Purification and Characterization of a Dictyostelium Protein Kinase Required for Actin Activation of the Mg2+ATPase Activity of Dictyostelium Myosin ID
1995; Elsevier BV; Volume: 270; Issue: 20 Linguagem: Inglês
10.1074/jbc.270.20.11776
ISSN1083-351X
AutoresSheu-Fen Lee, Graham P. Cô té,
Tópico(s)Force Microscopy Techniques and Applications
ResumoWe have isolated a protein from Dictyostelium with a molecular mass of 110 kDa as judged by SDS-gel electrophoresis that can stimulate the actin-activated MgATPase activity of Dictyostelium myosin ID (MyoD). In the presence of MgATP the 110-kDa protein incorporated phosphate into itself and into the heavy chain, but not the light chain, of MyoD. Phosphorylation to 0.5 mol of Pi/mol increased the MyoD actin-activated MgATPase rate from 0.2 to 3 μmol/min/mg. Renaturation following SDS-gel electrophoresis demonstrated that the 110-kDa protein contained intrinsic protein kinase and autophosphorylation activity. Autophosphorylation to 1 mol of Pi/mol enhanced the rate at which the 110-kDa protein kinase phosphorylated MyoD by 40-fold. The rate of autophosphorylation was strongly dependent on the 110-kDa protein kinase concentration, indicating an intermolecular reaction. Synthetic peptides of 9-11 residues corresponding to the heavy chain phosphorylation site of Acanthamoeba myosin IC and the homologous sites in Dictyostelium myosin IB (MyoB) and MyoD were poor substrates for the 110-kDa protein kinase. The 110-kDa protein kinase was unable to phosphorylate the MyoB isozyme suggesting that it may be specific for MyoD. We have isolated a protein from Dictyostelium with a molecular mass of 110 kDa as judged by SDS-gel electrophoresis that can stimulate the actin-activated MgATPase activity of Dictyostelium myosin ID (MyoD). In the presence of MgATP the 110-kDa protein incorporated phosphate into itself and into the heavy chain, but not the light chain, of MyoD. Phosphorylation to 0.5 mol of Pi/mol increased the MyoD actin-activated MgATPase rate from 0.2 to 3 μmol/min/mg. Renaturation following SDS-gel electrophoresis demonstrated that the 110-kDa protein contained intrinsic protein kinase and autophosphorylation activity. Autophosphorylation to 1 mol of Pi/mol enhanced the rate at which the 110-kDa protein kinase phosphorylated MyoD by 40-fold. The rate of autophosphorylation was strongly dependent on the 110-kDa protein kinase concentration, indicating an intermolecular reaction. Synthetic peptides of 9-11 residues corresponding to the heavy chain phosphorylation site of Acanthamoeba myosin IC and the homologous sites in Dictyostelium myosin IB (MyoB) and MyoD were poor substrates for the 110-kDa protein kinase. The 110-kDa protein kinase was unable to phosphorylate the MyoB isozyme suggesting that it may be specific for MyoD. INTRODUCTIONMyosins comprise a superfamily of motor proteins characterized by a conserved amino-terminal ∽80-kDa head domain that binds to actin filaments in an ATP-dependent manner. Phylogenetic analysis based on a sequence comparison of the head domains divides the currently known myosins into at least nine classes (1Bement W.M. Mooseker M.S. Nature. 1993; 365: 785-786Crossref PubMed Scopus (17) Google Scholar, 2Cheney R.E. Riley M.A. Mooseker M.S. Cell Motil. Cytoskeleton. 1993; 24: 215-223Crossref PubMed Scopus (235) Google Scholar). The members of the myosin I class are single-headed and have been identified in organisms ranging from amoebae to mammals. Myosin I isozymes are monomeric and do not assemble into filaments but have non-helical tail domains that can interact with phospholipids and, in some cases, actin filaments in an ATP-independent manner (3Hammer III, J.A. J. Muscle Res. Cell Motil. 