ABSTRACT
Selenoprotein W (SELENOW) is a 9.6 kDa protein containing selenocysteine (Sec, U) in a conserved Cys-X-X-Sec (CXXU) motif. Previously, we reported that SELENOW regulates various cellular processes by interacting with 14-3-3ß at the U of the CXXU motif. Thioredoxin (Trx) is a small protein that plays a key role in the cellular redox regulatory system. The CXXC motif of Trx is critical for redox regulation. Recently, an interaction between Trx1 and 14-3-3 has been predicted. However, the binding mechanism and its biological effects remain unknown. In this study, we found that Trx1 interacted with 14-3-3ß at the Cys32 residue in the CXXC motif, and SELENOW and Trx1 were bound at Cys191 residue of 14-3-3ß. In vitro binding assays showed that SELENOW and Trx1 competed for interaction with 14-3-3ß. Compared to control cells, Trx1-deficient cells and SELENOW-deficient cells showed increased levels of both the subG1 population and poly (ADP-ribose) polymerase (PARP) cleavage by etoposide treatment. Moreover, Akt phosphorylation of Ser473 was reduced in Trx1-deficient cells and was recovered by overexpression of SELENOW. These results indicate that SELENOW can protect Trx1-deficient cells from etoposide-induced cell death through its interaction with 14-3-3ß.
Subject(s)
14-3-3 Proteins/metabolism , Cell Death/drug effects , Etoposide/pharmacology , Selenoprotein W/pharmacology , Thioredoxins/metabolism , Animals , Cell Line , Cell Line, Tumor , HEK293 Cells , Humans , MCF-7 Cells , Mice , Oxidation-Reduction/drug effects , Phosphorylation/drug effects , Poly (ADP-Ribose) Polymerase-1/metabolism , Protein Binding/drug effects , Signal Transduction/drug effectsABSTRACT
Selenoprotein W (SelW) contains a selenocysteine (Sec, U) in a conserved CXXU motif corresponding to the CXXC redox motif of thioredoxin, suggesting a putative redox function of SelW. We have previously reported that the binding of 14-3-3 protein to its target proteins, including CDC25B, Rictor and TAZ, is inhibited by the interaction of 14-3-3 protein with SelW. However, the binding mechanism is unclear. In this study, we sought to determine the binding site of SelW to understand the regulatory mechanism of the interaction between SelW and 14-3-3 and its biological effects. Phosphorylated Ser(pS) or Thr(pT) residues in RSXpSXP or RXXXp(S/T)XP motifs are well-known common 14-3-3-binding sites, but Thr41, Ser59, and T69 of SelW, which are computationally predicted to serve are phosphorylation sites, were neither phosphorylation sites nor sites involved in the interaction. A mutant SelW in which Sec13 is changed to Ser (U13S) was unable to interact with 14-3-3 protein and thus did not inhibit the interaction of 14-3-3 to other target proteins. However, other Cys mutants of SelW(C10S, C33S and C37S) normally interacted with 14-3-3 protein. The interaction of SelW to 14-3-3 protein was enhanced by diamide or H2O2 and decreased by dithiothreitol (DTT). Taken together, these findings demonstrate that the Sec of SelW is involved in its interaction with 14-3-3 protein and that this interaction is increased under oxidative stress conditions. Thus, SelW may have a regulatory function in redox cell signaling by interacting with 14-3-3 protein.
Subject(s)
14-3-3 Proteins/metabolism , Oxidative Stress/physiology , Selenoprotein W/metabolism , 14-3-3 Proteins/genetics , Amino Acid Motifs , Dithiothreitol/pharmacology , Female , Humans , Hydrogen Peroxide/pharmacology , MCF-7 Cells , Mutation, Missense , Oxidation-Reduction/drug effects , Oxidative Stress/drug effects , Phosphorylation/drug effects , Phosphorylation/physiology , Protein Binding/drug effects , Protein Binding/physiology , Selenoprotein W/geneticsABSTRACT
Cytosolic valosin-containing protein (p97(VCP)) is translocated to the ER membrane by binding to selenoprotein S (SelS), which is an ER membrane protein, during endoplasmic reticulum-associated degradation (ERAD). Selenoprotein K (SelK) is another known p97(VCP)-binding selenoprotein, and the expression of both SelS and SelK is increased under ER stress. To understand the regulatory mechanisms of SelS, SelK, and p97(VCP) during ERAD, the interaction of the selenoproteins with p97(VCP) was investigated using N2a cells and HEK293 cells. Both SelS and SelK co-precipitated with p97(VCP). However, the association between SelS and SelK did not occur in the absence of p97(VCP). SelS had the ability to recruit p97(VCP) to the ER membrane but SelK did not. The interaction between SelK and p97(VCP) did not occur in SelS knockdown cells, whereas SelS interacted with p97(VCP) in the presence or absence of SelK. These results suggest that p97(VCP) is first translocated to the ER membrane via its interaction with SelS, and then SelK associates with the complex on the ER membrane. Therefore, the interaction between SelK and p97(VCP) is SelS-dependent, and the resulting ERAD complex (SelS-p97(VCP)-SelK) plays an important role in ERAD and ER stress.
