Cortactin is involved in transforming growth factor-β1-induced epithelial-mesenchymal transition in AML-12 cells

Cortactin is an F-actin binding protein, regulating cell movement and adhesive junction assembly. However, the function of cortactin in epithelial-mesenchymal transition （EMT） remains elusive. Here we found that during transforming growth factor-β1 （TGF-β1）- induced EMT in AML-12 murine hepatocytes, cortactin underwent tyrosine dephosphorylation. Inhibition of the dephosphorylation of eortactin by sodium vanadate blocked TGF-β1-induced EMT. Knockdown of cortactin by RNAi led to decrease of intercellular junction proteins E-cadherin and Zonula occludens-1 and induced expression of mesenchymal protein fibronectin. Additionally, knockdown of cortactin further promoted TGF-β1-induced EMT in AML-12 cells, as determined by EMT markers and cell morphological changes. Moreover, migration assay showed that cortactin knockdown promoted the migration of AML-12 cells, and also enhanced TGF-β1-induced migration. Our study showed the involvement of cortactin in the TGF- β1-induced EMT.     

Phosphorylation of a 27-kDa protein correlates with survival of protein-synthesis-inhibited MCF-7 cells

Summary Previously, we have shown that IGF-1, the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA) and aurintricarboxylic acid (ATA) protected MCF-7 cells against death induced by the protein synthesis inhibitor cycloheximide (CHX). We proposed that phosphorylation of a putative cellular protein(s) may be involved in this survival mechanism. In the present study we investigated the ability of several agents to induce phosphorylation of cellular proteins and correlated this ability to their survival effect. We found that TPA, ATA, and IGF-1 increased the degree of phosphorylation of a 27-kDa protein in a dose- and time-dependent manner in CHX-treated MCF-7 cells. The ED₅₀ values observed were 25 ng/ml, 40 μg/ml and 15 ng/ml for TPA, ATA, and IGF-1, respectively. The effect was measured upon 10 min of cell treatment with each agent; it reached maximum at 60 min and thereafter decreased continuously to control levels. The 27-kDa protein was found in the cytosolic fraction as a phosphorylated serine residue. Further characterization with two-dimensional electrophoresis indicated that the 27-kDa phosphoprotein was resolved into two isoforms with pI 5.7 and 5.9. Such characteristics were observed for the small molecular weight heat shock protein HSP27. Indeed, a single band of 27 kDa was detected immunologically with rabbit polyclonal anti-human HSP27. The inactive phorbol ester αTPA, epidermal growth factor (EGF), and 8-bromoadenosine 3′5′-cyclic monophosphate (Br-cAMP) did not increase phosphorylation of the 27-kDa protein. Cell survival was measured by exposure of the CHX-pretreated cells to increasing concentrations of the various agents for 60 min, followed by a further incubation for 48 h in the presence of CHX only. TPA, ATA, and IGF-1 were found to enhance cell survival, whereas αTPA, EGF, and Br-cAMP did not. Our results indicate a correlation between phosphorylation of a 27-kDa protein, probably HSP27, and enhanced cell survival, suggesting a role for this phosphoprotein in the survival mechanism.     

TGF-β promotes invasion and metastasis of gastric cancer cells by increasing fascinl expression via ERK and JNK signal pathways

Transforming growth factor-β（TGF-β） is involved in actin cytoskeleton reorganization and tumor progression. Fascinl, an actin-binding protein, increases cell invasiveness and motility in various transformed cells. To determine whether fascinl is an important mediator of the tumor response to TGF-β, we applied the small interfering RNA （siRNA） technique to silence fascinl in gastric cancer （GC） cells MKN45. Results showed that the effects of TGF-β1 on GC cells invasion and metastasis were mediated by tumor production of fascinl; furthermore, it was found that TGF-β1- induced fascinl expression was suppressed by the specific inhibitors of JNK and ERK pathways, SP6001125 and PD98059, respectively, but not by transient transfection of Smad2 and Smad4 siRNA. Our data for the first time demonstrated that fascin 1 is an important mediator of TGF-β1-induced invasion and metastasis of GC cells, which involves JNK and ERK signaling pathways.     

