Michio Yazawa

Molecular cloning of a novel phosphorylation‐dependent inhibitory protein of protein phosphatase‐1 (CPI17) in smooth muscle: its specific localization in smooth muscle1

The cDNA encoding a phosphorylation‐dependent inhibitory protein of protein phosphatase‐1 (PP1) was isolated from a porcine aorta library. The coding region represented the complete amino acid sequence of this protein comprised of a novel 147‐residue polypeptide, which we termed CPI17, a 17‐kDa PKC‐potentiated inhibitory protein of PP1. As well as the native CPI17 from porcine aorta, the recombinant protein completely suppressed the PP1 activity (IC50=0.18 nM) by the stoichiometric thiophosphorylation. The CPI17 mRNA is expressed in smooth muscle tissues such as aorta and bladder, whereas little expression was observed in heart, skeletal muscle, and non‐muscle tissues. These results suggest a specific regulatory mechanism of the PP1 activity through CPI17 in smooth muscle.

POSSIBLE INVOLVEMENT OF THE NOVEL CPI-17 PROTEIN IN PROTEIN KINASE C SIGNAL TRANSDUCTION OF RABBIT ARTERIAL SMOOTH MUSCLE

1 CPI‐17 has recently been identified as a novel protein in vascular smooth muscle. In vitro, its phosphorylation and thiophosphorylation by protein kinase C (PKC) specifically inhibits the type 1 class of protein phosphatases, including myosin light chain (MLC) phosphatase. 2 Both of the phosphorylated CPI‐17 states dose‐dependently potentiated submaximal contractions at constant [Ca2+] in β‐escin‐permeabilized and Triton X‐100‐demembranated arterial smooth muscle, but produced no effect in intact and less intensely permeabilized (α‐toxin) tissue. Thiophosphorylated CPI‐17 (tp‐CPI) induced large contractions even under Ca2+‐free conditions and decreased Ca2+ EC50 by more than an order of magnitude. Unphosphorylated CPI‐17 produced minimal but significant effects. 3 tp‐CPI substantially increased the steady‐state MLC phosphorylation to Ca2+ ratios in β‐escin preparations. 4 tp‐CPI affected the kinetics of contraction and relaxation and of MLC phosphorylation and dephosphorylation in such a manner that indicates its major physiological effect is to inhibit MLC phosphatase. 5 Results from use of specific inhibitors in concurrence with tp‐CPI repudiate the involvement of general G proteins, rho A or PKC itself in the Ca2+ sensitization by tp‐CPI. 6 Our results indicate that phosphorylation of CPI‐17 by PKC stimulates binding of CPI‐17 to and subsequent inhibition of MLC phosphatase. This implies that CPI‐17 accounts largely for the heretofore unknown signalling pathway between PKC and inhibited MLC phosphatase.

The amino acid sequence of the Tetrahymena calmodulin which specifically interacts with guanylate cyclase

Abstract The amino acid sequence of the Tetrahymena calmodulin was determined. The protein is composed of 147 amino acids and the amino-terminal is acetylated. Compared to bovine brain calmodulin, there were eleven substitutions and one deletion of amino acid residues. The substitutions and deletion were concentrated in the carboxyl-terminal half of the molecule. Among the substitutions, those at positions 86 (Arg → Ile), 135 (Gln → His) and 143 (Gln → Arg) may introduce the functional difference. The deletion occurred near the carboxyl-terminal, this region being assumed to be exposed to the surface area ( R.H. Kretsinger and C.D. Barry (1975) ). The change in the sequence at this terminal region may be attributable to the specific activation of guanylate cyclase.

The amino acid sequence of the calmodulin obtained from sea anemone (Metridium senile) muscle

Abstract The amino acid sequence of the calmodulin obtained from sea anemone muscle was determined. The calmodulin was composed of 148 amino acid residues and its amino terminal was blocked. When compared with bovine brain calmodulin, the number of amino acid residues per molecule was the same and there were 3 replacements at residues 99 (Tyr → Phe), 143 (Gln → Lys) and 147 (Ala → Ser).