Subject(s)
Adenosine Triphosphatases/metabolism , Cell Cycle Proteins/metabolism , Endoplasmic Reticulum/metabolism , Membrane Proteins/metabolism , Selenoproteins/metabolism , Animals , Cell Line , Humans , Mice , Protein Binding , Valosin Containing ProteinABSTRACT
During endoplasmic reticulum (ER)-associated degradation, p97(VCP) is recruited to the ER membrane through interactions with transmembrane proteins, such as selenoprotein S (SelS), selenoprotein K (SelK), hrd1, and gp78. SelS has a single-spanning transmembrane domain and protects cells from ER stress-induced apoptosis through interaction with p97(VCP). The cytosolic tail of SelS consists of a coiled-coil domain, a putative VCP-interacting motif (VIM), and an unpronounced glycine- and proline-rich secondary structure. To understand the regulatory mechanism of SelS during ER stress, we investigated the interaction of the protein with p97(VCP) using mouse neuroblastoma cells and human embryonic kidney 293 cells. The SelS expression level increased when ER stress was induced. In addition, the effect of ER stress was enhanced, and recruitment of p97(VCP) to the ER membrane was inhibited in SelS knockdown cells. The effect of SelS knockdown was rescued by ectopic expression of SelS U188C. p97(VCP) interacted with SelS U188C and was recruited to the ER membrane. The expression of SelS[ΔVIM], which is a VIM deletion mutant of SelS, also showed both a recovery effect and an interaction with p97(VCP) in cells. However, mutants in which the proline residue positions 178 or 183 of SelS were changed to alanine or were deleted did not interact with p97(VCP). The proline mutants did not rescue ER stress in SelS knockdown cells. These results suggest that both Pro(178) and Pro(183) of SelS play important roles in the translocation of p97(VCP) to the ER membrane and protect cells from ER stress.
Subject(s)
Adenosine Triphosphatases/metabolism , Cell Cycle Proteins/metabolism , Endoplasmic Reticulum-Associated Degradation , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Proline/metabolism , Selenoproteins/chemistry , Selenoproteins/metabolism , Amino Acid Sequence , Animals , Endoplasmic Reticulum Stress , Gene Silencing , HEK293 Cells , Humans , Intracellular Membranes/metabolism , Membrane Proteins/deficiency , Membrane Proteins/genetics , Mice , Molecular Sequence Data , Protein Binding , Protein Transport , Selenoproteins/deficiency , Selenoproteins/genetics , Valosin Containing ProteinABSTRACT
Selenoprotein W (SelW) is expressed in various tissues, particularly in skeletal muscle. We have previously reported that SelW is up-regulated during C2C12 skeletal muscle differentiation and inhibits binding of 14-3-3 to its target proteins. 14-3-3 reduces myogenic differentiation by inhibiting nuclear translocation of transcriptional co-activator with PDZ-binding motif (TAZ). Phosphorylation of TAZ at Ser89 is required for binding to 14-3-3, leading to cytoplasmic retention of TAZ and a delay in myogenic differentiation. Here, we show that myogenic differentiation was delayed in SelW-knockdown C2C12 cells. Down-regulation of SelW also increased TAZ binding to 14-3-3, which eventually resulted in decreasing translocation of TAZ to the nucleus. However, phosphorylation of TAZ at Ser89 was not affected. Although phosphorylation of TAZ at Ser89 was sustained by the phosphatase inhibitor okadaic acid, nuclear translocation of TAZ was increased by ectopic expression of SelW. This result was due to decreased binding of TAZ to 14-3-3. We also found that the interaction between TAZ and MyoD was increased by ectopic expression of SelW. Taken together, these findings strongly demonstrate that SelW enhances C2C12 cell differentiation by inhibiting TAZ binding to 14-3-3.