TGF-β1-promoted epithelial-to-mesenchymal transfor mation and cell adhesion contribute to TGF-β1-enhanced cell migration in SMMC-7721 cells

Transforming growth factor-bl (TGF-β1), a multi-function polypeptide, is a double-edged sword in cancer. For some tumor cells, TGF-β1 is a potent growth inhibitor and apoptosis inducer. More commonly, TGF-β1 losesits growth-inhibitory and apoptosis-inducing effects, but stimulates the metastatic capacity of tumor cells. It is currently little known about TGF-β1-promoted cell migration in hepatocellular carcinoma (HCC) cells, let alone its mechanism. In this study, we found that TGF-β1 lost its tumor-suppressive effects, but significantly stimulated cellmigration in SMMC-7721 human HCC cells. By FACS and Western blot analysis, we observed that TGF-β1 enhanced the expression of ct5131 integrin obviously, and subsequently stimulated cell adhesion onto fibronectin(Fn). Furthermore, we observed that TGF-β1 could also promote SMMC-7721 cells adhesion onto laminin (Ln).Our data also provided evidences that TGF-β1 induced epithelial-to-mesenchymal transformation (EMT) in SMMC-7721 cells. First, SMMC-7721 cells clearly switched to the spindle shape morphology after TGF-β1 treatment.Furthermore, TGF-β1 induced the down-regulation of E-cadherin and the nuclear translocation of β1-catenin. These results indicated that TGF-β1-promoted cell adhesion and TGF-β1-induced epithelial-to-mesenchymal transfor-mation might be both responsible for TGF-β1-enhanced cell migration.     

Enhancement of Metastatic and Invasive Capacity of Gastric Cancer Cells by Transforming Growth Factor-β1

Transforming growth factor-β (TGF-β),a multifunctional cytokine,exerts contradictory rolesin different kinds of cells.A number of studies have revealed its involvement in the progression of many typesof tumors.To investigate the effect of TGF-β on gastric carcinoma,SGC7901,BGC823 and MKN28 (aTGF-β-resistant cell line) adenocarcinoma clones were used.After pretreatment in serum-free medium withor without 10 ng/ml TGF-β1,their experimental metastatic potential,chemotaxis,and invasive and adhesiveability were measured.Furthermore,zymography for gelatinase was processed.Liver colonies were alsomeasured 4 weeks after inoculation of SGC7901,BGC823 and MKN28 in Balb/c nude mice,and an increasein the number of surface liver metastases was seen in SGC7901 (from 11.0±3.0 to 53.3±3.3) and BGC823(from 9.3±2.5 to 60.0±2.8) groups,whereas there was no difference between MKN28 groups (from 35.2±3.8 to 38.5±2.7).In vitro experiments showed that TGF-β1 increased the adhesion capacity of SGC7901and BGC823 cells to immobilized reconstituted basement membrane/fibronectin matrices and promoted theirpenetration through reconstituted basement membrane barriers.Zymography demonstrated that enhancedinvasive potential was partly due to the increased type Ⅳ collagenolytic (gelatinolytic) activity,but there wasno difference in type Ⅳ collagenolytic activity and other biological behaviors between MKN28 groups.Theseresults suggested that TGF-β1 might modulate the metastatic potential of gastric cancer cells by promotingtheir ability to break down and penetrate basement membrane barriers and their adhesive and motile activities.We speculated that TGF-β1 might act as a progression-enhancing factor in gastric cancer.Therefore blockageof TGF-β or TGF-β signaling might prevent gastric cancer cells from invading and metastasizing.     

Proliferation and tenogenic differentiation of bone marrow mesenchymal stem cells in a porous collagen sponge scaffold

BACKGROUND Collagen is one of the most commonly used natural biomaterials for tendon tissue engineering.One of the possible practical ways to further enhance tendon repair is to combine a porous collagen sponge scaffold with a suitable growth factor or cytokine that has an inherent ability to promote the recruitment,proliferation,and tenogenic differentiation of cells.However,there is an incomplete understanding of which growth factors are sufficient and optimal for the tenogenic differentiation of rat bone marrow mesenchymal stem cells(BMSCs)in a collagen sponge-based 3D culture system.AIM To identify one or more ideal growth factors that benefit the proliferation and tenogenic differentiation of rat BMSCs in a porous collagen sponge scaffold.METHODS We constructed a 3D culture system based on a type I collagen sponge scaffold.The surface topography of the collagen sponge scaffold was observed by scanning electron microscopy.Primary BMSCs were isolated from Sprague-Dawley rats.Cell survival on the surfaces of the scaffolds with different growth factors was assessed by live/dead assay and CCK-8 assay.The mRNA and protein expression levels were confirmed by quantitative real-time polymerase chain reaction and Western blot,respectively.The deposited collagen was assessed by Sirius Red staining.RESULTS Transforming growth factorβ1(TGF-β1)showed great promise in the tenogenic differentiation of BMSCs compared to growth differentiation factor 7(GDF-7)and insulin-like growth factor 1(IGF-1)in both the 2D and 3D cultures,and the 3D culture enhanced the differentiation of BMSCs into tenocytes well beyond the level of induction in the 2D culture after TGF-β1 treatment.In the 2D culture,the proliferation of the BMSCs showed no significant changes compared to the control group after TGF-β1,IGF-1,or GDF-7 treatment.However,TGF-β1 and GDF-7 could increase the cell proliferation in the 3D culture.Strangely,we also found more dead cells in the BMSC-collagen sponge constructs that were treated with TGF-β1.Moreover,TGF-β1 promoted more collagen deposition in both the 2D and 3D cultures.CONCLUSION Collagen sponge-based 3D culture with TGF-β1 enhances the responsiveness of the proliferation and tenogenic differentiation of rat BMSCs.     

