Assembly of Smooth Muscle Myosin by the 38K Protein, a Homologue Of

Assembly of Smooth Muscle Myosin by the 38k Protein, a Homologue of a Subunit of Pre-mRNA Splicing Factor-2 Tsuyoshi Okagaki,* Akio Nakamura,* Tomohiko Suzuki,‡ Kazuhiro Ohmi,§ and Kazuhiro Kohama* *Department of Pharmacology, Gunma University School of Medicine, Maebashi, Gunma 371-8511, Japan; ‡Department of Biology, Kochi University, Kochi 780-8072, Japan; and §National Children’s Hospital, Setagaya-ku, Tokyo 154-0004, Japan Abstract. Smooth muscle myosin in the dephosphory- bound to myosin with both COOH-terminal 20 and lated state does not form filaments in vitro. However, NH2-terminal 28 residues of the 38k protein being es- thick filaments, which are composed of myosin and my- sential for myosin binding. The amino acid sequence of osin-binding protein(s), persist in smooth muscle cells, the 38k protein was not homologous to telokin, but to even if myosin is subjected to the phosphorylation– human p32, which was originally found in nuclei as a Downloaded from dephosphorylation cycle. The characterization of subunit of pre-mRNA splicing factor-2. Western blot- telokin as a myosin-assembling protein successfully ex- ting showed that the protein was expressed in various plained the discrepancy. However, smooth muscle cells smooth muscles. Immunofluorescence microscopy with that are devoid of telokin have been observed. We ex- cultured smooth muscle cells revealed colocalization of pected to find another ubiquitous protein with a similar the 38k protein with myosin and with other cytoskeletal role, and attempted to purify it from chicken gizzard. elements. The absence of nuclear immunostaining was The 38k protein bound to both phosphorylated and de- discussed in relation to smooth muscle differentiation. jcb.rupress.org phosphorylated myosin to a similar extent. The effect of the myosin-binding activity was to assemble dephos- Key words: myosin assembly • myosin binding • myo- phorylated myosin into filaments, although it had no ef- sin filament • human p32 • smooth muscle fect on the phosphorylated myosin. The 38k protein on September 30, 2017 Introduction The thick filaments observed in various muscle cells are (for review, see Stull et al., 1991; Trybus, 1991; Gallagher composed of myosin filaments that are associated with et al., 1997). Smooth muscle myosin molecules, if not myosin-binding proteins. Assembly into filaments is an in- phosphorylated, are in a folded structure called the 10S herent property of myosin (Huxley, 1963). Myosin-binding form, and hardly assemble filaments. When the myosin is proteins, such as myosin-binding protein-C (C-protein)1, phosphorylated, its shape changes to the extended 6S form myosin-binding protein-H (H-protein), M-protein, titin, and filaments are assembled (Onishi and Wakabayashi, and myomesin are thought to stabilize the filaments (for 1982; Trybus et al., 1982; Craig et al., 1983). Thus, the fila- review, see Epstein and Fischman, 1991; Furst and Gautel, ments formed from the phosphorylated myosin become 1995). unstable upon dephosphorylation of the RLC and, as a re- In the case of skeletal muscle myosin, assembly is ob- sult, easily fall apart. served regardless of the phosphorylation state of myosin. These in vitro properties of smooth muscle myosin fila- However, smooth muscle myosin does not form filaments ments are of particular interest when we observe the per- unless its regulatory light chain (RLC) is phosphorylated sistent nature of thick filaments in smooth muscle cells. Their myosin is repeatedly subject to cycles of phosphorylation and dephosphorylation in response to contraction, Address correspondence to K. Kohama, Department of Pharmacology, Gunma University School of Medicine, Maebashi, Gunma 371-8511, followed by relaxation (for review, see Stull et al., 1991; Japan. Tel.: 81-27-220-7960. Fax: 81-27-220-7966. E-mail: gacho@sb. Somlyo and Somlyo, 1994). However, there is no sign of gunma-u.ac.jp assembly and disassembly of thick filaments, indicating T. Okagaki’s present address is Laboratories of Marine Food Science, that they are stable (Somlyo et al., 1981; Cooke et al., Faculty of Bioresources, Mie University, Tsu-city, Mie 514-0102, Japan. 1989; Xu et al., 1996). By raising an mAb to the 10S form, 1Abbreviations used in this paper: C-protein, myosin-binding protein-C; HMM, heavy meromyosin; LMM, light meromyosin; MHC, myosin heavy Horowitz et al. (1994) examined the intracellular form of chain; MLCK, myosin light chain kinase. myosin. The concentration of 10S myosin was very low, re- The Rockefeller University Press, 0021-9525/2000/02/653/11 $5.00 The Journal of Cell Biology, Volume 148, Number 4, February 21, 2000 653–663 http://www.jcb.org 653 gardless of contraction and relaxation states, most of the Figure 1. Purification of the myosin being in the 6S form. Therefore, myosin filaments 38k protein. Aliquots of sam- are expected to be stabilized as thick filaments by myosin- ples from the following puri- binding proteins in the