Research Article | Open Access
Yan Tang, JungWoo Yang, Wang Zheng, Jingfeng Tang, Xing-Zhen Chen, Jianzheng Yang, Zuocheng Wang, "Polycystin-1 Inhibits Cell Proliferation through Phosphatase PP2A/B56α", BioMed Research International, vol. 2019, Article ID 2582401, 8 pages, 2019. https://doi.org/10.1155/2019/2582401
Polycystin-1 Inhibits Cell Proliferation through Phosphatase PP2A/B56α
Autosomal dominant polycystic kidney disease (ADPKD) is associated with a number of cellular defects such as hyperproliferation, apoptosis, and dedifferentiation. Mutations in polycystin-1 (PC1) account for ∼85% of ADPKD. Here, we showed that wild-type (WT) or mutant PC1 composed of the last five transmembrane (TM) domains and the C-terminus (termed PC1-5TMC) inhibits cell proliferation and protein translation, as well as the downstream effectors of mTOR, consistent with previous reports. Knockdown of B56α, a subunit of the protein phosphatase 2A (PP2A) complex, or application of PP2A inhibitor okadaic acid or calyculin A, abolished the inhibitory effect of PC1 and PC1-5TMC on proliferation, indicating that PP2A/B56α mediates the regulation of cell proliferation by PC1. In addition to the phosphorylated S6 and 4EBP1, B56α was also downregulated by PC1 and PC1-5TMC. Furthermore, the downregulation of B56α, which may be mediated by mTOR but not AKT, can account for the dependence of PC1-inhibited proliferation on PP2A.
Autosomal dominant polycystic kidney disease (ADPKD) results from mutations in genes coding for polycystin 1 (PC1) and polycystin 2 (PC2), which account for about 85% and 15% of ADPKD, respectively . Both loss and gain of PC1 or PC2 function result in cystogenesis . PC1 is a 4302-amino acid (aa), 462-kDa protein containing 11 putative transmembrane (TM) spans. Its large extracellular N-terminus contains a number of motifs that suggest interaction with extracellular ligands and matrix proteins. The short C-terminus contains domains for G-protein activation and interaction with partner proteins. Thus, PC1 seems to function as a cell surface receptor that couples extracellular stimuli to intracellular signaling .
ADPKD is associated with several cellular abnormalities, including increased cell proliferation, apoptosis, and dedifferentiation. In addition to decreased Ca2+ signaling, several cell fate-related pathways are modulated by PC1/PC2, including cAMP, MAPK, Wnt, JAK-STAT, Hippo, Src, and mTOR . Overexpression of PC1 or PC2 in renal epithelial cells was shown to repress cell proliferation through the JAK-STAT signaling pathway . PC1 was found to function as a modulator of noncapacitive Ca2+ entry (NCCE) and Ca2+ oscillation, through which it affects cell proliferation . PC1 was also found to regulate proliferation in PKD1-depleted or -mutated epithelial cells through a CREB-AP1 pathway which upregulates the amphiregulin level . Moreover, the mammalian target of rapamycin (mTOR) pathway was shown to be abnormally activated in cyst-lining epithelial cells in human ADPKD patients and mouse models, which may result from loss of PC1 binding with tuberin, suggesting that PC1 inhibits cell proliferation by downregulating the activity of mTOR and its interaction with tuberin . It was later verified PC1 reduces cell size by negatively regulating mTOR and downstream effectors S6K1 and 4EBP1 in a tuberin-dependent manner . These studies together demonstrated that PC1 downregulates proliferation through different pathways and indicated that abnormalities of any of these pathways associated with ADPKD may account for the altered proliferation.
