Isolation of troponins from striated and smooth adductor muscles of Akazara scallop

Troponins which confer Ca-sensitivity to skeletal actomyosin ATPase were successfully isolated from striated and smooth adductor muscles of "Akazara" scallop (Chlamys nipponensis akazara). SDS-gel electrophoresis showed that striated and smooth adductor troponins were composed of three components having molecular weights of about 52K (52,000), 40K, and 20K, and about 40K, 21K, and 20K, respectively. The Mg-ATPase activity of actomyosin reconstituted from rabbit actin and either Akazara striated adductor myosin or smooth adductor myosin, along with the respective tropomyosin and troponin, indicated that the Ca2+ concentration required for the activation of actomyosin ATPase appeared to be favorable to myosin-linked regulation.     

Cyanogen bromide fragments of Akazara scallop Mr 52,000 troponin-I

Akazara scallop troponin-I of Mr 52,000 (52K) was cleaved into two fragments of 17K and 35K with cyanogen bromide. The 17K fragment, along with tropomyosin, inhibited weakly the rabbit actomyosin Mg-ATPase activity, however, the 35K fragment did not affect it at all. In the presence of Akazara scallop TnT (40K component), the 17K fragment, in turn, strongly inhibited the activity, while the 35K fragment did not. The amino acid composition and partial amino acid sequence suggested that the 17K and 35K fragments were derived from C- and N-terminal regions of the TnI, respectively, and that structural similarity to TnIs from other animals is present in the 17K region.     

Amino acid sequence of squid troponin C

The complete amino acid sequence of squid Todarodes pacificus troponin C (TnC), which was shown to bind only 1 mol Ca²⁺/mol, was determined by both the Edman and cDNA methods. The squid TnC is composed of 147 amino acids including an unblocked Pro at the N-terminus and the calculated molecular weight is 17 003.9. Among the four potential Ca²⁺-binding sites, namely sites I–IV from the N-terminus, only site IV completely satisfied the consensus amino acid sequence for the active Ca²⁺-binding loop. This indicates that squid TnC possesses a single Ca²⁺-binding site at the site IV as scallop TnCs [Nishita et al., J. Biol. Chem. 269 (1994) 3464–3468; Ojima et al., Arch. Biochem. Biophys. 311 (1994) 272–276). The sequence homology of squid TnC to TnCs of scallop, arthropods, and rabbit was 61%, 31–38%, and 31%, respectively. In the sequence of the central D/E-helix region of squid and scallop TnCs, a deletion of three amino acids was required to maximize the homology with the other TnCs.     

MAPKAPK-2-mediated LIM-kinase activation is critical for VEGF-induced actin remodeling and cell migration

Vascular endothelial growth factor-A (VEGF-A) induces actin reorganization and migration of endothelial cells through a p38 mitogen-activated protein kinase (MAPK) pathway. LIM-kinase 1 (LIMK1) induces actin remodeling by phosphorylating and inactivating cofilin, an actin-depolymerizing factor. In this study, we demonstrate that activation of LIMK1 by MAPKAPK-2 (MK2; a downstream kinase of p38 MAPK) represents a novel signaling pathway in VEGF-A-induced cell migration. VEGF-A induced LIMK1 activation and cofilin phosphorylation, and this was inhibited by the p38 MAPK inhibitor SB203580. Although p38 phosphorylated LIMK1 at Ser-310, it failed to activate LIMK1 directly; however, MK2 activated LIMK1 by phosphorylation at Ser-323. Expression of a Ser-323-non-phosphorylatable mutant of LIMK1 suppressed VEGF-A-induced stress fiber formation and cell migration; however, expression of a Ser-323-phosphorylation-mimic mutant enhanced these processes. Knockdown of MK2 by siRNA suppressed VEGF-A-induced LIMK1 activation, stress fiber formation, and cell migration. Expression of kinase-dead LIMK1 suppressed VEGF-A-induced tubule formation. These findings suggest that MK2-mediated LIMK1 phosphorylation/activation plays an essential role in VEGF-A-induced actin reorganization, migration, and tubule formation of endothelial cells.     