1994; 15: 1-10Crossref PubMed Scopus (57) Google Scholar, 4Pollard T.D. Doberstein S.K. Zot H.G. Annu. Rev. Physiol. 1991; 53: 653-681Crossref PubMed Google Scholar, 5Titus M.A. Curr. Opin. Cell Biol. 1993; 5: 77-81Crossref PubMed Scopus (83) Google Scholar).Five genes coding for myosin I isozymes (myoA-E) have so far been identified in the lower eukaryote Dictyostelium discoideum and the complete sequences of four of these isozymes (A, B, D, and E) have been reported (6Titus M.A. Warrick H.M. Spudich J.A. Cell Reg. 1989; 1: 55-63Crossref PubMed Scopus (73) Google Scholar, 7Jung G. Saxe III, C.L. Kimmel A.R. Hammer III, J.A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6186-6190Crossref PubMed Scopus (69) Google Scholar, 8Jung G. Fukui Y. Martin B. Hammer III, J.A. J. Biol. Chem. 1993; 268: 14981-14990Abstract Full Text PDF PubMed Google Scholar, 9Urrutia R.A. Jung G. Hammer III, J.A. Biochim. Biophys. Acta. 1993; 1173: 225-229Crossref PubMed Scopus (25) Google Scholar). Recent evidence suggests additional Dictyostelium myosin I isozymes probably also exist (8Jung G. Fukui Y. Martin B. Hammer III, J.A. J. Biol. Chem. 1993; 268: 14981-14990Abstract Full Text PDF PubMed Google Scholar, 10Titus M.A. Kuspa A. Loomis W.F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9446-9450Crossref PubMed Scopus (36) Google Scholar, 11Jung G. Hammer III, J.A. FEBS Lett. 1994; 342: 197-202Crossref PubMed Scopus (58) Google Scholar). The myosin I isozymes from Dictyostelium can be divided into two subfamilies; MyoB,1 1The abbreviations used are: MyoA-EDictyostelium myosins IA-IETesN-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acidFPLCfast protein liquid chromatographyPAGEpolyacrylamide gel electrophoresis. MyoC, and MyoD have heavy chains of ∽125 kDa in size (7Jung G. Saxe III, C.L. Kimmel A.R. Hammer III, J.A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6186-6190Crossref PubMed Scopus (69) Google Scholar, 8Jung G. Fukui Y. Martin B. Hammer III, J.A. J. Biol. Chem. 1993; 268: 14981-14990Abstract Full Text PDF PubMed Google Scholar, 11Jung G. Hammer III, J.A. FEBS Lett. 1994; 342: 197-202Crossref PubMed Scopus (58) Google Scholar), while MyoA and MyoE are smaller with heavy chains of ∽115 kDa (6Titus M.A. Warrick H.M. Spudich J.A. Cell Reg. 1989; 1: 55-63Crossref PubMed Scopus (73) Google Scholar, 9Urrutia R.A. Jung G. Hammer III, J.A. Biochim. Biophys. Acta. 1993; 1173: 225-229Crossref PubMed Scopus (25) Google Scholar). The tail regions of MyoB, MyoC, and MyoD contain a basic domain (TH1) at the head/tail junction that is implicated in membrane binding (12Doberstein S.K. Pollard T.D. J. Cell Biol. 1992; 117: 1241-1249Crossref PubMed Scopus (102) Google Scholar), a domain consisting of a repetitive GPX motif (TH2) that is responsible for ATP-independent actin binding (11Jung G. Hammer III, J.A. FEBS Lett. 1994; 342: 197-202Crossref PubMed Scopus (58) Google Scholar, 13Rosenfeld S.S. Rener B. Biochemistry. 1994; 33: 2322-2328Crossref PubMed Scopus (39) Google Scholar) and a domain with homology to SH3 domains (TH3) (14Hammer III, J.A. Trends Cell Biol. 1991; 1: 50-56Abstract Full Text PDF PubMed Scopus (54) Google Scholar, 15Drubin D.G. Mulholland J. Zhu Z. Botstein D. Nature. 