Protein-serine/threonine phosphatase 2A (PP2A) together with protein-serine/threonine phosphatase (PP1), PP2B, and PP4 to PP7 belongs to the phosphoprotein phosphatase family. Cellular PP2A exists in two forms, a core enzyme and a heterotrimeric holoenzyme . Each PP2A holoenzyme is formed by a combination of three subunits: a common catalytic subunit PP2Ac containing the active site, a variable regulatory (B) subunit PP2Ab, and a structural A subunit PP2Aa. The function of PP2A relies on the B subunit that determines the substrate specificity and the subcellular localization of the PP2A complex . The PP2A activity and subcellular localization are also regulated by posttranslational modifications of B56 that have homologous B56α and B56β isoforms subunit. Comparing with B56β, B56α is more highly expressed and has been widely studied . PP2A is known to dephosphorylate over 300 types of substrates involved in almost all major cellular signaling pathways including the Wnt, mTOR, and MAPK pathways .
Phosphorylation and dephosphorylation are two opposite processes that control the activity of numerous cellular events. The activation of p70s6k (also called 70-kDa S6 kinase) leads to an increase in the protein synthesis and cell proliferation through acting on its target substrate, the S6 ribosomal protein. Eukaryotic translation initiation factor 4E- (eIF4E-) binding protein 1 (4EBP1) is phosphorylated, resulting in its dissociation from eIF4E and activation of cap-dependent mRNA translation . Because S6 and 4EBP1 are the best known substrates of mTOR , while PP2A regulates translation initiation through dephosphorylating 4EBP1 and p70s6k , we speculate that PP2A is a major mTOR phosphatase to regulate downstream effectors. In this study, we employed culture cells transfected with PC1 or PC1 mutants to investigate the relationship between PC1-regulating cell proliferation/translation and PP2A (B56α).
2. Materials and Methods
2.1. Antibodies and Reagents
P-S6 and S6 were products of Cell Signaling Technology (NewEngland Biolabs, Pickering, ON). PP2A-B56α, 4EBP1, P-4EBP1, GFP (B-2), anti-β-actin, anti-Flag antibodies, and secondary antibodies were from Santa Cruz (Santa Cruz, Santa Cruz, CA). The PP2A inhibitor okadaic acid (OA) was from Calbiochem (EMD Chemicals Inc., Gibbstown, NJ); another PP2A inhibitor calyculin A (CA) was from Cell Signaling Technology (New England Bio labs). Rapamycin and puromycin were products of Sigma-Aldrich, Canada.
2.2. DNA Constructs, Cell Culture, and Transfection
Plasmid pcDNA3-GFP-PC1-5TMC (PC1-5TMC) comprising C-terminus of PC1 and last 5 transmembrane (TM) was constructed as described previously . HEK293T, HeLa, IMCD3, and MDCK cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum, L-glutamine, and penicillin-streptomycin in 5% CO2 and 37°C. HEK293T cells stably being transfected with mouse full-length PC1 was from one co-author Dr. J. Yang and was grown with 2 μg/ml of puromycin under above conditions . Transient transfection was carried out employing lipofectamine 2000 (Invitrogen, Burlington, ON).
2.3. Cell Proliferation Assay
HEK293T, HeLa, MDCK, or IMCD cells were transiently transfected in 100 mm dishes. At 24 hours (hr) after transfection, cell proliferation assay was performed as described previously [19, 20]. Cells were split and seeded into either separate wells of a 96-well plate labelling with or without OA or CA treatment or new 100 mm dishes for further knockdown experiments. After incubation for another 24 hr, luminescence activity was measured using Alarma-Blue (Invitrogen, Canada, Inc.) in serum-free medium and a microplate reader. HEK293T cells with stable transfection of wild-type (WT) mouse PC1 after 24 hr of culture revealed by WB were split and seeded into 96-well plates. The rest of cells in the 100 mm dishes were collected for immunoblotting at the same time point. The cell proliferation rate was calculated using the following formula: cell proliferation rate (%) = ODtest/ODcontrol × 100%.
