PLEIAD/SIMC1/C5orf25, a Novel Autolysis Regulator for a Skeletal-Muscle-Specific Calpain, CAPN3, Scaffolds a CAPN3 Substrate, CTBP1

AbstractCAPN3/p94/calpain-3 is a skeletal-muscle-specific member of the calpain protease family. Multiple muscle cell functions have been reported for CAPN3, and mutations in this protease cause limb-girdle muscular dystrophy type 2A. Little is known about the molecular mechanisms that allow CAPN3 to be so multifunctional. One hypothesis is that the very rapid and exhaustive autolytic activity of CAPN3 needs to be suppressed by dynamic molecular interactions for specific periods of time. The previously identified interaction between CAPN3 and connectin/titin, a giant molecule in muscle sarcomeres, supports this assumption; however, the regulatory mechanisms of non-sarcomere-associated CAPN3 are unknown. Here, we report that a novel CAPN3-binding protein, PLEIAD [Platform element for inhibition of autolytic degradation; originally called SIMC1/C5orf25 (SUMO-interacting motif containing protein 1/chromosome 5 open reading frame 25)], suppresses the protease activity of CAPN3. Database analyses showed that PLEIAD homologs, like CAPN3 homologs, are evolutionarily conserved in vertebrates. Furthermore, we found that PLEIAD also interacts with CTBP1 (C-terminal binding protein 1), a transcriptional co-regulator, and CTBP1 is proteolyzed in COS7 cells expressing CAPN3. The identified cleavage sites in CTBP1 suggested that it undergoes functional modification upon its proteolysis by CAPN3, as well as by conventional calpains. These results indicate that PLEIAD can shift its major function from CAPN3 suppression to CAPN3-substrate recruitment, depending on the cellular context. Taken together, our data suggest that PLEIAD is a novel regulatory scaffold for CAPN3, as reflected in its name

KLHDC10 Activates ASK1 by Suppressing PP5

Reactive oxygen species (ROS)-induced activation of Apoptosis signal-regulating kinase 1 (ASK1) plays crucial roles in oxidative stress-mediated cell death through the activation of the JNK and p38 MAPK pathways. However, the regulatory mechanism of ASK1 in the oxidative stress response remains to be elucidated. Here, we identified the kelch repeat protein, Slim, as an activator of ASK1 through a Drosophila misexpression screen. We also performed a proteomics screen and revealed that Kelch domain containing 10 (KLHDC10), a mammalian ortholog of Slim, interacted with Protein phosphatase 5 (PP5), which has been shown to inactivate ASK1 in response to ROS. KLHDC10 bound to the phosphatase domain of PP5 and suppressed its phosphatase activity. Moreover, KLHDC10 was required for H2O2-induced sustained activation of ASK1 and cell death in Neuro2A cells. These findings suggest that Slim/KLHDC10 is an activator of ASK1, contributing to oxidative stress-induced cell death through the suppression of PP5

