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  current news   Press   selected story    
     
  5 January 2011  
  Congratulations to IMCB’s recent PhD graduate
 
 



PhD Graduate: Li Fang KOH
Thesis Title: Regulation of NF-kappaB by MK2

Abstract
Activation of NF-κB in response to many stimuli is dependent upon signaling through the IKK complex. Although the core signaling components of IKK complex are well defined they are also subject to modulatory effects by parallel signaling pathways, which can act to fine-tune the NF-κB response, potentially through effects on IKK activity and targeting phosphorylation of the NF-κB subunits themselves.

MAPKAP kinase 2 (MK2) is highly homologous to the DNA damage induced checkpoint kinase Chk1, which has previously been shown to modulate NF-κB activity by direct phosphorylation of RelA at Thr 505. I have evaluated the role of MK2 on the NF-κB mediated stress response. Many inducers of NF-κB activity, such as LPS or the ribotoxic stress agent anisomycin, are also inducers of MK2 activity.

Anisomycin treatment results in the phosphorylation of IKK and RelA at Ser 468. Significantly, it was found that both of these processes are dependent upon MK2. Inhibition of MK2 results in a decrease in IKK phosphorylation and a loss of Ser 468 modification. EMSA analysis indicated that MK2 is required for NF-κB DNA binding, and MK2 is also required for the expression of NF-κB targets that are activated by anisomycin (A20, c-IAP2, MCP-1).

Analysis of NF-κB activation downstream of toll-like receptor ligands also shows MK2 dependency. Results demonstrated that MK2 modulates NF-κB and IKK function downstream of TLR4 (Toll-like receptor 4) following LPS treatment. IKK phosphorylation and RelA phosphorylation are reduced in cells lacking MK2. In addition, DNA binding and expression of NF-κB target genes are also reduced in cells lacking MK2. Therefore, MK2 modulates NF-κB and IKK function following LPS treatment. The activation of NF-κB occurs via two distinct pathways (MyD88 and Trif dependent) downstream of TLR4. To ascertain if MK2 affects both or either of these pathways, NF-κB signaling downstream of other TLRs that engage only one of the two pathways, was examined. This revealed that MK2 affect both the MyD88 dependent and Trif dependent pathway. Knocking down MK2 inhibits NF-κB signaling downstream of TLR3, which activates only the Trif dependent pathway. Similarly, knocking down MK2 also inhibited NF-κB signaling downstream of TLR 9, which activates only the MyD88 dependent pathway.

This suggests that MK2 does not act on the upstream activators of NF-κB, which vary between different stimuli but on the core NF-κB core signaling components, which are engaged in response to all NF-κB activating stimuli. Significantly, co-immunoprecipitation experiments indicate that MK2 interacts with TAB1, an important component of the TAK1 complex, which is required for downstream NF-κB activation.

Taken together, I propose that in certain contexts, MK2 can function as an important modulator of NF-κB/IKK activity and could be an attractive target to clinically modulate NF-κB mediated responses following physiologically relevant stimuli.

Figure legend: MK2 is a essential for NF-κB signaling.
HEK293-hTLR4/MD2-CD14 cells were transfected with a control siRNA oligonucleotide and a siRNA oligonucleotide targeting MK2. The cells were treated with 1µg/µl of LPS for the indicated times, then harvested for their total protein or RNA. (A) Whole cell extracts were prepared, subjected to SDS and western blotted with the indicated antibodies. (B) Total RNA was extracted using RNeasy Mini Kit (Qiagen) and total cDNA was prepared. Quantitative PCR was performed using primers specific to human A20 and c-IAP2. All results are normalized to Actin control and expressed as fold change relative to control samples. Knockdown of MK2 in HEK293-hTLR4/MD2-CD14 cells significantly reduced gene expression of A20, MCP-1 and c-IAP2 after 2 and 4 hours of LPS treatment (p<0.05, n=3/group) and is noted with *. All data are presented as mean +/- SD.

For more information on Vinay TERGAONKAR’s lab, please click here.