1990; 343: 288-290Crossref PubMed Scopus (204) Google Scholar). The shorter MyoA and MyoE tails consist of only the TH1 membrane-binding domain.It has been proposed that the Dictyostelium myosin I isozymes may be involved in vesicle movement and in the extension and contraction of pseudopods and filopodia (16Fath K.R. Burgess D.R. Curr. Opin. Cell Biol. 1994; 6: 131-135Crossref PubMed Scopus (45) Google Scholar, 17Coudrier E. Durrbach A. Louvard D. FEBS Lett. 1992; 307: 87-92Crossref PubMed Scopus (18) Google Scholar). Immunofluorescent studies have shown that MyoB, MyoC, and MyoD are localized mainly in the actin-rich regions at the leading edge of migrating cells (8Jung G. Fukui Y. Martin B. Hammer III, J.A. J. Biol. Chem. 1993; 268: 14981-14990Abstract Full Text PDF PubMed Google Scholar, 11Jung G. Hammer III, J.A. FEBS Lett. 1994; 342: 197-202Crossref PubMed Scopus (58) Google Scholar, 18Fukui Y. Lynch T.J. Brzeska H. Korn E.D. Nature. 1989; 341: 328-331Crossref PubMed Scopus (237) Google Scholar) but similar studies for MyoA and MyoE have not yet been performed. Attempts have been made to directly ascertain the role of the myosin I isozymes by analysis of cells in which myosin I genes have been rendered nonfunctional by homologous recombination. Cells lacking a functional myoA or myoB gene exhibit normal morphology and can complete development but display subtle abnormalities including slower translocation rates, an increase in the frequency of turning and lateral pseudopod formation and a slight delay in chemotactic aggregation (19Titus M.A. Wessels D. Spudich J.A. Soll D. Mol. Biol. Cell. 1993; 4: 233-246Crossref PubMed Scopus (117) Google Scholar, 20Jung G. Hammer III, J.A. J. Cell Biol. 1990; 110: 1955-1964Crossref PubMed Scopus (101) Google Scholar, 21Wessels D. Murray J. Jung G. Hammer III, J.A. Soll D.R. Cell Motil. Cytoskeleton. 1991; 20: 301-315Crossref PubMed Scopus (112) Google Scholar). A preliminary analysis of MyoD null cells similarly did not reveal any striking behavorial defects (8Jung G. Fukui Y. Martin B. Hammer III, J.A. J. Biol. Chem. 1993; 268: 14981-14990Abstract Full Text PDF PubMed Google Scholar). One explanation of these results is that there is considerable functional overlap between the multiple Dictyostelium myosin I isozymes, so that deletion of any one isozyme has only a minimal effect on cellular processes.At present, little is known concerning the mechanisms that regulate the motile activities of the Dictyostelium myosin I isozymes in vivo. Some evidence is available, though, to suggest that heavy chain phosphorylation, which stimulates the actin-activated MgATPase activity of the Acanthamoeba myosin I isozymes (22Maruta H. Korn E.D. J. Biol. Chem. 1977; 252: 8329-8332Abstract Full Text PDF PubMed Google Scholar, 23Lynch T.J. Brzeska H. Miyata H. Korn E.D. J. Biol. Chem. 1989; 264: 19333-19339Abstract Full Text PDF PubMed Google Scholar), and is required for these enzymes to support movement in in vitro motility assays (24Albanesi J.P. Fujisaki H. Hammer III, J.A. Korn E.D. Jones R. Sheetz M.P. J. Biol. Chem. 1985; 260: 8649-8652Abstract Full Text PDF PubMed Google Scholar, 25Zot H.G. Doberstein S.K. Pollard T.D. J. Cell Biol. 1992; 116: 367-376Crossref PubMed Scopus (83) Google Scholar), may also play a role in regulating the properties of some of the Dictyostelium myosin I isozymes. The strongest direct evidence is derived from a study showing that the purified Acanthamoeba myosin I heavy chain kinase (26Hammer III, J.A. Albanesi J.P. Korn E.D. J. Biol. Chem. 1983; 258: 10168-10175Abstract Full Text PDF PubMed Google Scholar) can stimulate the actin-activated MgATPase activity of one of the Dictyostelium myosin I isozymes (27Cô té G.P. Albanesi J.P. Ueno T. Hammer III, J.A. Korn E.D. J. Biol. Chem. 1985; 260: 4543-4546Abstract Full Text PDF PubMed Google Scholar) (later identified as MyoB (8Jung G. Fukui Y. Martin B. Hammer III, J.A. J. Biol. Chem. 1993; 268: 14981-14990Abstract Full Text PDF PubMed Google Scholar)). The studies described in this paper were undertaken in order to identify and isolate an endogenous