2.4. 35S Pulse Labelling
At 40 hr after transfection of HEK293T or HeLa cells, the cells were starved for 1 hr in L-methionine- and L-cysteine-free DMEM and then labelled with 50 μCi of (35S) methionine/cysteine (EXPRE 35S Protein Labeling Mix, PerkinElmer, Woodbridge, ON) for 10 minutes, and the cell extracts were employed for sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, as described previously .
2.5. RNA Interference
PP2A-B56α siRNA (Santa Cruz, Cat#: sc-39181) was used to interfere with the expression of B56α protein in HEK293T cells following product instruction. The efficiency of siRNA gene knockout was evaluated by blotting.
Densities of bands were quantified by Image J software. Values generated were presented as mean ± standard error (SEM). N represents the number of independent experiments. Data were statistically analyzed by Sigma Plot 12 soft ware program (Systat Software Inc., San Jose, CA). The paired t-test is used to determine the difference in data between two groups. value less than 0.05 was considered statistically significant.
3.1. PC1 Inhibits Proliferation/Translation and Downstream Effectors of mTOR
In order to clarify the effect of PC1 on cell proliferation, we used Alarma-Blue to label HeLa, human embryonic kidney (HEK293T), Madin-Darby canine kidney (MDCK), and mouse inner medullary collecting duct (IMCD3) cells after transfection of PC1-5TMC, a truncation mutant consisting of the last five TM domains (S7-S11), and the C-terminus of human PC1 and found that PC1-5TMC in HeLa, MDCK, and IMCD cells, as well as WT PC1 in HEK293T cells, suppresses proliferation (Figures 1(a) and 1(b)), consistent with previous reports [4–9]. The difference in magnitude of the responses can be explained mainly by the fact that different cell types can lead to different cell transfection efficiencies and thus different inhibition rates of cell proliferation.
Protein translation plays a critical role in the regulation of cellular processes associated with cell volume control and cell proliferation. We next utilized 35S labelling to determine whether PC1 modulates protein translation. We found that stably expressed WT PC1 and transiently expressed PC1-5TMC reduce protein synthesis in HEK293T and HeLa cells, respectively (Figures 1(c) and 1(d)). Therefore, PC1 repressed both the cell proliferation and mRNA translation.
PC1 is known to repress cell growth by downregulating mTOR and its downstream effectors S6 kinase 1 and 4EBP1/eIF4E in a tuberin-dependent manner [8, 9]. Our data from western blot (WB) assays showed PC1-5TMC inhibits phosphorylated S6 (P-S6) and 4EBP1 (P-4EBP1) (Figures 1(e) and 1(f)), which are in line with those of previous reports and supported that the mTOR pathway mediates the regulation of cell proliferation/protein translation by PC1.
3.2. PC1-Inhibited Proliferation Depends on PP2A/B56α
PP2A was shown to regulate translation initiation through dephosphorylating translational regulators 4EBP1 and p70s6k , suggesting that PP2A may act as a mTOR phosphatase to regulate downstream effectors in PC1-inhibited cell growth. To determine whether PC1 inhibits translation and/or proliferation through PP2A, we first examined cell proliferation using PP2A inhibitors OA and CA. After treating cells with 1 nM of OA or CA, which was reported to inhibit the PP2A activity while exhibiting no effect on PP1 , we found that PC1-5TMC (Figure 2(a), middle panel) and WT PC1 (Figure 2(b), right panel) no longer exhibit inhibitory effect on proliferation, suggesting that the inhibition of proliferation by PC1 is PP2A-dependent.
PP2A holoenzyme is regulated by its variable regulatory B subunit B56α . To determine whether the proliferation regulated by PC1 depends on PP2A-B56α, we knocked down B56α by siRNA. We found that knockdown of B56α abolished the effect of PC1 on proliferation when the expression of B56α was completely inhibited (Figure 2(c)), which further supported and verified that the PC1-inhibited cell proliferation is through PP2A (B56α).