STUDIES ON BONE MORPHOGENETICPROTEIN-BINDING PROTEINS IN THE EARLYDEVELOPMENT OF XENOPUS LAEVIS

　　　Almost all multicellular organisms arise from a single cell. After fertilization, it gives rise to diverse cell types, and then these differentiated cells are organized into tissues and organs of the body. In this embryonic development, a series of sequential embryonic inductions, especially mesoderm and neural induction, are essential processes for determining the individual basic body plan. Therefore, it is important to elucidate what signaling molecules are involved in the inductions, and how these molecules function in the cell-to-cell communication. To date, several signaling molecules acting in early developmental processes have been identified, and among them, bone morphogenetic protein (BMP), which is a member of the transforming growth factor-β (TGF-β) superfamily, is thought to play a key role in early events regulating morphogenesis. Recently, it has been demonstrated that BMP activity is antagonized by the factors (noggin and chordin) released by the Spemann\u27s organizer, and that such an antagonism takes place by the direct binding between BMP and noggin, or chordin. These findings revealed that such a negative regulation of BMP activity by its binding proteins is essential for the pattern formation of early embryos.　　　In this thesis, I will focus on the regulatory mechanisms of BMP activity by BMP-binding proteins at the protein level. To analyze the interaction of BMP and its binding proteins, I introduced a surface plasmon resonance (SPR) biosensor (BIACORE). This sensor was developed for monitoring biomolecular interactions in real time, using non-invasive optical detection principle based on SPR, and can detect a change in mass concentration at the sensor surface as molecules bind or dissociate. To perform SPR analysis on BMP and its receptor, firstly, large-scale expression and purification of xBMP-4 and its soluble form of type I receptor (sBMPR) was performed using a silkworm expression system (chapter 2). Secondly, the interaction between BMP-4 and follistatin, which is also expressed in the Spemann\u27s organizer region and suspected to bind directly to BMP-4, was analyzed (chapter 3). Lastly, the screening of the BMP-4-binding protein was performed, and the interaction between BMP-4 and isolated proteins was analyzed (chapter 4). In addition, the interaction was confirmed by in vivo functional analysis using mRNA microinjection into early Xenopus embryos.　　　In chapter 2, to enable the analysis of protein-protein interactions, large scale preparation of BMP-4 and sBMPR was performed using a silkworm expression system. From the hemolymph recovered from infected larvae (approximately 2,000 larvae), about 1 mg of xBMP-4 and 20 mg of sBMPR were purified by liquid chromatography. This receptor was in monomer form in solution, and bound to BMP-4 but not to activin A or TGF-β1. The SPR studies showed that the association rate constant(ka) of sBMPR for BMP-4 is 3.81×104 M-1s-1, and that the dissociation rate constant (kd) is 3.69×10-4s-1 (KD=9.6 nM). This affinity was similar to that of the intact membrane-associated receptor expressed on COS cells. The biological activity of expressed BMP-4 was confirmed by alkaline phosphatase (ALP) activity in BMP responsive cell lines such as mouse osteoblastic cells MC3T3-E1 and bone marrow stromal cells ST2. The BMP-binding ability of expressed sBMPR protein to BMP was confirmed as an inhibition of BMP-induced ALP activity by the addition of sBMPR protein.　　　In chapter 3, functional analyses of follistatin in the development of Xenopus embryos were performed using the SPR sensor and mRNA microinjection method. Follistatin, originally known as an activin-binding protein, is localized to the Spemann\u27s organizer of early Xenopus gastrula, as well as chordin and noggin. Until now, it is found that follistatin induces the secondary body axis when overexpressed in ventral blastomeres, and that it can induce neural tissue in ectoderm without affecting mesoderm. These observations indicate that follistatin might inhibit not only activin but also BMPs through direct binding.　　　To examine the antagonism between follistatin and BMPs (BMP-4 or BMP-7), mRNA microinjection assay was performed. BMP-4 and BMP-7 caused a ventralized embryo that lacked the anterior head structure and notochord when they were dorsally overexpressed by mRNA microinjection. These effects of BMPs were inhibited by coinjection with follistatin mRNA. On the contrary, the dorsalizing effect of follistatin in ventral side was repressed when coinjected with BMP mRNAs. These findings reveal that follistatin and BMPs inhibit each other.　　　Next, the interactions between follistatin(FS-288) and BMPs were analyzed by the SPR sensor. While the affinity of FS-288 for BMPs is lower than that for activin A, the results clearly indicated that FS-288 binds to BMP-4,-7 homodimers, and BMP- 4/7 heterodimer. In contrast to this, TGF-β1, other member of TGF-β superfamily, did not bind to FS-288. The kinetic parameters for the binding of FS-288 to BMP-4 were determined that the association rate constant（ka) is 1.16×105 M-1s-1, and that the dissociation rate constant (kd) is 2.7×10-3 s-1 (KD=23 nM).　　　The inhibitory mechanism of follistatin for BMP-4 was analyzed by the biosensor, and compared with those of noggin and chordin. As previously reported, noggin and chordin bind to BMP-4 directly, and inhibit the interaction between BMP-4 and its receptor. In contrast, it was suggested that follistatin, BMP-4, and sBMPR form a trimeric complex, but follistatin does not interfere the binding of BMP-4 to sBMPR.　　　In chapter 4, novel BMP-4-binding proteins, which may regulate the multipotent BMP activity in development, were screened using the SPR sensor as a specific monitor. Two BMP-4-binding proteins were isolated from Xenopus embryo extracts by 3-step chromatography. Comparisons of N-terminal amino acid sequences established that they are Ep45 and lipovitellin 1. Lipovitellin 1 is an egg yolk protein that is processed from vitellogenin, while Ep45 is a mermber of the serine protease inhibitor (serpin) superfamily. Both Ep45 and vitellogenin are synthesized under estrogen control in the liver. Because it has been reported that vitellogenin binds to both activin and BMP, subsequent functional analyses were performed for Ep45.　　　The binding specificity of Ep45 and the kinetic parameters for the binding of Ep45 to BMP-4 were demonstrated by SPR studies. The results indicated that Ep45 can interact only with BMP-4 among TGF-β family ligands, activin A, TGF-β1, and BMP-4 so far tested. Moreover, the kinetic parameters for the binding of Ep45 to BMP- 4 were calculated that the association rate constant (ka) and the dissociation rate constant (kd) are 1.06×104 M-1s-1 and 1.6×10-4s-1, respectively (KD=15.1 nM).　　　Next, functional analyses of Ep45 in the early development of Xenopus embryos were performed using the SPR biosensor and mRNA microinjection. When Ep45 mRNA was microinjected into embryos, it had no effect on the development. Furthermore, analysis using the SPR sensor indicated that Ep45 does not inhibit the binding of BMP- 4 to sBMPR. These results suggest that Ep45 is not a negative regulator of BMP-4. Subsequently, the influence of Ep45 on the interaction of BMP-4 with three organizer factors, which are noggin, chordin, and follistatin, was investigated. In microinjection assay, Ep45 had no effect on dorsalization induced by chordin or noggin, but inhibited dorsalization by follistatin in a dose-dependent manner. This result is further supported by the SPR biosensor. Namely, Ep45 inhibited the binding of BMP-4 to follistatin m a dose-dependent manner. Taking into consideration their affinities to BMP-4, it is assumed that Ep45 does not interfere with the high affinity binding of BMP to BMP-binding proteins such as noggin, or chordin. By contrast, because Ep45 binds to BMP-4 with higher affinity than follistatin, Ep45 is thought to be able to interfere the binding of follistatin to BMP-4. Taken together, it is likely that Ep45 is a specific inhibitor of follistatin against BMP.　　　In conclusion, this work demonstrates the differentially controlled mechanisms of regulation of BMP activity. Because the multipotent activities of polypeptide growth factors are essential for a variety of patterning events during not only early development, but also organogenesis, it is thought to be regulated through various mechanisms. Accordingly, the isolation of new molecules that bind to growth factors, and functional analyses of these growth factor-binding proteins would provide the key to understanding the mechanisms of development