Dictyostelium factor that could promote the actin-activated MgATPase activity of Dictyostelium myosin I. By assaying fractions for their ability to stimulate the actin-activated MgATPase activity of MyoD we have purified a protein with a molecular mass of 110 kDa that displays intrinsic protein kinase activity, is activated by autophosphorylation, and phosphorylates the MyoD heavy chain.EXPERIMENTAL PROCEDURESMaterialsATP (grade I), Tes, diisopropylfluorophosphate, bovine serum albumin, and histone 2A were obtained from Sigma; pepstatin, leupeptin, and antipain were supplied by Peptides International; and okadaic acid was from Calbiochem. [γ-32P]ATP was from DuPont NEN. The PC9 peptide was a gift from H. Brzeska (National Heart, Lung, and Blood Institute, NIH, Bethesda, MD) and the MyoB peptide was a gift from M. A. L. Atkinson (University of Texas Health Science Center, Tyler, TX). Dictyostelium myosin II (28Cô té G.P. Bukiejko U. J. Biol. Chem. 1987; 262: 1065-1072Abstract Full Text PDF PubMed Google Scholar) and skeletal muscle actin (29Spudich J.A. Watt S. J. Biol. Chem. 1971; 246: 4866-4871Abstract Full Text PDF PubMed Google Scholar) were prepared as described previously.Purification of Dictyostelium Myosin I IsozymesMyoD and MyoB were purified as described previously (30Lee S.F. Cô té G.P. J. Biol. Chem. 1993; 268: 20923-20929Abstract Full Text PDF PubMed Google Scholar), except that the actin-MyoD precipitate was resolubilized in 0.25 M KCl, 4 mM MgATP, 1 mM dithiothreitol, 20 mM Tes, pH 7.5. Soluble material obtained following centrifugation was immediately loaded onto an FPLC Mono S HR 5/5 column (Pharmacia LKB Biotech Inc.) and eluted with a 30-ml linear KCl gradient (0.25-0.55 M). The peak of MyoD activity was pooled, dialyzed against 30% glycerol, 10 mM KCl, 1 mM dithiothreitol, 20 mM Tes, pH 7.5, and stored at −20°C. Under these conditions MyoD was stable in terms of enzymatic activity and SDS-PAGE (31Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205998) Google Scholar) profile for several months. The MyoD was routinely >90% pure as judged by SDS-PAGE (see Fig. 6A) and displayed a K+EDTA-ATPase of 0.5 μmol/min/mg when assayed as described (30Lee S.F. Cô té G.P. J. Biol. Chem. 1993; 268: 20923-20929Abstract Full Text PDF PubMed Google Scholar). The MgATPase activity of the MyoD used in these studies was in the range of 0.06-0.07 μmol/min/mg and increased to no more than 0.20 μmol/min/mg in the presence of 10 μM F-actin.Purification of the 110-kDa Protein KinaseApproximately 200 g of D. discoideum AX-3 was grown and harvested as described (32Medley Q.G. Lee S.F. Cô té G.P. Methods Enzymol. 1991; 196: 23-34Crossref PubMed Scopus (10) Google Scholar). All subsequent procedures were performed at 0-4°C. The cell pellet was resuspended in 2 volumes of extraction buffer consisting of 12 mM sodium pyrophosphate, 1 mM EGTA, 2 mM EDTA, 1 mM dithiothreitol, 1.5 mM phenylmethylsulfonyl fluoride, 10 μg/ml pepstatin A, 2 μg/ml antipain, 2 μg/ml leupeptin, and 30 mM Tes, pH 7.5, and disrupted by 20 strokes in a tight-fitting Dounce homogenizer (Kontes). Diisopropylfluorophosphate and solid KCl were then added to give final concentrations of 1 and 100 mM, respectively. The homogenate was stirred for 30 min and centrifuged at 100,000 × g for 1 h (Beckman type Ti-60 rotor). The clear supernatant was collected, diluted with 2 volumes of Buffer A (1 mM EGTA, 1 mMβ-mercaptoethanol, 10 mM Tes, pH 7.5), adjusted to pH 7.5 with 1 M Tris, pH 11, and mixed with 200 ml of packed P-11 phosphocellulose (Whatman BioSystems) previously equilibrated with Buffer A containing 40 mM KCl. The slurry was gently stirred for 2 h, packed into a 5-cm