On the other hand, as shown in the left panel of Figure 2(c), the WB result of the first two bands suggested PC1 reduces the expression of B56α without knockdown while that of the third band showed B56α was depressed by knockdown and both of the two reduced B56α correspond to the decreased cell proliferation shown in the right panel of Figure 2(c), which indicated that PC1-inhibited proliferation could be caused by low expression of B56α. Moreover, the further reduction in cell proliferation is expected to be seen in the fourth band when there was no expression of B56α; however, cell proliferation was not decreased but increased or it was abolished in this condition, which implied that B56α expression or existence is required by PC1-inhibited proliferation.
3.3. PC1 Upregulates the PP2A Activity through Decreasing the B56α Expression
It was previously thought that overexpression of B56α results in an increased level of eIF4E phosphorylation, likely due to decreased PP2A activity , and reduced B56α expression increases cardiac PP2A activity ; that is, nonphosphorylated B56α inhibits the activity of purified PP2A . Based on the fact that PC1 upregulates the PP2A activity because PC1 inhibited P-S6 and P-4EBP1 and on the downregulation of the PP2A activity by B56α, we deduce that PC1 increases the PP2A activity on substrates through downregulating expression of B56α.
Our western blot experiments using HEK293T cells revealed that besides low levels of P-S6 and P-4EBP1 (Figures 1(e) and 1(f)), expression of PC1 results in a decrease in the expression of B56α (Figure 3(a)) which actually has been shown in the left panel of Figure 2(c). Similar results were observed in HeLa or HEK293T cells overexpressing PC1-5TMC (Figures 3(b) and 3(c)). The results showed that PC1 downregulates the expression of B56α, which indicated that the dependence of PC1-inhibited proliferation on PP2A is mediated by the negative regulation of B56α on the PP2A activity.
3.4. PC1 Downregulates B56α Expression Likely through mTOR
B56α expression is regulated via mTOR because the mTOR inhibitor rapamycin blocks B56α expression in REH cells . Our WB results also found that rapamycin inhibits both P-S6 and B56α in HEK293T cells which further supports that mTOR can promote B56α expression (Figure 4).
Similar to the potency of B56α for inhibition of PP2A, several reports have showed that mTOR negatively regulates the PP2A activity [26, 27]; in addition, the previous results have also showed that PC1 downregulates mTOR [9, 28] and that B56α inhibits the activity of PP2A [22, 23]. A model can be presented from our present results and those previously reported by others, which is based on the fact that downregulation of B56a and/or mTOR increases the PP2A activity that mediates the inhibition of cell proliferation/translation by PC1 (Figure 5). Therefore, it is highly possible that PC1 negatively regulates expression of B56α by negatively controlling mTOR.
The current evidence and research progress showed three main signaling pathways including B-Raf/methyl ethyl ketone (MEK)/extracellular regulated protein kinases (ERK) signaling cascade, mTOR, and nuclear factor of activated T-cell (NFAT) pathways involving increased cell proliferation, fluid secretion, and kidney cyst development seen in ADPKD which may arise due to either the loss of polycystin complex function or an imbalance in the PC1/PC2 ratio [7, 29]. However, PC1 was previously shown to slow cell proliferation and inhibit apoptosis [8, 9, 30]. In our study, the data showed that PC1 inhibits cell proliferation and/or protein translation and dephosphorylates downstream effectors of mTOR. These results were in line with those of previous reports about PC1-controlled cell growth (size) due to the downregulation of mTOR, S6K1, and 4EBP1 [8, 9]. The following results, with the inhibitors of PP2A and knockdown of PP2A/B56α, verified that PP2A/B56α causes PC1-inhibited cell proliferation. Furthermore, we first found PC1 downregulates expression of B56α. For decreased levels of S6 and 4EBP1 phosphorylation due to increased PP2A activity caused by either reduced B56α or PC1 [9, 22, 23], the dependence of PC1-inhibited proliferation on PP2A can be, at least partly, explained as low expression of B56α.
