RNAi screen to identify protein phosphatases that regulate the NF-kappaB signaling

Abstract

NF-kappaB plays a critical role in cell survival, apoptosis, and inflammatory responses. Serine/threonine-specific phosphatases (PPs) represent the second major class of enzymes that catalyze the dephosphorylation of proteins. The roles of PPs regulating NF-kappaB activities are poorly understood. Here we describe an RNAi-based screen to identify the PPs that involve in regulating NF-kappaB signaling. Thirty-four candidate PPs siRNAs were synthesized and primarily screened by NF-kappaB reporter gene assay in HeLa cells. PHLPP, one of the protein phosphatase type 2C family members (PP2C), was identified as a positive regulator of NF-kappaB signaling. Knock-down of PHLPP dramatically attenuated TNFα-stimulated NF-kappaB transcriptional activation. Knock-down of PHLPP led to enhancement of NF-kappaB/p65 nuclear import and retention, but decreased TNFα-induced phosphorylation at Ser276 on p65. This critical phosphorylation was also drastically reduced by knock-down of PKCalpha and Akt1, two important serine/threonine kinases dephosphorylated by PHLPP. The results together suggest that PHLPP-Akt-PKC may represent an important signaling loop that activates NF-kappaB/p65 signaling through critical serine phosphorylation.

Key words： nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappaB); protein serine/threonine phosphatases; PH domain leucine-rich repeat protein phosphatase (PHLPP); RNA interference

Acknowledgements

This research was supported by the National High Technology Research and Development Program of China (863 Program) (No.2006AA02Z191), the Bureau of Science and Technology of Guangzhou, China (No. 2007Z1-E4041) and Guangzhou Economic & Technological Development District (GETDD S & T Project) (2007G-P029).

References

1 Bohuslav J, Chen L F, Kwon H, Mu Y, Greene W C (2004). p53 induces NF-kappaB activation by an IkappaB kinase-independent mechanism involving phosphorylation of p65 by ribosomal S6 kinase 1. J Biol Chem, 279(25): 26115-26125

2 Brognard J, Newton A C (2008). PHLiPPing the switch on Akt and protein kinase C signaling. Trends Endocrinol Metab, 19(6): 223-230

3 Brognard J, Sierecki E, Gao T, Newton A C (2007). PHLPP and a second isoform, PHLPP2, differentially attenuate the amplitude of Akt signaling by regulating distinct Akt isoforms. Mol Cell, 25(6): 917-931

4 Gao T, Furnari F, Newton A C (2005). PHLPP: a phosphatase that directly dephosphorylates Akt, promotes apoptosis, and suppresses tumor growth. Mol Cell, 18(1): 13-24

5 Gao T, Brognard J, Newton A C (2008). The phosphatase PHLPP controls the cellular levels of protein kinase C. J Biol Chem, 283(10): 6300-6311

6 Hayden M S, Ghosh S (2004). Signaling to NF-kappaB. Genes Dev, 18(18): 2195-2224

7 Hayden M S, Ghosh S (2008). Shared principles in NF-kappaB signaling. Cell, 132(3): 344-362

8 Henmi T, Amano K, Nagaura Y, Matsumoto K, Echigo S, Tamura S, Kobayashi T (2009). A mechanism for the suppression of interleukin-1-induced nuclear factor kappaB activation by protein phosphatase 2Ceta-2. Biochem J, 423(1): 71-78

9 Hochrainer K, Racchumi G, Anrather J (2007). Hypo-phosphorylation leads to nuclear retention of NF-kappaB p65 due to impaired IkappaBalpha gene synthesis. FEBS Lett, 581(28): 5493-5499

10 Kuo Y C, Huang K Y, Yang C H, Yang Y S, Lee W Y, Chiang C W (2008). Regulation of phosphorylation of Thr-308 of Akt, cell proliferation, and survival by the B55alpha regulatory subunit targeting of the protein phosphatase 2A holoenzyme to Akt. J Biol Chem, 283(4): 1882-1892

11 Lee C H, Jeon Y T, Kim S H, Song Y S (2007). NF-kappaB as a potential molecular target for cancer therapy. Biofactors, 29(1): 19-35

12 Li H, Lin X (2008). Positive and negative signaling components involved in TNFalpha-induced NF-kappaB activation. Cytokine, 41(1): 1-8

13 Li S, Wang L, Berman M A, Zhang Y, Dorf M E (2006). RNAi screen in mouse astrocytes identifies phosphatases that regulate NF-kappaB signaling. Mol Cell, 24(4): 497-509

