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T Okagaki

Publications and source records attributed to T Okagaki.

At least 19 recordsLinked to original sources
PubMed1998

Structure and function of smooth muscle myosin light chain kinase.

Myosin light chain kinase (MLCK) plays a central role in regulating the actin-myosin interaction of smooth muscle. MLCK phosphorylates the light chain of myosin in the presence of Ca2+ and calmodulin (CaM) thereby activating myosin so that it can interact with actin. Besides this kinase activity, MLCK shows i) actin-binding activity that can assemble actin filaments into their bundles and ii) myosin-binding activity that can form myosin filaments. To localize the actin- and myosin-binding activities in the MLCK molecule and to examine their possible role in regulating the actin-myosin interaction, we expressed various fragments of cDNA encoding MLCK in Escherichia coli as recombinant proteins. We found that MLCK consists of an N-terminal actin-binding domain, a central kinase domain, and a C-terminal myosin-binding domain. The Met1-Pro41 sequence is responsible for Ca2+/CaM-sensitive binding to actin. This binding site exerts an inhibitory effect on the actin-myosin interaction only when myosin is phosphorylated. MLCK binds to myosin at the C-terminal domain, the sequence of which is identical to telokin, an abundant myosin-binding protein in smooth muscle cells. This domain itself has no regulatory role in the interaction. However, the interaction was stimulated when this domain was extended to include the sequence known to regulate the activity of the kinase domain. The stimulation was observed only when myosin was unphosphorylated.

Actins↗
PubMed1997

The structure and function of the actin-binding domain of myosin light chain kinase of smooth muscle.

In addition to its kinase activity, the myosin light chain kinase (MLCK) of smooth muscle has an actin binding activity through which it can regulate the actin-myosin interaction of smooth muscle (Kohama, K., Okagaki, T., Hayakawa, K., Lin, Y., Ishikawa, R., Shimmen, T., and Inoue, A. (1992) Biochem. Biophys. Res. Commun. 184, 1204-1211). In this study, we have analyzed the actin binding activity of MLCK and related it to its amino acid sequence by producing native and recombinant fragments of MLCK. Parent MLCK exhibited both calcium ion (Ca2+) and calmodulin (Ca2+/CaM)-sensitive and Ca2+/CaM-insensitive binding to actin filaments. The native fragment, which consists of the Met1-Lys114 sequence (Kanoh, S., Ito, M., Niwa, E., Kawano, Y., and Hartshorne, D. J. (1993) Biochemistry 32, 8902-8907), and the recombinant NN fragment, which contains this 1-114 sequence, showed only Ca2+/CaM-sensitive binding. An inhibitory effect of the NN fragment on the actin-myosin interaction was observed by assaying in vitro motility and by measuring the actin-activated ATPase activity of myosin. The recombinant NN/41 fragment, which is constructed without the Met1-Pro41 sequence of the NN fragment, lost both the actin binding activity and the inhibitory effect. We confirmed the importance of the 1-41 sequence by using a few synthetic peptides to compete against the NN fragment in binding to actin filaments. The experiments using recombinant fragments and synthetic peptides also revealed that the site for CaM-binding is the Pro26-Pro41 sequence. The site for the Ca2+/CaM-insensitive binding, which is shown to be localized between the Ca2+/CaM-sensitive site and the central kinase domain of MLCK, exerted no regulatory effects on the actin-myosin interaction.

Actins↗
PubMed1996

Mitotic count, nuclear atypia, and immunohistochemical determination of Ki-67, c-myc, p21-ras, c-erbB2, and p53 expression in granulosa cell tumors of the ovary: mitotic count and Ki-67 are indicators of poor prognosis.

In spite of extensive research, the behavior of granulosa cell ovarian tumors remains unpredictable and is complicated by the lack of prognostic factors in early-stage disease. Forty patients with granulosa cell tumors were identified from tumor registries and data were analyzed for patient outcome. Mitotic count and nuclear atypia were determined at time of histological review. Paraffin-embedded archival tumor tissues from 32 of 40 patients were available, and immunohistochemical testing for Ki-67, c-myc, p21-ras, c-erbB2, and p53 was performed on archival tissues. Results were correlated with patients' outcome. Mitotic count and Ki-67 were found to be negatively associated with survival in granulosa cell tumors. Nuclear atypia, c-myc, p21-ras, c-erbB2, and p53 were not found to be of prognostic significance.