Both B56α and mTOR negatively regulate the PP2A activity [22–24, 26, 27]. Our result also showed the inhibitor of mTOR rapamycin suppresses B56α expression, which indicated that PP2A is a major phosphatase of mTOR to regulate downstream effectors by B56α. Furthermore, previous WB results have showed that PC1 upregulates P-AKT  which indicates that B56α expression regulated by PC1 is independent of AKT, although experiments showed that inhibition of AKT also suppresses B56α expression in acute lymphoblastic leukemia-derived REH cells .
Also, Ruvolo et al. considered that although mTOR kinase seems to be involved in rapamycin-inhibited B56α expression, it is unlikely that mTOR regulates B56α directly by such kinase pathways, because expression of B56α can be restored with a proteasome inhibitor . They also found that protein kinase R (PKR) can protect B56α by suppressing proteasome-mediated proteolysis . Therefore, we speculate the possibility that mTOR supports B56α expression indirectly by promoting PKR that directly protects the B subunit from proteolysis.
B56α itself can also be regulated by reversible phosphorylation and dephosphorylation. Beside PKR, protein kinase A (PKA) , cyclin-dependent kinase (CDK), protein kinase C alpha (PKCα), checkpoint kinase (CHK), and PP2A itself can regulate the phosphorylation of B56α to promote or suppress B56α function [22, 32–34]. Moreover, the phosphorylation of B56α by PKA or PKR increased the PP2A activity, but conversely, the potency of B56α for PP2A inhibition was markedly increased by a phosphorylation of B56α at Ser41 by PKC . Therefore, more experiments will also be needed to determine the relationship between B56α phosphorylation and PP2A activity or mTOR in the inhibition of proliferation and translation by PC1.
In this study, we first demonstrated that PC1-inhibited proliferation depends on PP2A/B56α, which can be accounted for by the expression of B56α. The current and reported results reveal that PC1 is most likely to downregulate B56α expression through mTOR. Further experiments will explore the involvement of mTOR-dependent B56α expression and B56α phosphorylation in PC1-inhibited proliferation/translation.
The data used to support the findings of this study are available from the corresponding author upon request.
Conflicts of Interest
The authors declare no conflicts of interest.
The authors thank Dr. Yue Zhao for her important experimental work. This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada (to X. Z. C.), a grant from Jilin Provincial Science and Technology Department (no. 20180414084GH, to Y. T.) and a grant from the National Natural Science Foundation, People’s Republic of China (no. 81602448, to J. T.).
- W. J. Kimberling, S. Kumar, P. A. Gabow, J. B. Kenyon, C. J. Connolly, and S. Somlo, “Autosomal dominant polycystic kidney disease: localization of the second gene to chromosome 4q13-q23,” Genomics, vol. 18, no. 3, pp. 467–472, 1993.
- J. Zhou, “Polycystins and primary cilia: primers for cell cycle progression,” Annual Review of Physiology, vol. 71, no. 1, pp. 83–113, 2009.
- J. Hughes, C. J. Ward, B. Peral et al., “The polycystic kidney disease 1 (PKD1) gene encodes a novel protein with multiple cell recognition domains,” Nature Genetics, vol. 10, no. 2, pp. 151–160, 1995.
- F. O. Lemos and B. E. Ehrlich, “Polycystin and calcium signaling in cell death and survival,” Cell Calcium, vol. 69, no. 1, pp. 37–45, 2018.
- A. K. Bhunia, K. Piontek, A. Boletta et al., “PKD1 induces p21waf1 and regulation of the cell cycle via direct activation of the JAK-STAT signaling pathway in a process requiring PKD2,” Cell, vol. 109, no. 2, pp. 157–168, 2002.
- G. Aguiari, V. Trimi, M. Bogo et al., “Novel role for polycystin-1 in modulating cell proliferation through calcium oscillations in kidney cells,” Cell Proliferation, vol. 41, no. 3, pp. 554–573, 2008.
