The complete amino-acid sequence of actin of rabbit skeletal muscle was determined. The actin polypeptide chain is composed of 374 residues, including one residue of the unusual amino acid NT-methyl histidine, and has a calculated molecular weight of 41,785. The sequence of actin was determined by isolating the peptides produced by cleavage of the protein with cyanogen bromide, determining the sequence of these The location and function of actin is most clearly understood in vertebrate skeletal muscle; the actin units are assembled into double-stranded helices which, together with tropomyosin and the troponin complex, constitute the thin filaments. Extensions of myosin molecules in the thick filaments can interact with actin, forming crossbridges between the filaments. During the lifetimes of attachment of these crossbridges, force is generated, which results in sliding of the filaments past each other. Thus, actin serves as the site of crossbridge attachment and also as a support for the regulatory proteins, tropomyosin and troponin.Besides being found in skeletal, smooth, and cardiac muscle, actin-like proteins with properties that are very similar to those of muscle actin have been isolated from several other sources, including ameba (1), blood platelets (2), fibroblasts (3), brain (4), and cytoplasm of numerous embryonic cells (5). The exact role of actin in each of these cell types is under investigation, but the ability of cytoplasmic actin to interact with myosin from muscle (1, 2, 4) implies that in nonmuscle cells it is also involved in force generation.Actin clearly occupies a central role in biological movement, and, as a necessary step toward understanding its function on a molecular level, we have determined the amino-acid sequence of actin isolated from rabbit skeletal muscle. Actin Preparation. Rabbit back and leg muscle was used as a source of actin (6, 7). Purified actin gave a single band on Na dodecyl sulfate-gel electrophoresis. Reduction and alkylation or aminoethylation of sulfhydryl groups have been described (6, 7).Cyanogen Bromide Cleavage. Protein or peptides were dissolved in 70% formic acid and treated with a 50-to 125-fold molar excess of cyanogen bromide (CNBr) over methionine residues for 16-24 hr at 230. Under these conditions, the methionine was usually quantitatively converted to homoserine (6). In some cases complete conversion was insured by treatment of the protein with 2-mercaptoethanol, in order to convert residual methionine sulfoxide to methionine, and again treating the protein with cyanogen bromide (7). Separation of CNBr Peptides. Peptides CB-1 through CB-13 (see Table 1) were first fractionated on Sephadex G-50 equilibrated with 25% acetic (Fig. la) or 20% formic acid and were then purified by ion-exchange chromatography (6-8). The sequences of the purified cyanogen bromide fragments were determined by studying the products of enzymic digestion of the peptides with trypsin, chymotrypsin, and/or thermolysin.Peptides CB-15, CB-16, and CB-17 were separated from...
During the course of the expression of a 47-kDa COOH-terminal fragment of brain-type nonmuscle myosin heavy chain (MIIBF47), we found two closely related forms of MIIB, designated MIIB alpha and MIIB beta, in rabbit brains. The B alpha form corresponded to SMemb, described by Kuro-o et al. [(1991) J. Biol. Chem. 266, 3768] and was the more abundant form in rabbit brain, while the B beta form was novel. MIIB beta F47 differed from MIIB alpha F47 at six positions, three of which were within the carboxyl-terminal nonhelical domain; in MIIB beta F47, Ser, Pro, and Lys replaced Pro, Ser, and Glu, respectively. MIIB alpha F47 and MIIB beta F47 differed in filament assembly properties in the presence of various concentrations of salt, and a chimera containing the helical domain of MIIB beta F47 and the nonhelical domain of MIIB alpha F47 behaved very much like MIIB beta F47. Protein kinase C (PK C) incorporated 1 and 2 mol of phosphate/mol peptide of MIIB alpha F47 and MIIB beta F47, respectively, and caused similar levels of inhibition of assembly for both isoforms. Casein kinase II (CK II) incorporated 4 and 2 mol of phosphate/mol of MIIB alpha F47 and MIIB beta F47 peptides, respectively, and this caused strong inhibition of assembly for MIIB alpha F47 but only slight inhibition for MIIB beta F47. PK C sites in MIIB alpha F47 were localized within a region containing a cluster of Ser residues near the predicted junction of the helical and nonhelical domains: P-I-S(PO4)-F-S(PO4)-S(PO4)-S(PO4)-R-S(PO4)-. Out of the five potential PK C sites, only one site seemed to be phosphorylated per peptide. The PK C sites in MIIB beta F47 were localized as S(PO4)-I-S-F-S-S-(PO4)-R-S(PO4)-, with total incorporation of about 2 mol/mol of peptide. In addition, PK C phosphorylated a Ser within the predicted helical domain, E-V-S(PO4)-T-L, in both MIIB alpha F47 and MIIB beta F47. For CK II, five sites were identified within the COOH end of MIIB alpha F47: S(PO4)-L-E-L-S(PO4)-D-D-D-T(PO4)-E-S-K-T-S(PO4)-D-V-N-E-T-Q-P-P-Q-S(PO4) -E. The same sites were phosphorylated in MIIB beta F47 except for the first Ser, which was replaced by Pro in MIIB beta F47. An average of about two of the four potential sites were phosphorylated in MIIB beta F47, while in MIIB alpha F47 all five sites could be fully phosphorylated by CK II. Our results demonstrate that (1) the helical domains dictate the intrinsic salt dependence of assembly for nonmuscle myosin, (2) the isoforms are phosphorylatable by different kinases in an isoform specific manner mostly within the COOH-terminal nonhelical domain, and (3) the effects of the phosphorylation on assembly are isoform specific.