Adolescent↗
PubMed1996

Myosin light chain kinase: an actin-binding protein that regulates an ATP-dependent interaction with myosin.

Myosin light chain kinase (MLCK) is a key regulator of smooth muscle contraction. The most conspicuous form of regulation is achieved by phosphorylation of the myosin light chain, allowing myosin to interact with actin. This interaction is regulated by actin-binding proteins that modulate actin filaments. In this review Kazuhiro Kohama and colleagues consider MLCK as an actin-binding protein and attempt to shed light on the cross-talk between the different kinds of regulation of the actin-myosin interaction in smooth muscle. An understanding of these mechanisms will assist the development of compounds with therapeutic importance in muscular disorders.

Actins↗
PubMed1994

Malignant melanoma of the vulva treated by radical hemivulvectomy. A prospective study of the Gynecologic Oncology Group.

BACKGROUND: Beginning in 1983, the Gynecologic Oncology Group (GOG) conducted a prospective clinicopathologic study of primary malignant melanoma of the vulva. The objectives of this study were to determine the relationship of histopathologic parameters and microstaging to the International Federation of Gynaecology and Obstetrics (FIGO) staging and prognosis. METHODS: All patients with primary untreated malignant melanoma of the vulva and no history of previous or subsequent other primary invasive malignancy were eligible for study entry. All patients were required to have modified radical hemivulvectomy as minimal therapy. Groin dissection was optional. Histopathologic specimens were reviewed for capillary space involvement, Clark's level, Breslow's depth of invasion, cell type, and melanin distribution. Patient characteristics were analyzed in their relationship to groin node status and recurrence-free interval. RESULTS: Between 1983 and 1990, 81 patients were entered in the study. Of these, 71 were evaluable. Thirty-four patients underwent radical hemivulvectomy, and 37 patients underwent radical vulvectomy. In addition, 56 patients underwent groin node dissection. The factors that were independently correlated with groin node status were: capillary lymphatic space involvement (p = 0.0001) and central primary tumor location (i.e., bilateral/clitoral/T3) (P = 0.003). The other factors that were significant--clinical tumor size, vulvar staging (FIGO), GOG performance status, and Breslow's depth of invasion--were not independent predictors of positive nodes. The factor with the highest significant correlation with recurrence-free interval was the 1992 staging system of the American Joint Committee on Cancer (AJCC) for malignant melanoma of the skin. Using multiple regression, AJCC stage was the only independent prognostic factor. In the absence of AJCC stage, Breslow's depth of invasion was the most prognostic. CONCLUSION: The biologic behavior of vulvar melanoma is similar to other nongenital cutaneous malignant melanoma.

Adult↗
PubMed1994

Bundling of actin filaments by myosin light chain kinase from smooth muscle.

Myosin light chain kinase has an inhibitory effect on the interaction of actin filaments with phosphorylated smooth muscle myosin. Myosin light chain kinase binds to actin filaments, and the inhibition is attributable to the actin-binding activity and not the kinase activity of myosin light chain kinase [Kohama et al. (1992) Biochem. Biophys. Res. Commun. 184, 1204-1211]. We now report that myosin light chain kinase is able to assemble actin filaments into thick bundles, which can be visualized by optical and electron microscopy and can be monitored by measuring the sedimentation and flow birefringence of actin filaments. The bundling activity of myosin light chain kinase is abolished by calmodulin in the presence of Ca2+. The possibility is discussed that myosin light chain kinase has multiple actin-binding sites through which it can cross-link actin filaments.

Actins↗
PubMed1994

Stimulation of the ATP-dependent interaction between actin and myosin by a myosin-binding fragment of smooth muscle caldesmon.

