Shinji Yoshiyama

Calcium wave for cytoplasmic streaming of Physarum polycephalum

The plasmodium Physarum polycepharum exhibits periodic cycles of cytoplasmic streaming in association with those of contraction and relaxation movement. In the present study, we injected Calcium Green dextran as a fluorescent Ca2+ indicator into the thin‐spread living plasmodium. We found changes in the [Ca2+]i (intracellular concentration of Ca2+), which propagated in a wave‐like form in its cytoplasm. The Ca2+ waves were also detected when we used Fura dextran which detected [Ca2+]i by the ratio of two wavelengths. We prepared the plasmodial fragment from the thin‐spread and found that the cycles of the contraction–relaxation movement was so synchronized that the measurement of its area provided an indication of the movement. We observed that [Ca2+]i also synchronized in the entire fragment and that the relaxation ensued upon the reduction in [Ca2+]i. We suggest that the Ca2+ wave generated periodically is one of the major factors playing a crucial role in the relaxation of P. polycepharum.

APOBEC3B high expression status is associated with aggressive phenotype in Japanese breast cancers

BackgroundThe members of AID/APOBEC protein family possess cytidine deaminase activity that converts cytidine residue to uridine on DNA and RNA. Recent studies have shown the possible influence of APOBEC3B (A3B) as DNA mutators of breast cancer genome. However, the clinical significance of A3B expression in Japanese breast cancer has not been studied in detail.MethodsNinety-three primary breast cancer tissues (74 estrogen-receptor (ER) positive, 3 ER and HER2 positive, 6 HER2 positive, and 10 triple negative) including 37 tumor-normal pairs were assessed for A3B mRNA expression using quantitative real-time RT-PCR. We analyzed the relation between A3B expression, mutation analysis of TP53 and PIK3CA by direct sequencing, polymorphic A3B deletion allele and human papillomavirus (HPV) infection in tumors.ResultsA3B mRNA was overexpressed in tumors compared with normal tissue. Patients with high A3B expression were associated with subtype and progression of lymph node metastasis and pathological nuclear grade. However, the expression was not related to any other clinicopathological factors, including mutation of TP53 and PIK3CA, polymorphic A3B deletion allele, HPV infection and survival time.ConclusionThe expression of A3B in breast cancer was higher than in non-cancerous tissues and was related to the lymph node metastasis and nuclear grade, which are reliable aggressive phenotype markers in breast cancer. Evaluation of A3B expression in tumor may be a marker for breast cancer with malignant potential.

Myosin light chain kinase stimulates smooth muscle myosin ATPase activity by binding to the myosin heads without phosphorylating the myosin light chain.

Myosin light chain kinase (MLCK) is a multifunctional regulatory protein of smooth muscle contraction [IUBMB Life 51 (2001) 337, for review]. The well-established mode for its regulation is to phosphorylate the 20 kDa myosin light chain (MLC 20) to activate myosin ATPase activity. MLCK exhibits myosin-binding activity in addition to this kinase activity. The myosin-binding activity also stimulates myosin ATPase activity without phosphorylating MLC 20 [Proc. Natl. Acad. Sci. USA 96 (1999) 6666]. We engineered an MLCK fragment containing the myosin-binding domain but devoid of a catalytic domain to explore how myosin is stimulated by this non-kinase pathway. The recombinant fragment thus obtained stimulated myosin ATPase activity by V(max)=5.53+/-0.63-fold with K(m)=4.22+/-0.58 microM (n=4). Similar stimulation figures were obtained by measuring the ATPase activity of HMM and S1. Binding of the fragment to both HMM and S1 was also verified, indicating that the fragment exerts stimulation through the myosin heads. Since S1 is in an active form regardless of the phosphorylated state of MLC 20, we conclude that the non-kinase stimulation is independent of the phosphorylating mode for activation of myosin.

Nonkinase activity of MLCK in elongated filopodia formation and chemotaxis of vascular smooth muscle cells toward sphingosylphosphorylcholine