- G. Aguiari, F. Bizzarri, A. Bonon et al., “Polycystin-1 regulates amphiregulin expression through CREB and AP1 signalling: implications in ADPKD cell proliferation,” Journal of Molecular Medicine, vol. 90, no. 11, pp. 1267–1282, 2012.
- J. M. Shillingford, N. S. Murcia, C. H. Larson et al., “The mTOR pathway is regulated by polycystin-1, and its inhibition reverses renal cystogenesis in polycystic kidney disease,” Proceedings of the National Academy of Sciences, vol. 103, no. 14, pp. 5466–5471, 2006.
- G. Distefano, M. Boca, I. Rowe et al., “Polycystin-1 regulates extracellular signal-regulated kinase-dependent phosphorylation of tuberin to control cell size through mTOR and its downstream effectors S6K and 4EBP1,” Molecular and Cellular Biology, vol. 29, no. 9, pp. 2359–2371, 2009.
- C. Van Hoof and J. Goris, “Phosphatases in apoptosis: to be or not to be, PP2A is in the heart of the question,” Biochimica et Biophysica Acta (BBA)-Molecular Cell Research, vol. 1640, no. 2-3, pp. 97–104, 2003.
- U. S. Cho and W. Xu, “Crystal structure of a protein phosphatase 2A heterotrimeric holoenzyme,” Nature, vol. 445, no. 7123, pp. 53–57, 2007.
- B. McCright, A. M. Rivers, S. Audlin, and D. M. Virshup, “The B56 family of protein phosphatase 2A (PP2A) regulatory subunits encodes differentiation-induced phosphoproteins that target PP2A to both nucleus and cytoplasm,” Journal of Biological Chemistry, vol. 271, no. 36, pp. 22081–22089, 1996.
- N. Wlodarchak and Y. Xing, “PP2A as a master regulator of the cell cycle,” Critical Reviews in Biochemistry and Molecular Biology, vol. 51, no. 3, pp. 162–184, 2016.
- J. Chung, C. J. Kuo, G. R. Crabtree, and J. Blenis, “Rapamycin-FKBP specifically blocks growth-dependent activation of and signaling by the 70 kd S6 protein kinases,” Cell, vol. 69, no. 7, pp. 1227–1236, 1992.
- M. Shimobayashi and M. N. Hall, “Making new contacts: the mTOR network in metabolism and signalling crosstalk,” Nature Reviews Molecular Cell Biology, vol. 15, no. 3, pp. 155–162, 2014.
- R. T. Peterson, B. N. Desai, J. S. Hardwick, and S. L. Schreiber, “Protein phosphatase 2A interacts with the 70-kDa S6 kinase and is activated by inhibition of FKBP12-rapamycinassociated protein,” Proceedings of the National Academy of Sciences, vol. 96, no. 8, pp. 4438–4442, 1999.
- Y. Tang, Z. Wang, J. Yang et al., “Polycystin-1 inhibits eIF2α phosphorylation and cell apoptosis through a PKR-eIF2α pathway,” Scientific Reports, vol. 7, no. 1, p. 11493, 2017.
- Y. Yu, M. H. Ulbrich, M. H. Li et al., “Structural and molecular basis of the assembly of the TRPP2/PKD1 complex,” Proceedings of the National Academy of Sciences of the United States of America, vol. 106, no. 28, pp. 11558–11563, 2009.
- G. Liang, J. Yang, Z. Wang, Q. Li, Y. Tang, and X.-Z. Chen, “Polycystin-2 down-regulates cell proliferation via promoting PERK-dependent phosphorylation of eIF2 alpha,” Human Molecular Genetics, vol. 17, no. 28, pp. 3254–3262, 2008.
- Y. Tang, G. Shi, J. Yang et al., “Role of PKR in the inhibition of proliferation and translation by polycystin-1,” Biomed Research International, vol. 2019, Article ID 5320747, p. 8, 2019.