Ror2/Frizzled Complex Mediates Wnt5a-Induced AP-1 Activation by Regulating Dishevelled Polymerization

The receptor tyrosine kinase Ror2 acts as a receptor or coreceptor for Wnt5a to mediate Wnt5a-induced activation of the Wnt/JNK pathway and inhibition of the β-catenin-dependent canonical Wnt pathway. However, little is known about how Ror2 cooperates with another receptor component(s) to mediate Wnt5a signaling. We show here that Ror2 regulates Wnt5a-induced polymerization of Dishevelled (Dvl) and that this Ror2-mediated regulation of Dvl is independent of the cytoplasmic region of Ror2. Ror2 can associate with Frizzled7 (Fz7) via its extracellular cysteine-rich domain to form a receptor complex that is required for the regulation of Dvl and activation of the AP-1 promoter after Wnt5a stimulation. Suppressed expression of Fz7 indeed results in the inhibition of Wnt5a-induced polymerization of Dvl and AP-1 activation. Interestingly, both the DIX and the DEP domains of Dvl are indispensable for Dvl polymerization and subsequent AP-1 activation after Wnt5a stimulation. We further show that polymerized Dvl is colocalized with Rac1 and that suppressed expression of Rac1 inhibits Wnt5a-induced AP-1 activation. Collectively, our results indicate that Ror2/Fz receptor complex plays an important role in the Wnt5a/Rac1/AP-1 pathway by regulating the polymerization of Dvl.Wnt proteins can elicit β-catenin-dependent and -independent signaling pathways (2, 20, 46). Ror2 is a member of the Ror family of receptor tyrosine kinases and plays essential roles in developmental morphogenesis (21, 26, 31, 32, 44). Ror2 has been shown to act as a receptor or coreceptor for Wnt5a to activate the β-catenin-independent signaling pathway, involving JNK/c-Jun (AP-1), Src and Ca²⁺, which are essential for cell polarity, migration, and cancer cell invasion (8, 14, 28-31, 37). Wnt5a/Ror2 signaling also plays a crucial role in inhibiting the β-catenin-dependent signaling pathway (25). Structure-function analyses of Ror2 revealed that Ror2 mediates Wnt5a signaling through distinct mechanisms dependent on and independent of its kinase activity, i.e., Wnt5a-induced migration of fibroblast cells requires the cytoplasmic C-terminal portion of Ror2 but not its intrinsic kinase activity (28), whereas the intrinsic kinase activity of Ror2 is indispensable for extracellular matrix (ECM) degradation of osteosarcoma cells (8). In addition, inhibition of the β-catenin-dependent signaling pathway by Wnt5a also requires the intrinsic kinase activity of Ror2 (24). Importantly, the Caenorhabditis elegans ortholog of Ror2, CAM-1, also has the kinase activity-dependent and -independent functions (9, 12, 13). Furthermore, CAM-1 exhibits the cytoplasmic region-independent functions, including cell migration (17), synaptic transmission at the neuromuscular junction (10), and inhibition of the β-catenin-dependent signaling pathway (11), although their underlying molecular mechanisms remain to be determined. However, it is unknown whether or not Ror2 also exhibits the cytoplasmic region-independent functions in other organisms.Dishevelled (Dvl) is an essential mediator of both the β-catenin-dependent and -independent signaling pathways. We have previously reported that both Ror2 and Dvl are required for Wnt5a-induced cell migration (28). However, the relationship between Ror2 and Dvl in Wnt5a signaling remains unclear. It has been reported that Dvl has an ability to form dynamic polymers, which are crucial for activating the β-catenin-dependent signaling pathway probably by serving as a scaffold for Axin recruitment (39, 41). However, there is no direct evidence showing that Wnt stimulation indeed induces dynamic formation of Dvl polymers. In addition, it remains unclear whether or not the polymerization of Dvl is involved in the β-catenin-independent signaling pathway.In the present study we show that Wnt5a induces dynamic polymerization of Dvl2 via a receptor complex containing both Ror2 and Frizzled (Fz)7, even in the absence of the cytoplasmic region of Ror2. We further provide evidence indicating that Ror2/Fz7 receptor complex plays an important role in Wnt5a/Rac1/AP-1 pathway by regulating polymerization of Dvl2.     