Subject(s)
14-3-3 Proteins/metabolism , Muscle, Skeletal/metabolism , Myoblasts/metabolism , Selenoprotein W/metabolism , Transcription Factors/metabolism , 14-3-3 Proteins/genetics , Acyltransferases , Animals , Binding Sites , Cell Differentiation/drug effects , Cell Line , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Cytosol/drug effects , Cytosol/metabolism , Enzyme Inhibitors/pharmacology , Gene Expression Regulation, Developmental , HEK293 Cells , Humans , Mice , Muscle, Skeletal/cytology , Muscle, Skeletal/drug effects , MyoD Protein/genetics , MyoD Protein/metabolism , Myoblasts/cytology , Myoblasts/drug effects , Okadaic Acid/pharmacology , Phosphorylation , Protein Binding , Protein Transport/drug effects , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Selenoprotein W/antagonists & inhibitors , Selenoprotein W/genetics , Signal Transduction , Transcription Factors/geneticsABSTRACT
14-3-3 reduces cell proliferation by inhibiting the activity of proteins involved in the signaling pathway that includes Akt kinase. Activation of Akt is enhanced by activating the mammalian target of rapamycin complex 2 (mTORC2). 14-3-3 is also a negative regulator of the mTORC2/Akt pathway, by interacting with a component of mTORC2. Recently, we reported that selenoprotein W (SelW) regulated the interaction between 14-3-3 and its target protein, CDC25B. Here, we show that the binding of Rictor, a component of mTORC2, to 14-3-3, is regulated by the interaction of 14-3-3 with SelW. When SelW was down-regulated, mTORC2-dependent phosphorylation of Akt at Ser473 was decreased. However, the phosphorylation of Thr308 was not affected. The interaction of Rictor with 14-3-3 was increased in SelW-knockdown cells, as compared to control cells. SelW-knockdown cells were also more sensitive to DNA damage induced by etoposide, than control cells. This phenomenon was due to the decreased phosphorylation of Akt at Ser473. We also found that ectopic expression of SelW(U13C) reduced the interaction between Rictor and 14-3-3, leading to Akt phosphorylation at Ser473. Taken together, these findings demonstrate that SelW activates the mTORC2/Akt pathway for Akt phosphorylation at Ser473, by interrupting the binding of Rictor to 14-3-3.
Subject(s)
14-3-3 Proteins/metabolism , Breast Neoplasms/metabolism , Carrier Proteins/metabolism , Lung Neoplasms/metabolism , Multiprotein Complexes/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Selenoprotein W/metabolism , Serine/metabolism , TOR Serine-Threonine Kinases/metabolism , 14-3-3 Proteins/antagonists & inhibitors , 14-3-3 Proteins/genetics , Blotting, Western , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Carrier Proteins/genetics , Cell Proliferation , Flow Cytometry , Humans , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Mechanistic Target of Rapamycin Complex 2 , Multiprotein Complexes/genetics , Phosphorylation , Protein Binding , Proto-Oncogene Proteins c-akt/genetics , RNA, Messenger/genetics , Rapamycin-Insensitive Companion of mTOR Protein , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Selenoprotein W/genetics , Serine/genetics , Signal Transduction , TOR Serine-Threonine Kinases/genetics , Tumor Cells, Cultured , Tumor Stem Cell Assay , Wound HealingABSTRACT
Selenoprotein W (SelW) contains a highly reactive selenocysteine (Sec; U) in the CXXU motif corresponding to the CXXC motif in thioredoxin (Trx) and thus it appears to be involved in regulating the cellular redox state. Recent reports on the interaction between SelW and 14-3-3 suggest that SelW may be redox dependently involved in the cell cycle. However, the precise function of SelW has not yet been elucidated. Here, we show that SelW is involved in the G2-M transition, especially in the recovery from G2 arrest after deoxyribonucleic acid (DNA) damage. Knockdown of SelW significantly accumulated phosphorylated cyclin-dependent kinase (Cdk1), which eventually led to a delay in recovery from G2 arrest. We also found that inactive Cdk1 is caused by the sustained inactivation of CDC25B, which removes the inhibitory phosphate from Cdk1. Our observation from this study reveals that SelW activated CDC25B by promoting the dissociation of 14-3-3 from CDC25B through the reduction of the intramolecular disulfide bond during recovery. We suggest that SelW plays an important role in the recovery from G2 arrest by determining the dissociation of 14-3-3 from CDC25B in a redox-dependent manner.