Effect of 5-azacytidine on the protein expression of porcine bone marrow mesenchymal stem cells in vitro

Bone marrow-derived mesenchymal stem cells （MSCs） are pluripotent stem cells that show a vital potential in the clinical application for cell transplantation. In the present paper, proteomic techniques were used to approach the protein profiles associated with porcine bone marrow MSCs and investigate the regulation of MSC proteins on the effect of 5-azacytidine （5-aza）. Over 1,700 protein species were separated from MSCs according to gel analysis. Compared with the expression profiling of control MSCs, there were 11 protein spots up-regulated and 26 downregulated in the protein pattern of 5-aza-treated cells. A total of 21 proteins were successfully identified by MALDI-TOF-MS analysis, among which some interesting proteins, such as alpha B-crystallin, annexin A2, and stathmin 1, had been reported to involve in cell proliferation and differentiation through different signaling pathways. Our data should be useful for the future study of MSC differentiation and apoptosis.     

Heat shock protein 27 as a prognostic and predictive biomarker in pancreatic ductal adenocarcinoma

A role of heat shock protein 27 (HSP27) as a potential biomarker has been reported in various tumour entities, but comprehensive studies in pancreatic cancer are lacking. Applying tissue microarray (TMA) analysis, we correlated HSP27 protein expression status with clinicopathologic parameters in pancreatic ductal adenocarcinoma specimens from 86 patients. Complementary, we established HSP27 overexpression and RNA-interference models to assess the impact of HSP27 on chemo- and radiosensitivity directly in pancreatic cancer cells. In the TMA study, HSP27 expression was found in 49% of tumour samples. Applying univariate analyses, a significant correlation was found between HSP27 expression and survival. In the multivariate Cox-regression model, HSP27 expression emerged as an independent prognostic factor. HSP27 expression also correlated inversely with nuclear p53 accumulation, indicating either protein interactions between HSP27 and p53 or TP53 mutation-dependent HSP27-regulation in pancreatic cancer. In the sensitivity studies, HSP27 overexpression rendered HSP27 low-expressing PL5 pancreatic cancer cells more susceptible towards treatment with gemcitabine. Vice versa, HSP27 protein depletion in HSP27 high-expressing AsPC-1 cells caused increased gemcitabine resistance. Importantly, HSP27 expression was inducible in pancreatic cancer cell lines as well as primary cells. Taken together, our study suggests a role for HSP27 as a prognostic and predictive marker in pancreatic cancer. Assessment of HSP27 expression could thus facilitate the identification of specific patient subpopulations that might benefit from individualized treatment options. Additional studies need to clarify whether modulation of HSP27 expression could represent an attractive concept to support the incorporation of hyperthermia in clinical treatment protocols for pancreatic cancer.     

Absence of a human DnaJ protein hTid-1S correlates with aberrant actin cytoskeleton organization in lesional psoriatic skin

The biochemical mechanism by which the human tumorous imaginal disc1(S) (hTid-1(S)) interferes with actin cytoskeleton organization in keratinocytes of human skin epidermis was investigated. We found that hTid-1, specifically hTid-1(S), interacts with MK5, a p38-regulated/activated protein kinase, and inhibits the protein kinase activity of MK5 that phosphorylates heat shock protein HSP27 in cultured HeLa cells. Thus, hTid-1(S) expression inhibits the phosphorylation of HSP27 known to play important roles in F-actin polymerization and actin cytoskeleton organization. The interplay between MK5/HSP27 signaling and hTid-1(S) expression was supported by the inhibition of HSP27 phosphorylation and MK5 activity in HeLa cells in response to hypoxia during which hTid-1(S) expression was down-regulated. We also found that overexpression of hTid-1(S) results in the inhibition of HSP27 phosphorylation, F-actin polymerization, and actin cytoskeleton organization in transduced HaCaT keratinocytes. This study further proposes that the loss of hTid-1(S) expression in the basal layer of skin epidermis correlates with the enhanced HSP27 phosphorylation, keratinocyte hyperproliferation, and excess actin cytoskeleton organization in lesional psoriatic skin.     