14 Lin X, Duan X, Liang Y Y, Su Y, Wrighton K H, Long J, Hu M, Davis C M, Wang J, Brunicardi F C, Shi Y, Chen Y G, Meng A, Feng X H (2006). PPM1A functions as a Smad phosphatase to terminate TGFbeta signaling. Cell, 125(5): 915-928

15 Liu J, Weiss H L, Rychahou P, Jackson L N, Evers B M, Gao T (2009). Loss of PHLPP expression in colon cancer: role in proliferation and tumorigenesis. Oncogene, 28(7): 994-1004

16 Lu G, Wang Y (2008). Functional diversity of mammalian type 2C protein phosphatase isoforms: new tales from an old family. Clin Exp Pharmacol Physiol, 35(2): 107-112

17 Madrid L V, Wang C Y, Guttridge D C, Schottelius A J, Baldwin A S Jr, Mayo M W (2000). Akt suppresses apoptosis by stimulating the transactivation potential of the RelA/p65 subunit of NF-kappaB. Mol Cell Biol, 20(5): 1626-1638

18 Madrid L V, Mayo M W, Reuther J Y, Baldwin A S Jr (2001). Akt stimulates the transactivation potential of the RelA/p65 Subunit of NF-kappa B through utilization of the Ikappa B kinase and activation of the mitogen-activated protein kinase p38. J Biol Chem, 276(22): 18934-18940

19 Mumby M C, Walter G (1993). Protein serine/threonine phosphatases: structure, regulation, and functions in cell growth. Physiol Rev, 73(4): 673-699

20 Okazaki T, Sakon S, Sasazuki T, Sakurai H, Doi T, Yagita H, Okumura K, Nakano H (2003). Phosphorylation of serine 276 is essential for p65 NF-kappaB subunit-dependent cellular responses. Biochem Biophys Res Commun, 300(4): 807-812

21 Park K A, Byun H S, Won M, Yang K J, Shin S, Piao L, Kim J M, Yoon W H, Junn E, Park J, Seok J H, Hur G M (2007). Sustained activation of protein kinase C downregulates nuclear factor-kappaB signaling by dissociation of IKK-gamma and Hsp90 complex in human colonic epithelial cells. Carcinogenesis, 28(1): 71-80

22 Sakurai H, Chiba H, Miyoshi H, Sugita T, Toriumi W (1999). IkappaB kinases phosphorylate NF-kappaB p65 subunit on serine 536 in the transactivation domain. J Biol Chem, 274(43): 30353-30356

23 Song G, Ouyang G, Bao S (2005). The activation of Akt/PKB signaling pathway and cell survival. J Cell Mol Med, 9(1): 59-71

24 Sun Z, Andersson R (2002). NF-kappaB activation and inhibition: a review. Shock, 18(2): 99-106

25 Sun W, Yu Y, Dotti G, Shen T, Tan X, Savoldo B, Pass A K, Chu M, Zhang D, Lu X, Fu S, Lin X, Yang J (2009). PPM1A and PPM1B act as IKKbeta phosphatases to terminate TNFalpha-induced IKKbeta-NF-kappaB activation. Cell Signal, 21(1): 95-102

26 Vermeulen L, De Wilde G, Van Damme P, Vanden Berghe W, Haegeman G (2003). Transcriptional activation of the NF-kappaB p65 subunit by mitogen- and stress-activated protein kinase-1 (MSK1). EMBO J, 22(6): 1313-1324

27 Viatour P, Merville M P, Bours V, Chariot A (2005). Phosphorylation of NF-kappaB and IkappaB proteins: implications in cancer and inflammation. Trends Biochem Sci, 30(1): 43-52

28 Xiao L, Gong L L, Yuan D, Deng M, Zeng X M, Chen L L, Zhang L, Yan Q, Liu J P, Hu X H, Sun S M, Liu J, Ma H L, Zheng C B, Fu H, Chen P C, Zhao J Q, Xie S S, Zou L J, Xiao Y M, Liu W B, Zhang J, Liu Y, Li D W (2010). Protein phosphatase-1 regulates Akt1 signal transduction pathway to control gene expression, cell survival and differentiation. [Epub ahead of print] Cell Death Differ. DOI?

29 Yamamoto Y, Gaynor R B (2001). Therapeutic potential of inhibition of the NF-kappaB pathway in the treatment of inflammation and cancer. J Clin Invest, 107(2): 135-142

30 Zhong H, Voll R E, Ghosh S (1998). Phosphorylation of NF-kappa B p65 by PKA stimulates transcriptional activity by promoting a novel bivalent interaction with the coactivator CBP/p300. Mol Cell, 1(5): 661-671

31 Zhong H, May M J, Jimi E, Ghosh S (2002). The phosphorylation status of nuclear NF-kappa B determines its association with CBP/p300 or HDAC-1. Mol Cell, 9(3): 625-636