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.

Keywords

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

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

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