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The Role of RKIP in NF?B singaling pathway

by Tang, Huihui

Abstract (Summary)
RKIP (Raf Kinase Inhibitor Protein) was first identified as an inhibitor of Raf1 kinase in the Raf-MEK-ERK signaling pathway. It can interact with Raf1 and MEK and disrupt the interaction of Raf1 and MEK by competing with their binding. Since RKIP is a very abundant protein, it must have additional targets other than Raf-MEK-ERK pathway. Indeed, RKIP was also found to play a pivotal role in NF-?B pathway. NF-?B pathways are ubiquitous pathways that exist in many different tissues. NF-?B pathway is involved in different biological processes including inflammation and immune responses. NF-?B gets activated by phosphorylation and subsequent degradation of I?B in response to stimulation. Our studies show that RKIP interacts with multiple components of canonical NF-?B pathway in response to IL-1? stimulation. RKIP affected I?B degradation. Overexpression of RKIP inhibited I?B degradation, whereas downregulation of RKIP also inhibits I?B degradation. The inhibitory effect of I?B degradation in RKIP knockdown cells can be rescued by restoring RKIP. The effect of RKIP on I?B degradation was concentration dependent. The mode of action of RKIP on I?B degradation was consistent with the properties described for a scaffolding protein. A scaffolding protein’s main function is to bring other proteins together and allow them to interact. The signaling of the scaffolding cascade is dependent on the concentration of the scaffold protein. Both low and high concentration of scaffold protein will disrupt the interaction of the components of the scaffolding signaling cascade. Only intermediate concentration of scaffolding protein will allow for the optimal association of the different components in the signaling cascade, thus leads to the optimal signaling. One major feature of scaffolding protein is that it can interact with different components of the signaling cascade. Our studies show that RKIP interacts with TRAF6 and TAK1 of the canonical NF-?B pathway in both overexpression condition and physiological condition, and that the interaction of TAK1 and TRAF 6 was inhibited by knockdown of RKIP. Knockdown of RKIP inhibited phosphorylation of I?B, IKK? and TAK1. Ubiquitination of TRAF6 and TAK1 were also inhibited by the knockdown of RKIP. Once I?B was degraded, p50/p65 heterodimer would be released and then translocate to the nucleus. Our studies showed that both p50 and p65 nuclear translocation was inhibited in RKIP knockdown cells. Consistently, CHIP assays showed that binding of p65 with promoter of CIAP2, Rantes and I?B genes was also decreased in RKIP knockdown cells.
Bibliographical Information:

Advisor:

School:University of Toledo Health Science Campus

School Location:USA - Ohio

Source Type:Master's Thesis

Keywords:rkip pebp scaffold protein nfkb signaling pathway ubiquitination

ISBN:

Date of Publication:07/14/2009

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