diameter column, washed extensively with Buffer A containing 40 mM KCl and 0.4 mM ATP, and eluted using a 1.5-liter linear KCl gradient (0.04-0.85 M) in Buffer A containing 0.4 mM ATP (Fig. 1). Fractions were assayed for the ability to stimulate the actin-activated MgATPase activity of MyoD (as described below), and the most active fractions, eluting at a KCl concentration of 0.2 M, were pooled. The pooled material was immediately applied to a 2.5 × 4-cm column of hydroxylapatite HT (Bio-Rad) equilibrated in 5% sucrose, 0.15 M KCl, 1 mM EGTA, 1 mMβ-mercaptoethanol, and 2 mM KPO4, pH 7.0. The column was washed extensively with equilibration buffer and eluted with a 200-ml linear KPO4 gradient (0.002-0.3 M, pH 7.0) in the equilibration buffer (Fig. 2). The peak of activity eluting at 50 mM KPO4 was pooled, dialyzed overnight against 1 liter of 5% sucrose, 40 mM KCl, 1 mM dithiothreitol, 20 mM Tris, pH 7.8, and loaded onto an FPLC Mono Q HR 5/5 column (Pharmacia LKB Biotech Inc.) equilibrated with the same buffer. After extensive washing with the equilibration buffer the column was eluted with a 35-ml linear KCl gradient (0.04-0.2 M) (Fig. 3). The most active fractions, eluting at 0.13 M KCl, were immediately pooled and stored in liquid nitrogen in 50-μl aliquots.Figure 1:Phosphocellulose chromatography of the initial high speed supernatant. A high speed supernatant obtained from 200 g wet weight packed Dictyostelium was chromatographed over a phosphocellulose P-11 column as described under "Experimental Procedures." The flow rate was 80 ml/h and fractions of 13 ml were collected. The presence of ATP in the column buffer required that the elution of protein be monitored using the Bradford assay (...). Salt concentration was determined by conductivity measurements (Δ). Aliquots of 2 μl from every second fraction were assayed for the ability to stimulate the actin-activated MgATPase activity of MyoD (.) as described under "Experimental Procedures." The basal actin-activated MgATPase of the MyoD used in these experiments was 0.15 μmol/min/mg. In the absence of added MyoD, none of the column fractions displayed significant actin-activated MgATPase activity. Fractions that activated MyoD activity eluted at a KCl concentration of 0.2 M and were pooled as indicated by the solid bar.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 2:Chromatography of the phosphocellulose pool over hydroxylapatite. The pooled material from the phosphocellulose column was chromatographed over a 2.5 × 4-cm hydroxylapatite HT column as described under "Experimental Procedures." The flow rate was 20 ml/h and fractions of 2 ml were collected. The absorbance at 280 nm (...) and the conductivity (Δ) were monitored. Aliquots of 2 μl were assayed for the ability to activate the actin-activated MgATPase activity of MyoD (.) as described in the legend to Fig. 1. Active fractions eluted at a KPO4 concentration of 50 mM and were pooled as indicated by the solid bar.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3:Chromatography of the hydroxylapatite pool over Mono Q. The pooled material from the hydroxylapatite column was chromatographed over a Mono Q HR 5/5 as described under "Experimental Procedures." The flow rate was 20 ml/h and fractions of 0.5 ml were collected. The absorbance at 280 nm (...) and the conductivity (Δ) were monitored. Aliquots of 1 μl taken from 10 × diluted fractions were assayed for the ability to activate the actin-activated MgATPase activity of MyoD (.) as described in the legend to Fig. 1. Active fractions eluted at a KCl concentration of 0.13 M and were pooled as indicated by the solid bar.