We reported previously that smooth muscle caldesmon stimulates the ATP-dependent interaction between actin and phosphorylated smooth muscle myosin, as monitored by ATPase measurement and in vitro motility assay. Furthermore, this effect changes from stimulatory to inhibitory with increasing concentrations of caldesmon [Ishikawa et al., 1991: J. Biol. Chem. 266:21784-21790]. The N-terminal (myosin-binding) fragment and the C-terminal (actin-binding) fragment were purified from digests of caldesmon. The effects of the myosin-binding fragment and the actin-binding fragment on the interaction were stimulatory and inhibitory, respectively, indicating that stimulatory and inhibitory domains are localized in the myosin-binding domain and actin-binding domain of caldesmon, respectively. The effect of the myosin-binding fragment on the interaction was exclusively stimulatory when the interaction was challenged by caldesmon, both at lower and higher concentrations. However, the actin-binding fragment had no effect on the interaction at lower concentrations and inhibited the interaction at higher concentrations. Thus, the stimulatory effect of caldesmon that is observed at lower concentrations can be explained by the hypothesis that the stimulatory effect of the myosin-binding domain predominates over the inhibitory effect of the actin-binding domain when the concentration of caldesmon is low. With uncleaved caldesmon, we also emphasized the role of the myosin-binding domain in the stimulation as follows; the stimulatory effect of caldesmon became obscured when binding of caldesmon to myosin was competed by the exogenous caldesmon-binding fragment of myosin.

Actins↗
PubMed1994

The regulatory role of myosin light chain kinase as an actin-binding protein.

Myosin light chain kinase (MLCK) is present in muscle cells including those of smooth muscle as an actin-binding protein. By avoiding complication introduced as a result of kinase activity of MLCK, we have demonstrated regulatory role of MLCK through its actin-binding activity [Kohama et al. (1992) Biochem. Biophys. Res. Commun. 184, 1204-1211]. To analyze such a regulatory role of MLCK, we compared the effects of MLCK on the velocity of the movement of actin filaments on a surface coated with smooth muscle myosin with those of another actin-binding proteins in smooth muscle, namely, caldesmon (CaD) and calponin (CaP). Both CaD and CaP stimulated movement when their concentrations were low, but they inhibited movement as their concentrations were increased. Calmodulin (CaM) in the presence of Ca2+ (Ca-CaM) antagonized the inhibition but hardly affected the stimulation. The effect of MLCK, by contrast, was simply inhibitory when Ca-CaM was not present. No stimulation was observed until Ca-CaM was added. The inhibitory ability of these actin-binding proteins increased in the following order: CaD < CaP < MLCK. The effect of MLCK and CaD on movement was further examined on surfaces coated with skeletal muscle myosin. The basic effect was similar to that observed with smooth muscle myosin. However, 10-fold greater concentrations of MLCK and CaD were required for a comparable effect. Such an increase in the required concentration was also observed when the velocity of movement was increased by elevation of the temperature during the assay with smooth muscle myosin. Thus, it is the velocity of movement itself that determines the required concentrations of MLCK and CaD.

Actins↗
PubMed1994

The prognostic value of nuclear versus architectural grading in endometrial adenocarcinoma: a Gynecologic Oncology Group study.

The pathologic grade of endometrial adenocarcinoma is widely recognized as an important prognostic and therapeutic indicator. However, disagreement persists about the optimal method of determining grade. The pathology committee of the Gynecologic Oncology Group employs a system based on the proportion of tumor in glandular array; this system is both reproducible and predictive of outcome. Others have suggested that grading based on nuclear pleomorphism and the size of nucleoli provides better prognostication. We compared the three-level architectural grading system (AG) with a two-level nuclear grading system (NG) to determine reproducibility and prognostic value in 88 cases of stage 1 endometrial adenocarcinoma. Three pathologists made independent assessments of grade by each method. The division of tumors by architectural arrangement was superior for predicting survival (83%, 73%, and 44%, AG, p = 0.005; vs. 79% and 61%, NG, p = 0.14) and equivalent to nuclear grading for prediction of recurrence. Both systems were moderately reproducible (k = 0.49, AG; k = 0.57, NG). Assessment of NG was more tedious than that of AG. Subdivision of architectural grade based on high nuclear atypia, as recommended in current, International Federation of Gynecology and Obstetrics guidelines, did not improve prognostication. Because grading based on nuclear pleomorphism does not provide prognostic information superior to that resulting from architectural grading, we do not advocate its use in routine surgical pathology practice.

Adenocarcinoma↗
PubMed1993

Renal cell carcinoma extending into left side of double inferior vena cava.

A case of renal cell carcinoma extending into the left side of a double inferior vena cava is presented. En bloc excision of the left inferior vena cava and left kidney was performed without complications. Recognition of various venous anomalies, including double inferior vena cava, is important in the evaluation and surgical treatment of retroperitoneal disease.