Infrared studies of interaction between metal ions and Ca2+-binding proteins Marker bands for identifying the types of coordination of the side-chain COO− groups to metal ions in pike parvalbumin (pI = 4.10)

Metal‐ligand interactions in the Ca2+‐binding sites of pike parvalbumin (pI = 4.10) have been examined by Fourier‐transform infrared spectroscopy. The region of the COO− antisymmetric stretch provides useful information on the types of coordination of the COO− groups to the metal ions in the Mg2+‐, Mn2+‐, and Ca2+‐bound forms. In the spectrum of the Ca2+‐bound form, two bands are observed at 1,582 and 1,553 cm−, whereas, in the spectra of the Mg2+‐ and Mn2+‐bound forms, bands are observed only in the region around 1,582 cm−1 and no band is found in the region around 1,553 cm−1. The 1,553‐cm−1 band of the Ca2+‐bound form reflects the bidentate coordination of the COO− groups of both Glu‐62 in the CD site and Glu‐101 in the EF site to the Ca2+ ions, which has been made clear by X‐ray analysis as a feature of the Ca2+‐bound form. Absence of such a band in the spectrum of the Mn2+‐bound form is consistent with the X‐ray structure of this form where both of the two COO− groups are unidentate. These unidentate COO− groups of Glu‐62 and Glu‐101 in the Mn2+‐bound form seem to give rise to a band at 1,577‐1,574 cm−. The spectrum of the Mg2+‐bound form is also consistent with the ‘pseudo‐bridging’ coordination of the COO− group of Glu‐101 reported in the X‐ray structure of a form where the Mg2+ ion occupies only the EF site, and the same spectrum is further indicative of the ‘pseudo‐bridging’ coordination of the COO− group of Glu‐62.

Fortilin binds Ca2+ and blocks Ca2+-dependent apoptosis in vivo

Fortilin, a 172-amino-acid polypeptide present both in the cytosol and nucleus, possesses potent anti-apoptotic activity. Although fortilin is known to bind Ca2+, the biochemistry and biological significance of such an interaction remains unknown. In the present study we report that fortilin must bind Ca2+ in order to protect cells against Ca2+-dependent apoptosis. Using a standard Ca2+-overlay assay, we first validated that full-length fortilin binds Ca2+ and showed that the N-terminus (amino acids 1-72) is required for its Ca2+-binding. We then used flow dialysis and CD spectropolarimetry assays to demonstrate that fortilin binds Ca2+ with a dissociation constant (Kd) of approx. 10 mM and that the binding of fortilin to Ca2+ induces a significant change in the secondary structure of fortilin. In order to evaluate the impact of the binding of fortilin to Ca2+ in vivo, we measured intracellular Ca2+ levels upon thapsigargin challenge and found that the lack of fortilin in the cell results in the exaggerated elevation of intracellular Ca2+ in the cell. We then tested various point mutants of fortilin for their Ca2+ binding and identified fortilin(E58A/E60A) to be a double-point mutant of fortilin lacking the ability of Ca2+-binding. We then found that wild-type fortilin, but not fortilin(E58A/E60A), protected cells against thapsigargin-induced apoptosis, suggesting that the binding of fortilin to Ca2+ is required for fortilin to protect cells against Ca2+-dependent apoptosis. Together, these results suggest that fortilin is an intracellular Ca2+ scavenger, protecting cells against Ca2+-dependent apoptosis by binding and sequestering Ca2+ from the downstream Ca2+-dependent apoptotic pathways.