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Actin-activated MgATPase AssaysThe MgATPase activity of MyoD was assayed in a buffer containing 5 mM MgCl2, 1 mM [γ-32P]ATP (2 Ci/mol), 1 mM dithiothreitol, and 10 mM Tes, pH 7.5. Unless stated otherwise, actin-activated MgATPase assays were performed in the same buffer with 10 μM F-actin. Assays were carried out for 20 min at 25°C and ATPase activity was measured by the release of 32Pi from the [γ-32P]ATP (33Pollard T.D. Korn E.D. J. Biol. Chem. 1973; 248: 4682-4690Abstract Full Text PDF PubMed Google Scholar). Assays designed to measure the ability of fractions to stimulate the actin-activated MgATPase activity of MyoD were performed as follows. First, the fractions were dialyzed against 20 mM KCl, 1 mM dithiothreitol, and 20 mM Tes, pH 7.5, by spotting 10-μl samples of the fractions onto a 0.025-micron type VS filter (Millipore) floating on the dialysis solution. After 1 h an aliquot of the dialyzed sample was removed and added to a tube containing 10 μl of 0.25 mM ATP, 2 mM MgCl2, 1 mM dithiothreitol, 10 nM okadaic acid, 0.1 mg/ml bovine serum albumin, and 10 mM Tes, pH 7.0. In most cases a preincubation for 20 min at 25°C was performed and then a 10-μl sample of MyoD (0.5-1 μM) was added and incubation allowed to proceed for another 20 min at 25°C. At the end of this time 180 μl of MgATPase buffer containing actin was added and the MgATPase rate determined as described above. The volume of the column fractions to be assayed was chosen so that the most active fraction always produced less than the maximum possible MyoD actin-activated MgATPase (∽3 μmol/min/mg). Specific activities were determined by varying the length of time (from 1 to 20 min) that MyoD was incubated with the fractions. A linear relationship between the time of incubation and the percent enhancement of the MyoD actin-activated MgATPase activity was observed so long as MyoD was stimulated to less than 50% of its maximal activity. This result indicates that addition of the actin-containing MgATPase assay buffer essentially terminated further activation of MyoD. Activities determined by this assay are presented in units, with 1 unit being arbitrarily defined as the activity required to cause a 100% increase in the basal actin-activated MgATPase rate of MyoD.Protein Kinase and Phosphoamino Acid AssaysAssays were carried out by addition of substrate to an equal volume of 2 mM MgCl2, 2 mM dithiothreitol, 20 mM Tes, pH 7.0, and 0.5 mM [γ-32P]ATP (500 Ci/mol) followed by addition of the 110-kDa protein kinase to initiate the reaction. Kinase and substrate concentrations for each experiment are provided in the figure and table legends. When substrate concentrations were varied, the final ionic strength was kept constant by addition of the appropriate buffer. Autophosphorylation of the 110-kDa protein kinase was performed by diluting the kinase into 2 volumes of 0.4 mM [γ-32P]ATP (500 Ci/mol), 3 mM MgCl2, 1.5 mM dithiothreitol, 0.15 mg/ml bovine serum albumin, and 15 mM Tes, pH 7.0. All reactions were performed at 25°C. Protein phosphorylation or autophosphorylation activities were determined by removing aliquots of 10-20 μl from the assays at time intervals and immediately adding them to a one-fifth volume of boiling hot SDS sample buffer (5% SDS, 30% sucrose, 2.5%β-mercaptoethanol). Samples were subjected to SDS-PAGE, the gel stained with Coomassie Blue and the appropriate protein band excised and counted in liquid scintillation fluid in a scintillation counter. Kinase activity assays were carried out such that less than 0.1 mol of Pi was incorporated per mole of protein. Under these conditions the incorporation of 32P into substrate was linear with time and proportional to the amount of kinase. Phosphate incorporation into the synthetic peptides was determined by spotting 10-μl aliquots of the assay mixture onto squares of P-81 phosphocellulose paper (Whatman) that were then washed in 0.1% phosphoric acid as described (34Roskoski Jr., R. Methods Enzymol. 