cell. The first examination of this fication steps were applied to suggestion was by Shirinsky et al. (1993), who observed SDS-PAGE: 1, Total homo- that telokin binds to dephosphorylated smooth muscle genate of gizzard; 2, myosin extracted from gizzard; 3, myosin to stimulate myosin assembly. However, telokin is crude myosin preparation not present in all smooth muscle cells (Gallagher and Her- (supernatant in the presence ring, 1991), indicating the presence of another myosin- of 0.3 M NaCl and 5 mM binding protein(s) with a similar role in the cell. ATP); 4, ammonium sulfate We were interested in this indication and adopted a precipitate of partially puri- strategy to purify myosin-binding proteins from skeletal fied myosin preparation at muscles (Starr and Offer, 1971). We chose a crude prepa- 55–80% saturation; 5, the ration of smooth muscle myosin as a starting material and fraction of DEAE Toypearl purified a protein of 38 kD that bound to myosin, causing 650M with the highest myo- filament assembly. sin-assembling activity; and 6, the active principle eluted from Superdex HR75. The 38k to the right of lane 6 denotes the band of the 38k protein. For details, see Materials and Methods. Materials and Methods Preparation of Proteins supernatant was subjected to stepwise ammonium sulfate fractionation. Fresh chicken gizzards were minced within 2 h of killing, and kept frozen Aliquots of each step of the fractionation were desalted with Biogel P-6 Downloaded from at Ϫ80ЊC until use. Proteins other than myosin were purified from the fro- (BioRad Laboratories), equilibrated with buffer A (1 mM ATP, 2 mM zen mince, unless otherwise specified. Dephosphorylated myosin was pu- MgCl2, 0.1 M NaCl, 20 mM Tris-HCl, pH 7.5, 1 mM DTT, and 0.5 mM rified from chicken gizzard according to Ebashi (1976) with modifications PMSF) and mixed with unphosphorylated myosin at a final concentration (Ikebe and Hartshorne, 1985a) and used as unphosphorylated myosin. We of 0.2 ␮M in buffer A. The mixtures were subjected to the dark-field mi- checked that myosin preparations did not contain the 38k myosin-assem- croscopy to examine myosin-assembling activity (see Centrifugation As- bling protein by SDS-PAGE or Western blotting. When required, myosin say). The fractions precipitated at between 55 and 80% saturation (Fig. 1, was phosphorylated with myosin light chain kinase (MLCK) in the pres- lane 4) contained the myosin-assembling activity. The fractions were dis- ence of Ca2ϩ and calmodulin (Okagaki et al., 1991). Rod, light meromyo- solved in buffer B (20 mM Tris-HCl, pH 7.5, 1 mM DTT, 0.5 mM PMSF, and sin (LMM), and heavy meromyosin (HMM) of gizzard myosin were pre- 0.5 ␮g/ml leupeptin), the volume of which was selected to adjust its con- jcb.rupress.org pared proteolytically as described (Ikebe and Hartshorne, 1985b; King et ductivity to be as low as that of 0.2 M NaCl. Then the solution was clari- al., 1995). Myosin was also purified from chicken breast muscle and bo- fied by centrifugation at 100,000 g for 2 h. The supernatant was applied to vine heart and used as skeletal and cardiac myosin, respectively (Okagaki DEAE Toyopearl 650M (Tosoh), equilibrated with buffer B supple- et al., 1993). In this paper, myosin denotes smooth muscle myosin unless mented with 0.2 M NaCl, followed by elution with a linear gradient of otherwise mentioned. MLCK, telokin, and tropomyosin were purified NaCl from 0.2–0.5 M. Aliquots of each eluate were desalted and tested for myosin-assembling activity as described. The activity was detected in the from chicken gizzard according to Hayakawa et al. (1994), Ito et al. (1989), on September 30, 2017 and Ishikawa et al. (1989), respectively. Monomeric actin was purified fractions eluted with 0.3–0.4 M NaCl. The fractions containing activity from chicken breast muscle and used as actin filaments after polymeriza- were composed of polypeptides of 38 kD, 17 kD, and minor contaminants tion (Okagaki et al., 1991). The concentration of actin filaments was ex- (Fig. 1, lane 5). They were pooled, concentrated with Centricon-30 (Milli- pressed as that of monomeric actin. pore), and applied to a Superdex HR75 column (Amersham-Pharmacia) equilibrated with buffer B supplemented with 0.3 M NaCl. Fractions cor- responding to 15–30 kD, as estimated by a molecular weight marker for Preparation of the 38k Protein gel filtration (Amersham-Pharmacia), showed the myosin-assembling ac- Following our experience with C-protein, a myosin-binding protein of tivity. The major protein band of these fractions was 38 kD on SDS- skeletal muscle (Okagaki et al., 1993), we used a crude myosin prepara- PAGE (Fig. 1, lane 6). We used this fraction for the most of biochemical tion (see below) as the starting material. From this, we purified a protein experiments. During column chromatography, the 38k protein often de- of 38 kD in SDS-PAGE using myosin-assembling activity under dark-field graded to polypeptides of 20 and 17 kD if leupeptin was absent.

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