Phosphorylation of heat shock protein 27 antagonizes TNF-α induced HeLa cell apoptosis via regulating TAK1 ubiquitination and activation of p38 and ERK signaling

Tumor necrosis factor (TNF)-α is a potent cytokine that regulates critical cellular processes including apoptosis. TNF-α usually triggers both survival and apoptotic signals in various cell types. Heat shock protein 27 (HSP27), an important cellular chaperone, is believed to protect cells from apoptosis. HSP27 can be phosphorylated and changed its cellular function according to different stimuli. However, available reports on the role of HSP27 phosphorylation in apoptosis remain elusive. In this study, we investigated the role of HSP27 phosphorylation in TNF-α induced apoptosis in human cervical carcinoma (HeLa) cells. We found that TNF-α induced apoptosis was enhanced if we suppressed the TNF-α induced HSP27 phosphorylation by specific inhibitor CMPD1 or MAPKAPK2 (MK2) knockdown or by overexpression of non-phosphorylatable mutant HSP27-3A. Through co-immunoprecipitation and confocal microscopy, we observed that HSP27 associated with transforming growth factor-β (TGF-β)-activated kinase 1 (TAK1) in response to TNF-α stimulation. By blocking MK2 activity or overexpressing phospho-mimetic mutant Hsp27-3D, we further showed that HSP27 phosphorylation facilitated the TNF-α induced ubiquitination and phosphorylation of TAK1 and the activations of p38 MAPK and ERK, the TAK1 downstream pro-survival signaling. In addition, we also found that increased HSP27 phosphorylation inhibited TRADD ubiquitination but did not influence the binding between TRADD and FADD in a pro-apoptotic complex. Taken together, our data indicated that HSP27 phosphorylation was involved in modulating the TNF-α induced apoptosis via interacting with TAK1 and regulating TAK1 post-translational modifications in HeLa cells. This study demonstrates that HSP27 phosphorylation serves as a novel regulator in TNF-α-induced apoptosis, and provides a new insight into the cytoprotective role of HSP27 phosphorylation.     

Proteomic Characterization of Annexin l (ANX1) and Heat Shock Protein 27 (HSP27) as Biomarkers for Invasive Hepatocellular Carcinoma Cells

To search for reliable biomarkers and drug targets for management of hepatocellular carcinoma (HCC), we performed a global proteomic analysis of a pair of HCC cell lines with distinct differentiation statuses using 2-DE coupled with MALDI-TOF MS. In total, 106 and 55 proteins were successfully identified from the total cell lysate and the cytosolic, nuclear and membrane fractions in well-differentiated (HepG2) and poorly differentiated (SK-Hep–1) HCC clonal variants, respectively. Among these proteins, nine spots corresponding to proteins differentially expressed between HCC cell types were selected and confirmed by immunofluorescence staining and western blotting. Notably, Annexin 1 (ANX1), ANX–2, vimentin and stress-associated proteins, such as GRP78, HSP75, HSC–70, protein disulfide isomerase (PDI), and heat shock protein–27 (HSP27), were exclusively up-regulated in SK-Hep–1 cells. Elevated levels of ANX–4 and antioxidant/metabolic enzymes, such as MnSOD, peroxiredoxin, NADP-dependent isocitrate dehydrogenase, α-enolase and UDP-glucose dehydrogenase, were observed in HepG2 cells. We functionally demonstrated that ANX1 and HSP27 were abundantly overexpressed only in highly invasive types of HCC cells, such as Mahlavu and SK-Hep–1. Knockdown of ANX1 or HSP27 in HCC cells resulted in a severe reduction in cell migration. The in-vitro observations of ANX1 and HSP27 expressions in HCC sample was demonstrated by immunohistochemical stains performed on HCC tissue microarrays. Poorly differentiated HCC tended to have stronger ANX1 and HSP27 expressions than well-differentiated or moderately differentiated HCC. Collectively, our findings suggest that ANX1 and HSP27 are two novel biomarkers for predicting invasive HCC phenotypes and could serve as potential treatment targets.     

