HSP27 Phosphorylation Studies Against TNF-α-induced Apoptosis In HeLa Molecular Mechanisms

Tumour necrosis factor (TNF)-a is a multifunction cytokine that regulates critical cellular processes, including apoptosis. TNF-a itself exerts its biological effects interacting with two different receptors:TNFR1and TNFR2, TNFR1is the major receptor which expressed in almost all cell types, after binding to its TNFR1, TNF-a usually trigger both survival and apoptotic signals in various cell types. Heat shock protein27(HSP27), an important cellular chaperone, belongs to the small molecular weight heat shock protein (HSP) family (12-43kD), is believed to protect cells from apoptosis, regulate cytoskeletal remodeling, involve in regulating cell proliferation, differentiation signal. HSP27can be phosphorylation at serine residues15,78and82according to different stimuli. Once HSP27was phosphorylated it disaggregated from large oligomeric complexes to small oligomers, dimer or monomers, subsequently the biological functions of Hsp27were also changed. Many studies have shown that the phosphorylation status of HSP27correlates with the metastatic potential and chemotherapy and radiotherapy resistance of cancer cells. However, it has also been reported HSP27phosphorylation levels can also be inversely correlated with the progression of tumours. Thus, the role of HSP27phosphorylation in tumor is still contradiction.In this study, we investigated the role of phosphorylated HSP27in TNF-α induced human cervical carcinoma (HeLa) cells apoptosis and found that HSP27was phosphorylated upon TNF-a stimulation and suppression of HSP27phosphorylation by specific inhibitor CMPD1or MAPKAPK2(MK2) knockdown and by overexpression of nonphosphorylated mutant and phospho-mimetic mutants demonstrated that attenuated HSP27phosphorylation enhanced the TNF-a-induced apoptosis. Then, we analyzed the molecular mechanism of HSP27phosphorylation in the antagonism of TNF-a induced apoptosis in HeLa cells. By coimmunoprecipitation, we observed that HSP27associated with transforming growth factor-β (TGF-β)-activated kinase1(TAK1) in response to TNF-a stimulation. and, interestingly, CMPD1pretreatment resulted in the reduced complex formation of HSP27and TAK1, suggesting that phosphorylated HSP27could be composed into the complex. The co-localization of HSP27and TAK1was also detected by confocal microscopy. Result showed that HSP27and TAK1distributed in cytoplasm in the untreated HeLa cells, while their immunostainings obviously superposed and congregated in a compartment of cytoplasm upon stimulation of TNF-a. By contrast, suppression of HSP27phosphorylation largely reduced the congregation of HSP27and TAK1. By using CMPD1to block MK2activity or direct knockdown of MK2in order to hinder HSP27phosphorylation and by overexpression phospho-mimetic mutants Hsp27-3D, we further founded that phosphorylation of HSP27regulated TAK1ubiquitination, phosphorylation, and activation of downstream pro-survival molecules p38MAPK and ERK. In addition, our data also showed inhibition HSP27phosphorylation can promote ubiquitination of TRADD, but did not affect the binding of TRADD and FADD in HeLa cells. These results suggested HSP27phosphorylation not only regulated the ubiquitination of TAK1but also TRADD ubiquitination, and, the effects of HSP27phosphorylation on the ubiquitianation of TAK1and TRADD was opposite. Moreover, HSP27phosphorylation only regulated the pro-survival signal pathway, but did not affect the association of TRADD and FADD in apoptotic complex induced by TNF-a in HeLa cells. Taken together, our study revealed that HSP27phosphorylation regulating TAK1ubiquitination, phosphorylation and activation of p38and ERK pro-survival signaling pathway via interacting with TAK1in HeLa cells. This study demonstrates that HSP27phosphorylation serves as a novel regulator in TNF-a-induced apoptosis, and provides a new insight into the cytoprotective role of HSP27phosphorylation.