-396 oriented into a shallow, positively charged pocket at the inner rim of the β-propeller channel of FBXW7 (Figure 4m). Together, these
findings strongly suggested that ZNF322A phosphorylation at Ser-391/396 by GSK3β/CK1δ is crucial for FBXW7α-ZNF322A interaction thus targeting ZNF322A protein for ubiquitination and degradation.
Figure 4. FBXW7α promotes ZNF322A ubiquitination and degradation in a CK1δ/GSK3β-dependent manner. (a, b) IB analysis of whole-cell lysates and anti-GFP (a) or anti-HA (b) IP from H1299 cells expressing GFP-ZNF322A with or without HA-tagged FBXW7α (HA-FBXW7α). (c, d) IB analysis of the endogenous ZNF322A protein level (c) and ectopically expressed HA-ZNF322A level (d) in H1299 cells with or without Flag-FBXW7α expression. (e) IB analysis of cell lysates from H1299 sh-Ctrl and sh-FBXW7α (#1 and #2 clones) cells expressing HA-ZNF322A for 24 h before CHX treatment at the indicated times. (f) RT-PCR analysis of mRNA level of FBXW7α (left) and ZNF322A (right) in H1299 sh-Ctrl and sh-FBXW7α cells. Data are presented as mean ± s.e.m. P-values determined using two-tailed Student’s t-test. ***Po0.001. (g, h) IB analysis of anti-GFP IP products from H1299 cells expressing Flag-FBXW7α (g) or from H1299 sh-Ctrl or sh-FBXW7α cells (#1 and #2 clones) (h). Where
Figure 4. FBXW7α promotes ZNF322A ubiquitination and degradation in a CK1δ/GSK3β-dependent manner. (a, b) IB analysis of whole-cell lysates and anti-GFP (a) or anti-HA (b) IP from H1299 cells expressing GFP-ZNF322A with or without HA-tagged FBXW7α (HA-FBXW7α). (c, d) IB analysis of the endogenous ZNF322A protein level (c) and ectopically expressed HA-ZNF322A level (d) in H1299 cells with or without Flag-FBXW7α expression. (e) IB analysis of cell lysates from H1299 sh-Ctrl and sh-FBXW7α (#1 and #2 clones) cells expressing HA-ZNF322A for 24 h before CHX treatment at the indicated times. (f) RT-PCR analysis of mRNA level of FBXW7α (left) and ZNF322A (right) in H1299 sh-Ctrl and sh-FBXW7α cells. Data are presented as mean ± s.e.m. P-values determined using two-tailed Student’s t-test. ***Po0.001. (g, h) IB analysis of anti-GFP IP products from H1299 cells expressing Flag-FBXW7α (g) or from H1299 sh-Ctrl or sh-FBXW7α cells (#1 and #2 clones) (h). Where