Soo-A Kim

Regulation of myotubularin-related (MTMR)2 phosphatidylinositol phosphatase by MTMR5, a catalytically inactive phosphatase

The myotubularin (MTM) family constitutes one of the most highly conserved protein-tyrosine phosphatase subfamilies in eukaryotes. MTM1, the archetypal member of this family, is mutated in X-linked myotubular myopathy, whereas mutations in the MTM-related (MTMR)2 gene cause the type 4B1 Charcot–Marie-Tooth disease, a severe hereditary motor and sensory neuropathy. In this study, we identified a protein that specifically interacts with MTMR2 but not MTM1. The interacting protein was shown by mass spectrometry to be MTMR5, a catalytically inactive member of the MTM family. We also demonstrate that MTMR2 interacts with MTMR5 via its coiled-coil domain and that mutations in the coiled-coil domain of either MTMR2 or MTMR5 abrogate this interaction. Through this interaction, MTMR5 increases the enzymatic activity of MTMR2 and dictates its subcellular localization. This article demonstrates an active MTM member being regulated by an inactive family member.

Epigallocatechin-3-gallate suppresses the expression of HSP70 and HSP90 and exhibits anti-tumor activity in vitro and in vivo

BackgroundEpigallocatechin-3-gallate (EGCG), one of the major catechins in green tea, is a potential chemopreventive agent for various cancers. The aim of this study was to examine the effect of EGCG on the expression of heat shock proteins (HSPs) and tumor suppression.MethodsCell colony formation was evaluated by a soft agar assay. Transcriptional activity of HSP70 and HSP90 was determined by luciferase reporter assay. An EGCG-HSPs complex was prepared using EGCG attached to the cyanogen bromide (CNBr)-activated Sepharose 4B. In vivo effect of EGCG on tumor growth was examined in a xenograft model.ResultsTreatment with EGCG decreased cell proliferation and colony formation of MCF-7 human breast cancer cells. EGCG specifically inhibited the expression of HSP70 and HSP90 by inhibiting the promoter activity of HSP70 and HSP90. Pretreatment with EGCG increased the stress sensitivity of MCF-7 cells upon heat shock (44°C for 1 h) or oxidative stress (H2O2, 500 μM for 24 h). Moreover, treatment with EGCG (10 mg/kg) in a xenograft model resulted in delayed tumor incidence and reduced tumor size, as well as the inhibition of HSP70 and HSP90 expression.ConclusionsOverall, these findings demonstrate that HSP70 and HSP90 are potent molecular targets of EGCG and suggest EGCG as a drug candidate for the treatment of human cancer.

Sorting Nexin 9 Interacts with Dynamin 1 and N-WASP and Coordinates Synaptic Vesicle Endocytosis

Sorting nexin 9 (SNX9) is a member of the sorting nexin family of proteins, each of which contains a characteristic Phox homology domain. SNX9 is widely expressed and plays a role in clathrin-mediated endocytosis, but it is not known if it is present in neuronal cells. We report that SNX9 is expressed in the presynaptic compartment of cultured hippocampal neurons, where it binds to dynamin-1 and N-WASP. Overexpression of full-length SNX9 or a C-terminal truncated version caused severe defects in synaptic vesicle endocytosis during, as well as after, stimulation. Knockdown of SNX9 with short interfering RNA also reduced synaptic vesicle endocytosis, and the W39A mutation of SNX9 abolished the inhibitory effect of SNX9 on endocytosis. Rescue experiments showed that most of the effect of SNX9 on endocytosis results from its interaction with dynamin 1, although its interaction with N-WASP contributes in some degree. We further showed that SNX9 dimerizes through its C-terminal domain, suggesting that it may interact simultaneously with dynamin 1 and N-WASP. We propose that SNX9 interacts with dynamin-1 and N-WASP in presynaptic terminals, where it links actin dynamics and synaptic vesicle endocytosis.