Cdc37 Interacts with the Glycine-Rich Loop of Hsp90 Client Kinases

Recently, we identified a client-binding site of Cdc37 that is required for its association with protein kinases. Phage display technology and liquid chromatography-tandem mass spectrometry (which identifies a total of 33 proteins) consistently identify a unique sequence, GXFG, as a Cdc37-interacting motif that occurs in the canonical glycine-rich loop (GXGXXG) of protein kinases, regardless of their dependence on Hsp90 or Cdc37. The glycine-rich motif of Raf-1 (GSGSFG) is necessary for its association with Cdc37; nevertheless, the N lobe of Raf-1 (which includes the GSGSFG motif) on its own cannot interact with Cdc37. Chimeric mutants of Cdk2 and Cdk4, which differ sharply in their affinities toward Cdc37, show that their C-terminal portions may determine this difference. In addition, a nonclient kinase, the catalytic subunit of cyclic AMP-dependent protein kinase, interacts with Cdc37 but only when a threonine residue in the activation segment of its C lobe is unphosphorylated. Thus, although a region in the C termini of protein kinases may be crucial for accomplishing and maintaining their interaction with Cdc37, we conclude that the N-terminal glycine-rich loop of protein kinases is essential for physically associating with Cdc37

Serine Phosphorylation by mTORC1 Promotes IRS-1 Degradation through SCFβ-TRCP E3 Ubiquitin Ligase

Summary: The insulin receptor substrate IRS-1 is a key substrate of insulin and insulin-like growth factor (IGF) receptor tyrosine kinases that mediates their metabolic and growth-promoting actions. Proteasomal degradation of IRS-1 is induced following activation of the downstream kinase mTOR complex 1 (mTORC1) to constitute a negative feedback loop. However, the underlying mechanism remains poorly understood. Here we report that Ser 422 of IRS-1 is phosphorylated by mTORC1 and required for IRS-1 degradation induced by prolonged IGF stimulation. Phosphorylation of Ser 422 then recruits the SCFβ-TRCP E3 ligase complex, which catalyzes IRS-1 ubiquitination. Phosphorylation-dependent IRS-1 degradation contributes to impaired growth and survival responses to IGF in cells lacking TSC2, a negative regulator of mTORC1. Inhibition of IRS-1 degradation promotes sustained Akt activation in IGF-stimulated cells. Our work clarifies the nature of the IRS-1-mTORC1 feedback loop and elucidates its role in temporal regulation of IGF signaling. : Biochemistry; Biochemical Mechanism; Molecular Physiology; Molecular Interaction Subject Areas: Biochemistry, Biochemical Mechanism, Molecular Physiology, Molecular Interactio