1983; 99: 3-6Crossref PubMed Scopus (690) Google Scholar). Phosphoamino acid analysis was performed essentially as described (35Kamps M.P. Sefton B.M. Anal. Biochem. 1989; 176: 22-27Crossref PubMed Scopus (322) Google Scholar).Renaturation Following SDS-PAGEThe 110-kDa protein was subjected to SDS-PAGE and renatured in the polyacrylamide gel following incubation in 6 M guanidine HCl as described (36Kameshita I. Fujisawa H. Anal. Biochem. 1989; 183: 139-143Crossref PubMed Scopus (434) Google Scholar). The gel was then equilibrated in 2 mM MgCl2, 1 mM dithiothreitol, 10 mM Tes, pH 7.5, and incubated for 1 h at 25°C in 3 ml of the same buffer containing 0.1 mM [γ-32P]ATP (200 Ci/mol). Radioactivity was removed by washing the gel extensively in 5% trichloroacetic acid, 1% sodium pyrophosphate. The gel was then stained with Coomassie Blue, dried, and subjected to autoradiography.Miscellaneous MethodsProtein concentrations were determined by the colorimetric assay of Bradford (37Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (213288) Google Scholar). In some cases the concentrations of MyoD and the 110-kDa protein kinase were determined following SDS-PAGE. The gels were stained with Coomassie Blue, scanned at 596 nm using a laser densitometer (LKB 2202 Ultro), and the areas of the peaks calculated. For both methods bovine serum albumin was used as the standard. For autoradiography dried gels and thin layer sheets were exposed at −80°C to x-ray Hyperfilm (Amersham Corp.) with an intensifying screen (Du Pont, Cronex Lightning Plus).RESULTSPurification of a 110-kDa Protein That Stimulates the Actin-activated MgATPase Activity of MyoDAs isolated, MyoD displays an actin-activated MgATPase activity of less than 0.2 μmol/min/mg, which is considerably below the 2-8 μmol/min/mg actin-activated MgATPase rates reported for other Acanthamoeba and Dictyostelium myosin I isozymes (23Lynch T.J. Brzeska H. Miyata H. Korn E.D. J. Biol. Chem. 1989; 264: 19333-19339Abstract Full Text PDF PubMed Google Scholar, 26Hammer III, J.A. Albanesi J.P. Korn E.D. J. Biol. Chem. 1983; 258: 10168-10175Abstract Full Text PDF PubMed Google Scholar, 27Cô té G.P. Albanesi J.P. Ueno T. Hammer III, J.A. Korn E.D. J. Biol. Chem. 1985; 260: 4543-4546Abstract Full Text PDF PubMed Google Scholar, 30Lee S.F. Cô té G.P. J. Biol. Chem. 1993; 268: 20923-20929Abstract Full Text PDF PubMed Google Scholar, 38Albanesi J.P. Fujisaki H. Korn E.D. J. Biol. Chem. 1985; 260: 11174-11179Abstract Full Text PDF PubMed Google Scholar). Initial experiments in which MyoD was incubated with crude Dictyostelium homogenates were unsuccessful in detecting any stimulation of the MyoD actin-activated MgATPase activity. The crude homogenates exhibited a high background level of actin-activated MgATPase activity (perhaps resulting from the presence of myosin II) that tended to obscure any activity contributed by the added MyoD. However, following chromatography over a phosphocellulose P-11 column fractions that were capable of stimulating the actin-activated MgATPase activity of MyoD were readily detected (Fig. 1). Control assays demonstrated that the column fractions alone displayed negligible actin-activated MgATPase activity and that the observed activity was dependent on the addition of both MyoD and actin. The most active column fractions were capable of stimulating the actin-activated MgATPase of MyoD greater