Antitumor Activity of Novel Indirubin Derivatives in Rat Tumor Model

Purpose: The novel indirubin derivatives 5′-nitro-indirubinoxime, 5′-fluoro-indirubinoxime, and 5′-trimethylacetamino-indirubinoxime were designed and tested for antitumor activity both in vitro and in vivo using rat tumor model. Experimental Design: Three-week-old male Sprague-Dawley rats were inoculated s.c. on the left flank with 107 RK3E-ras rat kidney epithelial cells harboring k-ras gene. Alternatively, 5 × 106 RK3E-ras cells were injected into the oral mucosa. Indirubin derivative treatment began on the 3rd or 6th day after oral or s.c. cell injection, respectively. Indirubin derivatives were directly injected into the tumor every other day for a total of five times. Animals were monitored daily and tumor volume was measured by caliper. Results: Indirubin derivatives showed potent antiproliferative activity on various human cancer cells and oncogenic RK3E-ras rat kidney cells, with IC50 ranging from 1 to 12 μmol/L. Treatment with indirubin derivatives induced the activation of caspase-7 followed by apoptosis in RK3E-ras cells. Indirubin derivatives showed strong antitumor activity in rat solid and oral tumor models. Direct injection of indirubin derivatives every other day for 10 days induced significant inhibition of tumor growth in Sprague-Dawley rats bearing RK3E-ras-induced tumors. Histologically, treatment with indirubin derivatives caused significant inhibition of tumor formation with increased apoptosis and decreased tumor cell proliferation. Conclusions: Our data showed that novel indirubin derivatives 5′-nitro-indirubinoxime, 5′-fluoro-indirubinoxime, and 5′-trimethylacetamino-indirubinoxime effectively arrested the tumor growth by inhibiting cell proliferation and inducing apoptosis. These findings provide the potential value of indirubin derivatives as novel candidates for antitumor agents.

Crystal structure of the Golgi casein kinase

The family with sequence similarity 20 (Fam20) kinases phosphorylate extracellular substrates and play important roles in biomineralization. Fam20C is the Golgi casein kinase that phosphorylates secretory pathway proteins within Ser-x-Glu/pSer motifs. Mutations in Fam20C cause Raine syndrome, an osteosclerotic bone dysplasia. Here we report the crystal structure of the Fam20C ortholog from Caenorhabditis elegans. The nucleotide-free and Mn/ADP-bound structures unveil an atypical protein kinase-like fold and highlight residues critical for activity. The position of the regulatory αC helix and the lack of an activation loop indicate an architecture primed for efficient catalysis. Furthermore, several distinct elements, including the presence of disulfide bonds, suggest that the Fam20 family diverged early in the evolution of the protein kinase superfamily. Our results reinforce the structural diversity of protein kinases and have important implications for patients with disorders of biomineralization.

Toll-like receptor 5 activation promotes migration and invasion of salivary gland adenocarcinoma.

BACKGROUND Toll-like receptor (TLR) signaling has been found to be closely associated with tumor development. The aim of this study was to examine whether activation of TLRs promote migration and invasion of salivary gland adenocarcinoma. MATERIALS AND METHODS TLR expression in SGT and HSG cells was examined by RT-PCR. Wound scratch and chemotaxis cell migration assay were performed. Invasiveness was determined by Matrigel invasion assay. RESULTS All the tested TLRs including TLR1, TLR2, TLR4, and TLR5 and myeloid differentiation factor-2 (MD-2) were expressed on SGT and HSG cells. Treatment of flagellin, but not Pam(3) CSK(4) and LPS, led to the production of IL-6 and IL-8, suggesting TLR5 is functional in both cells. Stimulation by flagellin also accelerated wound closure of SGT and HSG cells in a dose-dependent manner. In addition, flagellin promoted migration and invasion ability of SGT cells. Blocking of TLR5 using antibody restored the promoting effect of flagellin on migration and invasion of SGT cells. CONCLUSION These findings suggest that TLR5 activation by flagellin can promote migration and invasion of salivary gland adenocarcinoma.

Sox4 stimulates ß-catenin activity through induction of CK2

ß-catenin is a key component of the Wnt signaling pathway and the abnormal accumulation of ß-catenin is characteristic of various types of cancer. Here we demonstrate that overexpression of Sox4 enhances ß-catenin/TCF activity by increasing the stability of ß-catenin. Sox4 increased the protein level of ß-catenin and its target gene cyclin D1 in a dose-dependent manner. An siRNA experiment for Sox4 also demonstrated that Sox4 increases the protein levels of ß-catenin and thus activates the Wnt signaling pathway. We found that induction of ß-catenin/TCF activity by Sox4 is caused by stabilization of the ß-catenin protein, but not by induction of ß-catenin transcription. We further demonstrate that the increased level of ß-catenin is caused by induction of CK2. In light of recent evidence that Sox4 expression is activated in the colon and in other tumors with ß-catenin dysregulation, our findings suggest that Sox4 acts as an agonist of Wnt signaling in cancer cells.

