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Phosphorylation of Subunit Proteins of Intermediate Filaments from Chicken Muscle and Nonmuscle Cells (Desmin/Tropomyosin/Two-Dimensional Gel Electrophoresis) CLARE M

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Phosphorylation of Subunit Proteins of Intermediate Filaments from Chicken Muscle and Nonmuscle Cells (Desmin/Tropomyosin/Two-Dimensional Gel Electrophoresis) CLARE M Proc. Natl. Acad. Sci. USA Vol. 76, No. 2, pp. 819-823, February 1979 Cell Biology Phosphorylation of subunit proteins of intermediate filaments from chicken muscle and nonmuscle cells (desmin/tropomyosin/two-dimensional gel electrophoresis) CLARE M. O'CONNOR, DAVID R. BALZER, JR., AND ELIAS LAZARIDES* Division of Biology, California Institute of Technology, Pasadena, California 91125 Communicated by James F. Bonner, November 16, 1978 ABSTRACT The phosphorylation of the subunit proteins reactivity (6, 9-13). Except for the keratin filaments that as- of intermediate (10-nm) filaments has been investigated in sociate with in and chicken muscle and nonmuscle cells by using a two-dimensional desmosomes epithelial epidermal cells, the gel electrophoresis system. Desmin, the 50,000-dalton subunit nonmuscle 10-nm filaments are not associated with well-de- rotein of the intermediate filaments of muscle, had previously fined cytoplasmic structures. The cytoskeletal networks formed been shown to exist as two major isoelectric variants-a and by the intermediate filaments are stable, however, and remain a-in smooth, skeletal, and cardiac chicken muscle. Incubation insoluble after Triton X-100 extraction of the cells (14). The of skeletal and smooth muscle tissue with 32P043- reveals that muscle intermediate filaments also remain insoluble after the the acidic variant, a-desmin, and three other desmin variants removal of actin are phosphorylated in vivo and in vitro. Under the same con- and myosin filaments with 1 M KI (15). Col- ditions, minor components of a- and fl-tropomyosin from skel- cemid treatment of nonmuscle and muscle cells induces a re- etal muscle, but not smooth muscle, are also phosphorylated. arrangement of the intermediate filaments into large perinu- Both the phosphorylated desmin variants and the nonphos- clear caps (6, 16-18). Biochemical characterization of the in- phorylatedB-desmin variant remain insoluble under conditions termediate filaments present in the Triton cytoskeletons and that solubilize actin and myosin filaments, but leave Z-discs and Colcemid-induced of fibroblasts has intermediate filaments insoluble. Primary cultures of embryonic caps identified the subunit chicken muscle labeled with 32PO43- possess, in addition to the as a protein with a molecular weight of 52,000-55,000 (14, desmin variants described above, a major nonphosphorylated 18). and multiple phosphorylated variants of the 52,000-dalton, fi- Several contractile and structural proteins have been shown broblast-type intermediate filament protein (IFP). Filamentous to be phosphorylated in muscle and nonmuscle cells, including cytoskeletons, prepared from primary myogenic cultures by the myosin light chains, a-tropomyosin, filamin, and spectrin Triton X-100 extraction, contain actin and all of the phos- In the case of phorylated and nonphosphorylated variants of both desmin and (19-22). myosin, phosphorylation is required for the IFP. Similarly, these proteins are the major components of the activation of myosin ATPase by actin (19), while phos- the caps of aggregated 10-nm filaments isolated from the same phorylation of spectrin is required for its association with actin cell cultures previously exposed to Colcemid. These results (22). In this paper, we show that desmin is present as a single demonstrate that a nonphosphorylated and several phos- nonphosphorylated and several phosphorylated variants in phorylated variants of desmin and IFP are present in assembled chicken smooth and skeletal muscle. All of these desmin variants structures in muscle and nonmuscle cells. are also present together with a nonphosphorylated and several Muscle cells contain a cytoskeletal network of filaments, com- phosphorylated variants of the fibroblast-type intermediate monly referred to as 10-nm or intermediate filaments, that are filament protein in primary cultures of embryonic thigh muscle. intermediate in diameter to the actin and myosin filaments We have also observed that minor components of both the a involved in contraction (1-3). Biochemical and immunological and ,B forms of tropomyosin are phosphorylated in skeletal, but studies have demonstrated that the intermediate filaments from not in smooth, muscle cells. smooth muscle are comprised largely of a 50,000-dalton protein, desmin (4), which occurs as the same two isoelectric variants MATERIALS AND METHODS in chicken skeletal, smooth, and cardiac muscle (5). Immu- Protein Phosphorylation in Intact and Excised Muscle nofluorescent staining of muscle cells with a desmin-specific Tissues. Muscle proteins were labeled in vivo by injecting the antibody has shown that desmin is present