than 10-fold, to a rate approaching 2.5 μmol/min/mg. It should be noted that in order to obtain this high degree of activation it was found necessary to preincubate the column fractions with MgATP prior to the addition of MyoD.The pooled material from the phosphocellulose column was subsequently chromatographed over a hydroxylapatite column (Fig. 2) and then a Mono Q column (Fig. 3). In both cases single peaks of an activity capable of greatly stimulating the actin-activated MgATPase activity of MyoD were obtained. An SDS-PAGE analysis of fractions collected throughout the purification procedure (Fig. 4) indicated that the hydroxylapatite pool consisted primarily of two proteins with molecular masses of 110 and 55 kDa (Fig. 4, lane d). Chromatography over Mono Q removed the 55-kDa protein, which eluted in the flow-through, while the 110-kDa protein bound to the column and eluted with those fractions capable of stimulating the actin-activated MgATPase activity of MyoD (Fig. 4, lane e). The approximate degree of purification of the 110-kDa band at each chromatographic step in the purification procedure was estimated by densitometry of Coomassie Blue-stained SDS gels (Table I). The 110-kDa band represented less than 1% of the total protein in the phosphocellulose pool and about 10% of the total protein in the hydroxylapatite pool. The recovery of the 110-kDa protein from the Mono Q column was quite low (about 10%) and reflects, in part, the fact that the Mono Q column was pooled very narrowly. Usually only the two most active fractions from the column were retained for further study. The 110-kDa band comprised 95% of the total protein in the Mono Q pool (average of five separate preparations), while no other band represented more than 1% of the total protein.Figure 4:SDS gel analysis of fractions obtained during purification. Samples were electrophoresed on an 8% SDS-polyacrylamide gel and stained with Coomassie Blue. The samples and the amount of proteins loaded in each lane are: a, initial homogenate, 20 μg; b, high speed supernatant, 20 μg; c, phosphocellulose pool, 20 μg; d, hydroxylapatite pool, 10 μg; and e, Mono Q pool, 1 μg. The molecular mass standards in kDa are indicated to the left of the gel and were, from top to bottom: skeletal muscle myosin heavy chain, β-galactosidase, phosphorylase b, bovine serum albumin, and ovalbumin.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IPurification of Dictyostelium 110-kDa protein kinaseStarting material was 200 g of wet packed Dictyostelium. Open table in a new tab The specific activities of the fractions obtained following each column chromatography step were determined by performing time courses of the stimulation of MyoD actin-activated MgATPase activity as described under "Experimental Procedures." The specific activity of the phosphocellulose pool was determined to be 11.5 units/min/mg, giving a total activity at this stage of the purification of 2150 units/min (Table I). Chromatography over hydroxylapatite resulted in an ∽50-fold elevation in specific activity and a 2-fold rise in total activity. The last step in the purification yielded a 1350-fold increase in specific activity, despite the fact that the purity of the 110-kDa protein increased less than 10-fold. The total activity at this step jumped to 72,000 units/min. It is apparent that some factor that acts to prevent stimulation of the actin-activated MgATPase activity of MyoD is removed as the purification procedure proceeds.Gel filtration chromatography of the 110-kDa protein on a Bio-Gel A-0.5m column resulted in the elution of the protein at a Stokes radius es
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