A Phytochemical in the Edible Tamogi-take Mushroom (Pleurotus cornucopiae), D-Mannitol, Inhibits ACE Activity and Lowers the Blood Pressure of Spontaneously Hypertensive Rats

D-Mannitol, one of the main phytochemicals of the edible Tamogi-take mushroom (Pleurotus cornucopiae), was found to inhibit an angiotensin I converting enzyme (ACE). The antihypertensive effect of D-mannitol and a hot water extract of Tamogi-take mushroom was demonstrated in spontaneously hypertensive rats (SHR) by oral administration.

Inhibition of myosin/moesin phosphatase by expression of the phosphoinhibitor protein CPI-17 alters microfilament organization and retards cell spreading

Cell migration and cytokinesis require reorganization of the cytoskeleton, involving phosphorylation and dephosphorylation of proteins such as myosin II and moesin. Myosin and moesin bind directly to a regulatory subunit of myosin/moesin phosphatase (MMP) that contains a protein type-1 phosphatase (PP1) catalytic subunit. Here we examined the role of MMP in cytoskeletal dynamics using a phosphorylation-dependent inhibitor protein specific for MMP, called CPI-17. Fibroblasts do not express CPI-17, making them a null background to study effects of expression. Wild type CPI-17 in rat embryo fibroblasts caused (1) abnormal accumulation of cortical F-actin fibers, distinct from the stress fibers induced by expression of active RhoA; (2) progressive contraction of cell area, leaving behind filamentous extensions that stained for F-actin and moesin, but not myosin; and (3) significantly retarded spreading of fibroblasts on fibronectin with elevated myosin II light chain phosphorylation. A phosphorylation site mutant CPI-17(T38A) and inhibitor-2 (Inh2), another PP1-specific inhibitor protein, served as controls and did not elicit these same responses when expressed at the same level as CPI-17. Inhibition of myosin light chain kinase by ML-9 prevented the abnormal accumulation of cortical microfilaments by CPI-17, but did not reverse shrinkage in area, whereas kinase inhibitors HA1077 and H7 prevented CPI-17-induced changes in microfilament distribution and cell contraction. These results highlight the physiological importance of myosin/moesin phosphatase regulation to dynamic remodeling of the cytoskeleton.

Sequence‐specific assignments of downfield‐shifted amide proton resonances of calmodulin Use of two‐dimensional NMR analysis of its tryptic fragments

Two‐dimensional NMR methods were applied to assign the extremely downfield‐shifted amide‐proton resonances in the 500‐MHz 1H‐NMR spectra of the NH2‐terminal fragment of residues 1–75 of calmodulin. The low‐field resonances of the 1H‐NMR spectra of intact calmodulin were assigned to specific amino acid residues by comparison with spectra of the tryptic fragments of residues 1–75 and 78–148, in both the Ca2+‐free and Ca2+‐bound states. The hydrogen bonding of glycine residues connecting the two amino acid residues at the Z and − Y positions in the octahedral Ca2+ coordination site was investigated. The Gly 134 in site IV showed a different property from the other glycines, 25, 61 and 98, involved in sites I, II and III, respectively.

Structural organization of lower marine nonvertebrate calmodulin genes

The troponin C (TnC) superfamily genes generally possess five introns, and the positions where they are inserted are well conserved except for the fourth intron. Based on a structural comparison of TnC genes, we proposed that the common ancestor of TnC or TnC superfamily genes had no intron corresponding to the modern fourth intron, and therefore members of the superfamily have gained the fourth intron independently within each lineage. Here, we cloned calmodulin (CaM, one of the members of the TnC superfamily) cDNAs from two lower marine nonvertebrates, the sea anemone, Metridium senile, belonging to the Cnidaria, and the sponge, Halichondria okadai, belonging to the Porifera, and also determined their genomic organization. Chordate CaM genes generally possess five introns, but neither sea anemone nor sponge CaM has anything corresponding to the fourth intron of chordate CaMs, suggesting that the early metazoan CaM must have had only four introns. The modern fourth intron of chordate CaMs was acquired within the chordate lineage after nonvertebrate/chordate divergence. This notion concurs with our proposal explaining the evolution of the TnC superfamily genes.