Regulated expression of cofilin and the consequent regulation of p27kip1 are essential for G1 phase progression

Cofilin, a ubiquitously expressed actin binding protein, is responsible for the formation of the actin cytoskeleton and is indispensable for cell cycle control. However, the association between cofilin expression and the cell cycle remains to be elucidated. In this study, we found that the expression level of cofilin up-regulated in G1 phase-arrested confluent cells, while knockdown of cofilin expression by small interference RNA (siRNA) in these cells led to a reduction in the population of G1 cells. To investigate the role of cofilin in the control of G1 phase progression, a tet-on gene expression system was introduced to over-express different concentrations of cofilin in cells. The results showed that G1 phase progression was blocked following induction of exogenous cofilin. A survey of the cell cycle proteins controlling the G1 phase progression revealed that the cyclin-dependent kinase inhibitor (CKI) p27kip1 was the primary molecule induced by over-expressed cofilin in a time and dose dependent manner. Up-regulated p27kip1 repressed phosphorylation of the retinoblastoma protein (Rb) mediated by cyclin D1/CDK4 activity. Conversely, siRNA against p27kip1 expression in the cofilin over-expressing cells released the G1 phase arrest. Furthermore, we found that over-expression of cofilin led to induction of p27kip1 gene promoter transactivation using luciferase reporter gene assay. This effect was associated with increase of p27kip1 mRNA transiently. In addition, inhibition of threonine-187 phosphorylation of p27kip1 protein for ubiquitinyl-proteasomal mediated degradation was also involved in up-regulation of p27 kip1. These data suggest that cofilin expression and its regulation of p27kip1 expression is important for the control of G1 phase progression.     

Contrasting effects of midazolam on induction of heat shock protein 27 by vasopressin and heat in aortic smooth muscle cells.

We previously showed that vasopressin stimulates the induction of heat shock protein (HSP) 27, a low molecular-weight HSP, through protein kinase C activation in aortic smooth muscle A10 cells. In the present study, we examined the effects of midazolam, an intravenous anesthetic, on the HSP27 induction stimulated by vasopressin, heat, or sodium arsenite (arsenite) in A10 cells. Midazolam inhibited the accumulation of HSP27 induced by vasopressin or 12-O-tetradecanoylphorbol 13-acetate (TPA), a direct activator of protein kinase C. Midazolam also reduced the vasopressin-induced level of the mRNA for HSP27. In contrast, midazolam enhanced the HSP27-accumulation induced by heat or arsenite. Midazolam also enhanced the heat-increased level of the mRNA for HSP27. However, midazolam had no effect on the dissociation of the aggregated form of HSP27 following stimulation by vasopressin, heat, or arsenite. These results suggest that midazolam suppresses vasopressin-stimulated HSP27 induction in vascular smooth muscle cells, and that this inhibitory effect is exerted at a point downstream from protein kinase C. In contrast, midazolam enhanced heat- or arsenite-stimulated HSP27 induction. Thus, midazolam has dual effects on the HSP27 induction stimulated by various stresses in vascular smooth muscle cells.     

The small heat shock-related protein, HSP20, is a cAMP-dependent protein kinase substrate that is involved in airway smooth muscle relaxation

Activation of the cAMP/cAMP-dependent PKA pathway leads to relaxation of airway smooth muscle (ASM). The purpose of this study was to examine the role of the small heat shock-related protein HSP20 in mediating PKA-dependent ASM relaxation. Human ASM cells were engineered to constitutively express a green fluorescent protein-PKA inhibitory fusion protein (PKI-GFP) or GFP alone. Activation of the cAMP-dependent signaling pathways by isoproterenol (ISO) or forskolin led to increases in the phosphorylation of HSP20 in GFP but not PKI-GFP cells. Forskolin treatment in GFP but not PKI-GFP cells led to a loss of central actin stress fibers and decreases in the number of focal adhesion complexes. This loss of stress fibers was associated with dephosphorylation of the actin-depolymerizing protein cofilin in GFP but not PKI-GFP cells. To confirm that phosphorylated HSP20 plays a role in PKA-induced ASM relaxation, intact strips of bovine ASM were precontracted with serotonin followed by ISO. Activation of the PKA pathway led to relaxation of bovine ASM, which was associated with phosphorylation of HSP20 and dephosphorylation of cofilin. Finally, treatment with phosphopeptide mimetics of HSP20 possessing a protein transduction domain partially relaxed precontracted bovine ASM strips. In summary, ISO-induced phosphorylation of HSP20 or synthetic phosphopeptide analogs of HSP20 decreases phosphorylation of cofilin and disrupts actin in ASM, suggesting that one possible mechanism by which HSP20 mediates ASM relaxation is via regulation of actin filament dynamics.     