The actin-myosin interaction of vascular smooth muscle cells (VSMCs) is regulated by myosin light chain kinase (MLCK), which is a fusion protein of the central catalytic domain with the N-terminal actin-binding and C-terminal myosin-binding domains. In addition to the regulatory role of kinase activity mediated by the catalytic domain, nonkinase activity that derives from both terminals is able to exert a regulatory role as reviewed by Nakamura et al. (32). We previously showed that nonkinase activity mediated the filopodia upon the stimulation by sphingosylphosphorylcholine (SPC) (25). To explore the regulatory role of nonkinase activity in chemotaxis, we constructed VSMCs where the expression of MLCK was totally abolished by using a lentivirus-mediated RNAi system. We hypothesized that the MLCK-downregulated VSMCs were unable to form filopodia and to migrate upon SPC stimulation and confirmed the hypothesis. We further constructed a kinase-inactive mutant from bovine cDNA coding wild-type (WT) MLCK by mutating the ATP-binding sites located in the catalytic domain, followed by confirming the presence (absence) of the kinase activity of WT (kinase-inactive mutant). We transfected WT and the mutant into MLCK-downregulated VSMCs. We expected that the transfected VSMCs will recover the ability to induce filopodia and chemotaxis toward SPC and found both constructs rescued the ability. Because they share the actin- and myosin-binding domains, we concluded nonkinase activity plays a major role for SPC-induced migration.

Stimulatory effects of arachidonic acid on myosin ATPase activity and contraction of smooth muscle via myosin motor domain

We have been searching for a mechanism to induce smooth muscle contraction that is not associated with phosphorylation of the regulatory light chain (RLC) of smooth muscle myosin (Nakamura A, Xie C, Zhang Y, Gao Y, Wang HH, Ye LH, Kishi H, Okagaki T, Yoshiyama S, Hayakawa K, Ishikawa R, Kohama K. Biochem Biophys Res Commun 369: 135-143, 2008). In this article, we report that arachidonic acid (AA) stimulates ATPase activity of unphosphorylated smooth muscle myosin with maximal stimulation (R(max)) of 6.84 +/- 0.51 relative to stimulation by the vehicle and with a half-maximal effective concentration (EC(50)) of 50.3 +/- 4.2 microM. In the presence of actin, R(max) was 1.72 +/- 0.08 and EC(50) was 26.3 +/- 2.3 microM. Our experiments with eicosanoids consisting of the AA cascade suggested that they neither stimulated nor inhibited the activity. Under conditions that did not allow RLC to be phosphorylated, AA stimulated contraction of smooth muscle tissue with an R(max) of 1.45 +/- 0.07 and an EC(50) of 27.0 +/- 4.4 microM. In addition to the ATPase activities of the myosin, AA stimulated those of heavy meromyosin, subfragment 1 (S1), S1 from which the RLC was removed, and a recombinant heavy chain consisting of the myosin head. The stimulatory effects of AA on these preparations were about twofold. The site of AA action was indicated to be the step-releasing inorganic phosphate (P(i)) from the reaction intermediate of the myosin-ADP-P(i) complex. The enhancement of P(i) release by AA was supported by computer simulation indicating that AA docked in the actin-binding cleft of the myosin motor domain. The stimulatory effect of AA was detectable with both unphosphorylated myosin and the myosin in which RLC was fully phosphorylated. The AA effect on both myosin forms was suggested to cause excess contraction such as vasospasm.

Calcium regulation of non‐kinase and kinase activities of recombinant myosin light‐chain kinase and its mutants

Myosin light‐chain kinase (MLCK) comprised of N‐terminal actin‐binding domain, central catalytic domain, and C‐terminal myosin‐binding domain. It exerted not only kinase activity to phosphorylate 20 kDa regulatory light chain of smooth muscle but also exerted non‐kinase activity on myosin motor and myosin ATPase activities (Nakamura et al., Biochem. Biophys. Res. Commun. 2008, 369, 135). The previous studies on the multiple MLCK functions were done using MLCK fragments. The present study reported the expression of whole MLCK molecules in Escherichia coli in a large amount. The construct in which the calmodulin (CaM) binding domain for regulating kinase activity was mutated lost the kinase activity. However, the mutant exerted non‐kinase activity and inhibited both myosin motor and ATPase activities. The domain that regulated kinase activity was also shown to be involved in the Ca2+ regulation of non‐kinase activity. The deletion mutants of actin‐binding domain which located at N‐terminal 1–41 amino acids demonstrated that non‐kinase activity was mediated through actin filaments.

Calcium-dependent regulation of the motor activity of recombinant full-length Physarum myosin

We successfully synthesized full-length and the mutant Physarum myosin and heavy meromyosin (HMM) constructs associated with Physarum regulatory light chain and essential light chain (PhELC) using Physarum myosin heavy chain in Sf-9 cells, and examined their Ca(2+)-mediated regulation. Ca(2+) inhibited the motility and ATPase activities of Physarum myosin and HMM. The Ca(2+) effect is also reversible at the in vitro motility of Physarum myosin. We demonstrated that full-length myosin increases the Ca(2+) inhibition more effectively than HMM. Furthermore, Ca(2+) did not affect the motility and ATPase activities of the mutant Physarum myosin with PhELC that lost Ca(2+)-binding ability. Therefore, we conclude that PhELC plays a critical role in Ca(2+)-dependent regulation of Physarum myosin.