Aged↗
PubMed1993

The major myosin-binding domain of skeletal muscle MyBP-C (C protein) resides in the COOH-terminal, immunoglobulin C2 motif.

A common feature shared by myosin-binding proteins from a wide variety of species is the presence of a variable number of related internal motifs homologous to either the Ig C2 or the fibronectin (Fn) type III repeats. Despite interest in the potential function of these motifs, no group has clearly demonstrated a function for these sequences in muscle, either intra- or extracellularly. We have completed the nucleotide sequence of the fast type isoform of MyBP-C (C protein) from chicken skeletal muscle. The deduced amino acid sequence reveals seven Ig C2 sets and three Fn type III motifs in MyBP-C. alpha-chymotryptic digestion of purified MyBP-C gives rise to four peptides. NH2-terminal sequencing of these peptides allowed us to map the position of each along the primary structure of the protein. The 28-kD peptide contains the NH2-terminal sequence of MyBP-C, including the first C2 repeat. It is followed by two internal peptides, one of 5 kD containing exclusively spacer sequences between the first and second C2 motifs, and a 95-kD fragment containing five C2 domains and three fibronectin type III motifs. The C-terminal sequence of MyBP-C is present in a 14-kD peptide which contains only the last C2 repeat. We examined the binding properties of these fragments to reconstituted (synthetic) myosin filaments. Only the COOH-terminal 14-kD peptide is capable of binding myosin with high affinity. The NH2-terminal 28-kD fragment has no myosin-binding, while the long internal 100-kD peptide shows very weak binding to myosin. We have expressed and purified the 14-kD peptide in Escherichia coli. The recombinant protein exhibits saturable binding to myosin with an affinity comparable to that of the 14-kD fragment obtained by proteolytic digestion (1/2 max binding at approximately 0.5 microM). These results indicate that the binding to myosin filaments is mainly restricted to the last 102 amino acids of MyBP-C. The remainder of the molecule (1,032 amino acids) could interact with titin, MyBP-H (H protein) or thin filament components. A comparison of the highly conserved Ig C2 domains present at the COOH-terminus of five MyBPs thus far sequenced (human slow and fast MyBP-C, human and chicken MyBP-H, and chicken MyBP-C) was used to identify residues unique to these myosin-binding Ig C2 repeats.

Amino Acid Sequence↗
PubMed1992

A novel regulatory effect of myosin light chain kinase from smooth muscle on the ATP-dependent interaction between actin and myosin.

The actin-binding activity of myosin light chain kinase (MLCK) from smooth muscle was studied with special reference to the ATP-dependent interaction between actin and myosin. MLCK in the presence of calmodulin endowed sensitivity to Ca2+ on the movement of actin filaments on phosphorylated myosin from smooth muscle that was fixed on a coverslip. This regulatory effect was not attributable to the kinase activity of MLCK but could be explained by its actin-binding activity. The importance of the actin-binding activity was further substantiated by results of an experiment with Nitellopsis actin-cables in which MLCK regulated the interaction under conditions where MLCK was exclusively associated with the actin-cables.

Actins↗
PubMed1992

Regulation by Ca(2+)-calmodulin of the actin-bundling activity of Physarum 210-kDa protein.

From the plasmodia of a lower eukaryote, Physarum polycephalum, we have previously purified a 210-kDa protein that showed similar properties to those of smooth muscle caldesmon. Further characterization of the 210-kDa protein revealed that it bundled actin filaments. This bundling activity was inhibited by calmodulin in the presence of Ca2+. Unlike smooth muscle caldesmon, the 210-kDa protein bundled actin filaments whether or not a reducing agent, such as dithiothreitol, was present. The protein was shown to have two (or more) different actin-binding sites which were classified into salt-sensitive and salt-insensitive sites. Electron microscopy revealed that the 210-kDa protein was an elongated molecule (mean length, 97 +/- 25 nm) which was bent in the middle. The Stokes radius and sedimentation coefficient of the 210-kDa protein were 130 A and 2.9 S, respectively. An immunofluorescence study revealed that the 210-kDa protein colocalized with the bundles of actin filaments in thin-spread preparations of Physarum plasmodia, suggesting that the 210-kDa protein was regulating the appearance and disappearance of the actin bundles that are associated with the contraction-relaxation cycle of the plasmodia.

Actins↗