Transforming Growth Factor β1–Induced Heat Shock Protein 27 Activation Promotes Migration of Mouse Dental Papilla–derived MDPC-23 Cells

INTRODUCTION Transforming growth factor beta1 (TGFbeta1) regulates cellular functions including cell growth, differentiation, angiogenesis, migration, and metastasis. The TGFbeta1 signal transduction pathways are mostly undefined in mouse dental papilla-derived MDPC-23 cells. In this study, we investigated TGFbeta1-induced migration focusing on heat shock protein 27 (Hsp27) activation. METHODS Cellular responses mediated by TGFbeta1 in MDPC-23 cells were measured by Western blot and MTT assays. Cell migration was determined by counting migrated cells using the chemotaxis cell migration assay. RESULTS TGFbeta1 induced cell migration and increased the phosphorylation of Hsp27 and p38 MAPK in MDPC-23 cells. However, TGFbeta1 did not affect Akt/NF-kappaB signaling to regulate the migration of MDPC-23 cells. Inhibiting p38 MAPK with SB203580 blocked TGFbeta1-induced Hsp27 activation and cell migration. CONCLUSION Hsp27 phosphorylation followed by p38 MAPK activation was required for TGFbeta1-induced migration, and Hsp27 itself contributed to MDPC-23 cell migration.

TAT-Hsp40 inhibits oxidative stress-mediated cytotoxicity via the inhibition of Hsp70 ubiquitination.

Heat shock protein 40 (Hsp40) functions as a co‐chaperone of mammalian Heat shock protein 70 (Hsp70) and facilitates the ATPase activity of Hsp70, and also promotes the cellular protein folding and renaturation of misfolded proteins. In an effort to assess the effects of Hsp40, we generated TAT‐fused Hsp40 (TAT‐Hsp40). The cells were transduced with TAT‐Hsp40 and exposed to H2O2. We demonstrated that the TAT‐Hsp40‐transduced cells were more resistant to cellular cytotoxicity and cell death. In particular, the degradation of Hsp70 was significantly reduced in TAT‐Hsp40‐containing cells as a consequence of reduced ubiquitin–proteasome activity after oxidative injury. These data support the notion that Hsp40 may confer resistance to oxidative stress via the prevention of proteasome activity.

HOXC6 is deregulated in human head and neck squamous cell carcinoma and modulates Bcl-2 expression

Background: HOXC6 is involved in malignant progression, yet its signaling pathways in HNSCC remain largely unknown. Results: HOXC6 induces Bcl-2 expression by directly increasing promoter activity. Conclusion: The role of HOXC6 in HNSCC is associated with the aberrant cell growth and anti-apoptotic role. Significance: These studies provide a new mechanistic link between HOXC6 and Bcl-2 in HNSCC cell lines. Homeobox C6 (HOXC6) genes belong to the homeoprotein family of transcription factors, which play an important role in morphogenesis and cellular differentiation during embryonic development. The aim of this study was to explore the role of HOXC6 in the regulation of Bcl-2 in human head and neck squamous cell carcinoma (HNSCC). The HOXC6 and Bcl-2 gene were identified as being overexpressed in HNSCC tissue and cell lines. Transfection assays demonstrated that HOXC6 increased the levels of Bcl-2 mRNA and protein. A luciferase reporter assay suggested that HOXC6 induced activity of the Bcl-2 promoter. A series of Bcl-2 promoter deletion mutants were examined and the minimal HOXC6-responsive region was identified to be in the TAAT motif (-420 bp) of the Bcl-2 promoter. Interestingly, the inhibition of HOXC6 using siRNA led to the repression of Bcl-2 expression and induced caspase-3-dependent apoptosis; overexpression of HOXC6 in HNSCC cells increased the resistance to paclitaxel-induced apoptosis. Together, our findings suggest that HOXC6 is an important mechanism of the anti-apoptotic pathway via regulation of Bcl-2 expression.