Calcium regulation of the ATPase activity of Physarum and scallop myosins using hybrid smooth muscle myosin: The role of the essential light chain

To examine the role of two light chains (LCs) of the myosin II on Ca2+ regulation, we produced hybrid heavy meromyosin (HMM) having LCs from Physarum and/or scallop myosin using the smooth muscle myosin heavy chain. Ca2+ inhibited motility and ATPase activity of hybrid HMMs with LCs from Physarum myosin but activated those of hybrid HMM with LCs from scallop myosin, indicating an active role of LCs. ATPase activity of hybrid HMMs with LCs from different species showed the same effect by Ca2+ even though they did not support motility. Our results suggest that communication between the original combinations of LC is important for the motor function.

STXBP4 Drives Tumor Growth and Is Associated with Poor Prognosis through PDGF Receptor Signaling in Lung Squamous Cell Carcinoma

Purpose: Expression of the ΔN isoform of p63 (ΔNp63) is a diagnostic marker highly specific for lung squamous cell carcinoma (SCC). We previously found that Syntaxin Binding Protein 4 (STXBP4) regulates ΔNp63 ubiquitination, suggesting that STXBP4 may also be an SCC biomarker. To address this issue, we investigated the role of STXBP4 expression in SCC biology and the impact of STXBP4 expression on SCC prognosis. Experimental Design: We carried out a clinicopathologic analysis of STXBP4 expression in 87 lung SCC patients. Whole transcriptome analysis using RNA-seq was performed in STXBP4-positive and STXBP4-negative tumors of lung SCC. Soft-agar assay and xenograft assay were performed using overexpressing or knockdown SCC cells. Results: Significantly higher levels of STXBP4 expression were correlated with accumulations of ΔNp63 in clinical lung SCC specimens (Spearman rank correlation ρ = 0.219). Notably, STXBP4-positive tumors correlated with three important clinical parameters: T factor (P < 0.001), disease stage (P = 0.030), and pleural involvement (P = 0.028). Whole transcriptome sequencing followed by pathway analysis indicated that STXBP4 is involved in functional gene networks that regulate cell growth, proliferation, cell death, and survival in cancer. Platelet-derived growth factor receptor alpha (PDGFRα) was a key downstream mediator of STXBP4 function. In line with this, shRNA mediated STXBP4 and PDGFRA knockdown suppressed tumor growth in soft-agar and xenograft assays. Conclusions: STXBP4 plays a crucial role in driving SCC growth and is an independent prognostic factor for predicting worse outcome in lung SCC. These data suggest that STXBP4 is a relevant therapeutic target for patients with lung SCC. Clin Cancer Res; 23(13); 3442–52. ©2017 AACR.

Mutant TP53 modulates metastasis of triple negative breast cancer through adenosine A2b receptor signaling.

Purpose The identification of genes with synthetic lethality in the context of mutant TP53 is a promising strategy for the treatment of basal-like triple negative breast cancer (TNBC). This study investigated regulators of mutant TP53 (R248Q) in basal-like TNBC and their impact on tumorigenesis. Experimental Design TNBC cells were analyzed by RNA-seq, and synthetic-lethal shRNA knock-down screening, to identify genes related to the expression of mutant TP53. A tissue microarray of 232 breast cancer samples, that included 66 TNBC cases, was used to assess clinicopathological correlates of tumor protein expression. Functional assays were performed in vitro and in vivo to assess the role of ADORA2B in TNBC. Results Transcriptome profiling identified ADORA2B as up-regulated in basal-like TNBC cell lines with R248Q-mutated TP53, with shRNA-screening suggesting the potential for a synthetic-lethal interaction between these genes. In clinical samples, ADORA2B was highly expressed in 39.4% (26/66) of TNBC patients. ADORA2B-expression was significantly correlated with ER (P < 0.01), PgR (P = 0.027), EGFR (P < 0.01), and tumor size (P = 0.037), and was an independent prognostic factor for outcome (P = 0.036). In line with this, ADORA2B-transduced TNBC cells showed increased tumorigenesis, and ADORA2B knockdown, along with mutant p53 knockdown, decreased metastasis both in vitro and in vivo. Notably, the cytotoxic cyclic peptide SA-I suppressed ADORA2B expression and tumorigenesis in TNBC cell lines. Conclusions ADORA2B expression increases the oncogenic potential of basal-like TNBC and is an independent factor for poor outcome. These data suggest that ADORA2B could serve as a prognostic biomarker and a potential therapeutic target for basal-like TNBC.