The beta-thymosins are a family of highly polar peptides which serve in vivo to maintain a reservoir of unpolymerized actin monomers. In vitro, beta-thymosins form 1:1 complexes with actin monomers and inhibit both polymerization and exchange of the bound nucleotide. Circular dichroism data indicate that free thymosin beta 4 is predominantly unstructured, containing at most six residues of alpha-helix, and that up to six additional residues may adopt an alpha-helical conformation upon binding actin. NMR data indicate that many parts of thymosin beta 4 are not in tight contact with actin. Contacts between specific residues in actin and thymosin beta 4 were identified by zero-length cross-linking followed by isolation and sequencing of cross-linked peptides. After carbodiimide-mediated cross-linking, Lys-3 of thymosin beta 4 was cross-linked to Glu-167 of actin, and Lys-18 of thymosin beta 4 was cross-linked to one of the the N-terminal acidic residues of actin (Asp-1-Glu-4); the cross-linked actin residues lie within subdomains 3 and 1, respectively. These two contacts flank the alpha-helical region of thymosin beta 4 and place it on the barbed end; thymosin beta 4 can thus block actin polymerization sterically. After transglutaminase-mediated cross-linking, Lys-38 of thymosin beta 4 was cross-linked to Gln-41 of actin, placing the C-terminal region of thymosin beta 4 in contact with subdomain 2 on the pointed end; thymosin beta 4 may sterically block actin polymerization at the pointed end as well as the barbed end of the monomer. The distance between the pointed-end and barbed-end contacts requires that the C-terminal half of thymosin beta 4 be in a predominantly extended conformation.
The minibrain kinase (Mnbk)/dual specificity Yak 1-related kinase 1A (Dyrk1A) gene is implicated in the mental retardation associated with Down's syndrome. It encodes a proline-directed serine/threonine kinase whose function has yet to be defined. We have used a solid-phase Mnbk/Dyrk1A kinase assay to aid in the search for the cellular Mnbk/Dyrk1A substrates. The assay revealed that rat brain contains two cytosolic proteins, one with a molecular mass of 100 kDa and one with a molecular mass of 140 kDa, that were prominently phosphorylated by Mnbk/Dyrk1A. The 100-kDa protein was purified and identified as dynamin 1. The conclusion was further supported by evidence that a recombinant glutathione S-transferase fusion protein containing dynamin isoform 1aa was phosphorylated by Mnbk/ Dyrk1A. In addition to isoform 1aa, Mnbk/Dyrk1A also phosphorylated isoforms 1ab and 2aa but not human MxA protein when analyzed by the solid-phase kinase assay. Upon Mnbk/Dyrk1A phosphorylation, the interaction of dynamin 1 with the Src homology 3 domain of amphiphysin 1 was reduced. However, when Mnbk/ Dyrk1A phosphorylation was allowed to proceed more extensively, the phosphorylation enhanced rather than reduced the binding of dynamin 1 to amphiphysin 1. The result suggests that Mnbk/Dyrk1A can play a dual role in regulating the interaction of dynamin 1 with amphiphysin 1. Mnbk/Dyrk1A phosphorylation also reduced the interaction of dynamin with endophilin 1, whereas the same phosphorylation enhanced the binding of dynamin 1 to Grb2. Nevertheless, the dual function of Mnbk/ Dyrk1A phosphorylation was not observed for the interaction of dynamin 1 with endophilin 1 or Grb2. The interactions of dynamin with amphiphysin and endophilin are essential for the formation of endocytic complexes; our results suggest that Mnbk/Dyrk1A may function as a regulator controlling the assembly of endocytic apparatus.Minibrain kinase (Mnbk) 1 was originally identified in Drosophila as a mutation affecting neurogenesis (1). Mnbk mutant flies, which have low levels of kinase expression, possess fewer neuroblasts and a reduced brain volume as compared with the wild type (WT), especially in the optic lobes and central brain (1). The reduction in brain size leads to several distinct learning and behavioral defects. Because the Mnbk mutation does not appear to affect the development of Drosophila until late in the third instar, it was postulated that the Mnbk gene is required for the proliferation of neuroblasts during postembryonic neurogenesis (1).Dual specificity Yak 1-related kinase (Dyrk) 1A was subsequently cloned (2-5) and identified as the mammalian homologue of the Drosophila Mnbk gene. The Mnbk/Dyrk1A gene is a member of a growing family of Dyrk-related genes (6) whose members include Yak1 (7), several Dyrks (8), ANPK (9), HIPK2 (10), Mirk (11), Myak (12), and Pom1p (13). Mammalian Mnbk/Dyrk1A gene contains either 763 or 754 amino acid residues as a result of alternative splicing (2). The kinase domain of Mnbk/Dyrk1A, consisting of ϳ320 residues, is...
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