- B. Favre, P. Turowski, and B. A. Hemmings, “Differential inhibition and posttranslational modification of protein phosphatase 1 and 2A in MCF7 cells treated with calyculin-A, okadaic acid, and tautomycin,” Journal of Biological Chemistry, vol. 272, no. 21, pp. 13856–13863, 1997.
- Z. Xu and B. R. G. Williams, “The B56alpha regulatory subunit of protein phosphatase 2A is a target for regulation by double-stranded RNA-dependent protein kinase PKR,” Molecular and Cellular Biology, vol. 20, no. 14, pp. 5285–5299, 2000.
- S. C. Little, J. Curran, M. A. Makara et al., “Protein phosphatase 2A regulatory subunit B56α limits phosphatase activity in the heart,” Science Signaling, vol. 8, no. 386, p. ra72, 2015.
- Z. Mao, C. Liu, X. Lin, B. Sun, and C. Su, “PPP2R5A: a multirole protein phosphatase subunit in regulating cancer development,” Cancer Letters, vol. 414, pp. 222–229, 2018.
- V. R. Ruvolo, S. M. Kurinna, K. B. Karanjeet et al., “PKR regulates B56(alpha)-mediated BCL2 phosphatase activity in acute lymphoblastic leukemia-derived REH cells,” Journal of Biological Chemistry, vol. 283, no. 51, pp. 3544–3585, 2008.
- C. J. Carlson, M. F. White, and C. M. Rondinone, “Mammalian target of rapamycin regulates IRS-1 serine 307 phosphorylation,” Biochemical and Biophysical Research Communications, vol. 316, no. 2, pp. 533–539, 2004.
- S. Gao, C. Duan, G. Gao, X. Wang, and H. Yang, “Alpha-synuclein overexpression negatively regulates insulin receptor substrate 1 by activating mTORC1/S6K1 signaling,” The International Journal of Biochemistry & Cell Biology, vol. 64, no. 7, pp. 25–33, 2015.
- A. N. Gargalionis, P. Korkolopoulou, E. Farmaki et al., “Polycystin-1 and polycystin-2 are involved in the acquisition of aggressive phenotypes in colorectal cancer,” International Journal of Cancer, vol. 136, no. 7, pp. 1515–1527, 2015.
- A. Mangolini, L. de Stephanis, and G. Aguiari, “Role of calcium in polycystic kidney disease: from signaling to pathology,” World Journal of Nephrology, vol. 5, no. 1, pp. 76–83, 2016.
- M. Boca, G. Distefano, F. Qian, A. K. Bhunia, G. G. Germino, and A. Boletta, “Polycystin-1 induces resistance to apoptosis through the phosphatidylinositol 3-kinase/Akt signaling pathway,” Journal of the American Society of Nephrology, vol. 17, no. 3, pp. 637–647, 2006.
- J.-H. Ahn, T. McAvoy, S. V. Rakhilin, A. Nishi, P. Greengard, and A. C. Nairn, “Protein kinase A activates protein phosphatase 2A by phosphorylation of the B56 subunit,” Proceedings of the National Academy of Sciences, vol. 104, no. 8, pp. 2979–2984, 2007.
- U. Kirchhefer, A. Heinick, S. König et al., “Protein phosphatase 2A is regulated by protein kinase cα (PKCα)-dependent phosphorylation of its targeting subunit B56α at Ser41,” Journal of Biological Chemistry, vol. 289, no. 1, pp. 163–176, 2014.
- J. Falck, N. Mailand, R. G. Syljuåsen, J. Bartek, and J. Lukas, “The ATM-Chk2-Cdc25A checkpoint pathway guards against radioresistant DNA synthesis,” Nature, vol. 410, no. 6830, pp. 842–847, 2001.
- F. W. Syljuåsen, M. O. Collins, A. Lichawska, P. Zegerman, J. S. Choudhary, and J. Pines, “Quantitative proteomics reveals the basis for the biochemical specificity of the cell-cycle machinery,” Molecular Cell, vol. 43, no. 3, pp. 406–417, 2011.
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