Identification of breast cancer metastasis-associated proteins in an isogenic tumor metastasis model using two-dimensional gel electrophoresis and liquid chromatography-ion trap-mass spectrometry

To better understand the molecular mechanisms underlying breast cancer metastasis and search for potential markers for metastatic progression, we have developed a highly metastatic variant of human MDA-MB-435 breast cancer cell line through in vivo stepwise selection of pulmonary metastatic cells caused by parental MDA-MB-435 cells in the athymic mice. Comparative proteomic analysis using 2-DE and LC-IT-MS revealed that 102 protein spots were reproducibly altered more than three-fold between the selected variant and its parental counterpart. Eleven differentially expressed protein spots were identified with high confidence using SEQUEST with uninterpreted tandem mass raw data. Cathepsin D precursor, peroxiredoxin 6 (PDX6), heat shock protein 27 (HSP27), HSP60, tropomyosin 1 (TPM1), TPM2, TPM3, TPM4, 14-3-3 protein epsilon, and tumor protein D54 were up-regulated in the highly metastatic variant, whereas alpha B-crystalline (CRAB) was only detected in its parental counterpart. Differential expression was confirmed for four proteins including PDX6, CRAB, TPM4, and HSP60 by real-time quantitative PCR and Western blotting analysis in our model. Immunohistochemical analysis in 80 breast cancer donors demonstrated a significant association of TPM4 (p = 0.002), HSP60 (p = 0.001), PDX6 (p = 0.002) but not CRAB (p = 0.113) staining with the presence of lymph node metastasis. In addition, TPM4 staining was also associated with clinical stage (p = 0.000), but no significant association was found between TPM4, PDX6, CRAB, and HSP60 expression and tumor size, hormone receptor, and HER-2 status (p > 0.05). The functional implication of these identified proteins was also discussed. These proteomic data are valuable and informative for understanding breast cancer metastasis and searching for potential markers for metastatic progression.     

HSP27 phosphorylation modulates TRAIL-induced activation of Src-Akt/ERK signaling through interaction with β-arrestin2

Heat shock protein 27 (HSP27) regulates critical cellular functions such as development, differentiation, cell growth and apoptosis. A variety of stimuli induce the phosphorylation of HSP27, which affects its cellular functions. However, most previous studies focused on the role of HSP27 protein itself in apoptosis, the particular role of its phosphorylation state in signaling transduction remains largely unclear. In the present study, we reported that HSP27 phosphorylation modulated TRAIL-triggered pro-survival signaling transduction. In HeLa cells, suppression of HSP27 phosphorylation by specific inhibitor KRIBB3 or MAPKAPK2 (MK2) knockdown and by overexpression of non-phosphorylatable HSP27(3A) mutant demonstrated that hindered HSP27 phosphorylation enhanced the TRAIL-induced apoptosis. In addition, reduced HSP27 phosphorylation by KRIBB3 treatment or MK2 knockdown attenuated the TRAIL-induced activation of Akt and ERK survival signaling through suppressing the phosphorylation of Src. By overexpression of HSP27(15A) or HSP27(78/82A) phosphorylation mutant, we further showed that phosphorylation of HSP27 at serine 78/82 residues was essential to TRAIL-triggered Src-Akt/ERK signaling transduction. Co-immunoprecipitation and confocal microscopy showed that HSP27 interacted with Src and scaffolding protein β-arrestin2 in response of TRAIL stimulation and suppression of HSP27 phosphorylation apparently disrupted the TRAIL-induced interaction of HSP27 and Src or interaction of HSP27 and β-arrestin2. We further demonstrated that β-arrestin2 mediated HSP27 action on TRAIL-induced Src activation, which was achieved by recruiting signaling complex of HSP27/β-arrestin2/Src in response to TRAIL. Taken together, our study revealed that HSP27 phosphorylation modulates TRAIL-triggered activation of Src-Akt/ERK pro-survival signaling via interacting with β-arrestin2 in HeLa cells.