N-terminal modification and its effect on the biochemical characteristics of Akazara scallop tropomyosins expressed in Escherichia coli

Akazara scallop striated muscle tropomyosin mutants without a fused amino acid (nf-Tm), and with Ala- (A-Tm) or Asp-Ala- (DA-Tm) fused at the N-terminus were expressed in Escherichia coli cells. Among them, nf-Tm alone has an initial methionine. The native Akazara scallop tropomyosin and DA-Tm showed similar alpha-helix contents and intrinsic viscosity, but nf-Tm and A-Tm exhibited lower values than those of the native tropomyosin. According to the relative viscosity, all the expressed tropomyosins appear to have lost head-to-tail polymerization ability. Though nf-Tm has extremely low actin-binding ability, the ability was almost completely recovered with a two amino acid fusion but incompletely with a one amino acid fusion. On the other hand, an amino acid fusion, irrespective of the number, seemed to inhibit the Mg-ATPase activity of actomyosin. However, the bacterially expressed tropomyosins together with Akazara scallop troponin do not confer the full Ca(2+)-regulation ability of Mg-ATPase activity of actomyosin. These results support that N-terminal blocking probably by an acetyl group of Akazara scallop tropomyosin plays an important role not only in head-to-tail polymerization and actin-binding, as known for vertebrate tropomyosin, but also in maintaining the secondary or higher structure and Ca(2+)-regulation together with troponin.     

Fourier transform infrared spectroscopic study on the binding of Mg2+ to a mutant Akazara scallop troponin C (E142Q)

Troponin C (TnC) is the Ca(2+)-binding regulatory protein of the troponin complex in muscle tissue. Vertebrate fast skeletal muscle TnCs bind four Ca(2+), while Akazara scallop (Chlamys nipponensis akazara) striated adductor muscle TnC binds only one Ca(2+) at site IV, because all the other EF-hand motifs are short of critical residues for the coordination of Ca(2+). Fourier transform infrared (FTIR) spectroscopy was applied to study coordination structure of Mg(2+) bound in a mutant Akazara scallop TnC (E142Q) in D(2)O solution. The result showed that the side-chain COO(-) groups of Asp 131 and Asp 133 in the Ca(2+)-binding site of E142Q bind to Mg(2+) in the pseudo-bridging mode. Mg(2+) titration experiments for E142Q and the wild-type of Akazara scallop TnC were performed by monitoring the band at about 1600 cm(-1), which is due to the pseudo-bridging Asp COO(-) groups. As a result, the binding constants of them for Mg(2+) were the same value (about 6 mM). Therefore, it was concluded that the side-chain COO(-) group of Glu 142 of the wild type has no relation to the Mg(2+) ligation. The effect of Mg(2+) binding in E142Q was also investigated by CD and fluorescence spectroscopy. The on-off mechanism of the activation of Akazara scallop TnC is discussed on the basis of the coordination structures of Mg(2+) as well as Ca(2+).     

Roles for Hedgehog signaling in androgen production and prostate ductal morphogenesis

Previous studies have demonstrated that the Hedgehog (Hh) signaling pathway plays a critical role in the development and patterning of many endodermally derived tissues. We have investigated the role of Sonic hedgehog (Shh) in formation of the prostate gland by examining the urogenital phenotype of Shh mutant fetuses. Consistent with earlier work reporting an essential role for Shh in prostate induction, we have found that Shh mutant fetuses display abnormal urogenital development and fail to form prostate buds. Unexpectedly, however, we have discovered that this prostate defect could be rescued by three different methods: renal grafting, explant culture in the presence of androgens, and administration of dihydrotestosterone (DHT) to pregnant mice, indicating that the prostate defect in Shh mutants is due to insufficient levels of androgens. Furthermore, we find that the inhibition of Hh pathway signaling by treatment with cyclopamine does not block prostate formation in explant culture, but instead produces morphological defects consistent with a role for Hh signaling in ductal patterning. Taken together, our studies indicate that the initial organogenesis of the prostate proceeds independently of Shh, but that Shh or other Hh ligands may play a role in subsequent events that pattern the prostate.     

Highly potent inhibitors of TNF-alpha production. Part II: metabolic stabilization of a newly found chemical lead and conformational analysis of an active diastereoisomer

Design and synthesis of metabolically stabilized inhibitors of TNF-alpha production, which could be new drug candidates, are reported. Conformational analysis of an active diastereoisomer was performed based on biological evaluations of the conformationally fixed indane derivatives 17 and 18. Structure-activity relationships (SARs) based on biological evaluations of the optically active derivatives are also discussed. Full details including chemistry are reported.