both in cytoplasmic right thigh muscle of a young chick (1 to 10 days old) with 0.5 filaments and in areas where actin filaments associate with the mCi (1 Ci = 3.7 X 1010 becquerels) of neutralized [32P]phos- plasma membrane, such as the cardiac muscle intercalated discs phoric acid (New England Nuclear, carrier-free). After 24 hr, and the peripheries of skeletal muscle Z-discs (4, 6-8). Thus, it the tissue was excised, minced, and homogenized in 3 vol of 8 was proposed that desmin-containing structures in muscle cells M urea/2% (vol/vol) Nonidet P-40/0.5% 2-mercaptoethanol are involved in the mechanical integration of contractile with a ground-glass homogenizer. The homogenized tissue was units. solubilized for 1 hr at room temperature. Insoluble material was Cytoskeletal networks of 10-nm filaments also exist in many removed by centrifugation at 500 X g for 5 min, and the su- nonmuscle cells. Recent studies have demonstrated that, al- pernatant was applied directly to isoelectric focusing gels. though the 10-nm filaments from various nonmuscle and Small pieces of excised thigh or gizzard muscle were equili- muscle cells are morphologically similar, their subunit proteins brated for 30-60 min in phosphate-free minimal essential may be cell-type specific. Neurofilaments, glial filaments, medium (GIBCO) and subsequently incubated in 0.5 ml of epidermal keratin filaments, the 10-nm filaments from fibro- phosphate-free minimal essential medium to which 32P043- blasts, and muscle intermediate filaments are each composed was added to give a final concentration of 100-200 ,uCi/ml. of biochemically distinct subunits with little serological cross- After 4- to 5-hr incubation, the tissue pieces were removed, rinsed in phosphate-free minimal essential medium, and pro- The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "ad- Abbreviations: EGTA, ethylene glycol bis(fl-aminoethyl ether)- vertisement" in accordance with 18 U. S. C. §1734 solely to indicate N,N,N',N'-tetraacetic acid; IFP, intermediate filament protein. this fact. * To whom reprint requests should be addressed. 819 Downloaded by guest on September 29, 2021 820 Cell Biology: O'Connor et al. Proc. Natl. Acad. Sci. USA 76 (1979) W.. -I.. W-' * . I 0 TM s A. a- a a. ..k B C 4 , .n __ I Ei .:T. I.- I iiIt la a * :-. ...": F _ ._"' ~~~~~. .1.1. 1-0-1111M i. wi .fl- _. + m'", v _ a''. z. .;,K" .+r. # .. '. ti, .11.i .: ... ::. .. .. ,. n .,,:, I':. _ _ __ ._ * a0 *<JQ At: '........48r. 't ,: tRS,, ':: .:;. .:::. -W .,. for .g. Or u a 4w |_.sg--s ..: .r _ a *.. *f P-D A:-.4 A..*. ...;.. *. n S S FIG. 1. Two-dimensional gel electrophoresis of the urea extracts of 32P-labeled chicken muscle. Isoelectric focusing is in the horizontal di- mension, with the acidic end of the gradient on the left. Electrophoresis in the presence of sodium dodecyl sulfate is in the vertical dimension, with the anode on the bottom. The Coomassie blue-stained gels are on the left and the corresponding autoradiograms are on the right. (A andC) Thigh muscle labeled in vivo. (B and D). Tropomyosin region from a gel with an expanded pH gradient from pH 4 to 6. Arrows point to the Downloaded by guest on September 29, 2021 Cell Biology: O'Connor et al. Proc. Natl. Acad. Sci. USA 76 (1979) 821 cessed as above. In some cases, actomyosin was extracted from (vol/vol). After destaining in 12.5% ethanol/5% acetic acid/ the minced gizzard or thigh tissue by homogeniizing tli ti 82.59 hwater,the'gels were photographed and dried onto in half-strength phosphate-buffered saline (full-strength Whatman no. 1 paper for autoradiography. Autoradiography phosphate-buffered saline is 0.14 M NaCl/3 mM KCI/1.5 mM was carried out by using either Kodak NS-2T No-screen film KH2PO4/8.1 mM Na2HPO4, pH 7.2) and extracting the residue at room temperature or Kodak X-omat XR5 film with a Du at 40C with 0.6 M KI/0.01 M Na4P207/0.02 M Na2S203/0.01 Pont Cronex intensifier screen at -70°C for various lengths of M NaH2PO4, pH 7.0, overnight. This final residue was prepared time. No-screen film was developed with Kodak D-19 devel- for gel electrophoresis as described above. operand X-omat film with Kodak x-ray developer. Protein Phosphorylation in Myogenic Cells Grown in Tissue Culture. Primary myogenic cell cultures were prepared as described by O'Neill and Stockdale (23), except that trypsin RESULTS was used at a final concentration of 0.05% and preplating was Skeletal muscle done for 25 min. Cells were plated at an initial density of 0.6-0.8 X 106 per 35-mm plate. They were labeled in 0.8 ml of phos- Fig. 1 A and C show the stained gel and corresponding auto- phate-free minimal essential medium without serum or chicken radiogram obtained from the solubilized thigh tissue of a 10- embryo extract to which 32P043- was added to give a final day-old chick injected with 32P043-. Several phosphorylated concentration of 60-100 tCi/ml. After 4-6 hr of incubation at proteins can be identified on the basis of their comigration with 370C, the cells were washed twice with phosphate-free MEM known muscle proteins (5).
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