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

doi:10.1007/s11515-010-0050-5

Front Biol

2010, Vol. 5

Issue (3) : 263-271 https://doi.org/10.1007/s11515-010-0050-5

RESEARCH ARTICLE

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

Guoxin WANG, Suping LI, Feifei WANG, Shufang HUANG, Xian LI, Wei XIONG, Biliang ZHANG(

)

Laboratory of RNA Chemical Biology, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China

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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

Corresponding Author(s): ZHANG Biliang,Email:zhang_biliang@gibh.ac.cn

Issue Date: 01 June 2010

Cite this article:

Guoxin WANG,Suping LI,Feifei WANG, et al. RNAi screen to identify protein phosphatases that regulate the NF-kappaB signaling[J]. Front Biol, 2010, 5(3): 263-271.

URL:

https://academic.hep.com.cn/fib/EN/10.1007/s11515-010-0050-5
https://academic.hep.com.cn/fib/EN/Y2010/V5/I3/263

Fig.1 Effects of protein serine/threonine phosphatases (partial) siRNAs on NF-kB transcriptional activities. A: The NF-kB transcriptional activity in HeLa cells following TNFa stimulation was determined by the luciferase reporter gene assay as described in section. The data were normalized to the percentage of the no targeting siRNA control (Notarget), and the means of three independent experiments were presented. B: The NF-kB reporter assay in A549 cells transfected with positive candidates in primary screen was measured. A value of <0.05, compared with Notarget, was considered significant. C: The transcriptional activity of NF-kB was determined by qRT-PCR detection of the expression of a well-known NF-kB target gene COX-2 in HeLa cells stimulated by TNFa. The NF-kB/p65 siRNA was used as positive control. And the efficiency of corresponding siRNAs was shown in Fig. S1 in the supplemental data. The results were normalized against the control, and presented as mean±SE of three experiments. **: <0.001; *: <0.05.

Tab.1 Identification of phosphatase genes regulating NF-κB activity in HeLa cells

Fig.2 Effects of PHLPP siRNA on nuclear translocation of p65. The distribution of p65 in HeLa cells transfected with control siRNA or candidate siRNAs for 48 h, followed by treatment with or without TNFa for an indicated time, was determined by p65 nuclear translocation assay (A) and by Western blot analysis of nuclear-cytoplasmic fractions of p65 (B) as described in section. Histone H3 (H3) was used as a marker of the nuclear extract. The data were shown as mean±SE of three experiments. *: <0.05.

Fig.3 Effects of PHLPP siRNA on cellular IkBa content and phosphorylation of IkBa. HeLa cells were transfected with the control siRNA and PHLPP siRNA for 48 h, followed by treatment with or without TNFa for an indicated time. Phosphorylation of IkBa and total IkBa were respectively detected by Western blot as described in section.

Fig.4 Effects of PHLPP and candidate kinases siRNAs on p65 phosphorylation and cellular IkBa. A: Effects of PHLPP and IKKb on TNFa-induced phosphorylation of p65 Ser276. The efficiency of IKKb siRNA was shown in Fig. S1 in the supplemental data. B: Effects of candidate kinases siRNAs on phosphorylation of p65 Ser276 and cellular IkBa exposed to TNFa.

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