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Functional Analysis of Tail Domains of AcanthamoebaMyosin IC by Characterization of Truncation and Deletion Mutants

2000; Elsevier BV; Volume: 275; Issue: 32 Linguagem: Inglês

10.1074/jbc.m004287200

1083-351X

Xiong Liu, Hanna Brzeska, Edward D. Korn,

Cellular Mechanics and Interactions

Acanthamoeba myosin IC has a single 129-kDa heavy chain and a single 17-kDa light chain. The heavy chain comprises a 75-kDa catalytic head domain with an ATP-sensitive F-actin-binding site, a 3-kDa neck domain, which binds a single 17-kDa light chain, and a 50-kDa tail domain, which binds F-actin in the presence or absence of ATP. The actin-activated MgATPase activity of myosin IC exhibits triphasic actin dependence, apparently as a consequence of the two actin-binding sites, and is regulated by phosphorylation of Ser-329 in the head. The 50-kDa tail consists of a basic domain, a glycine/proline/alanine-rich (GPA) domain, and aSrc homology 3 (SH3) domain, often referred to as tail homology (TH)-1, -2, and -3 domains, respectively. The SH3 domain divides the TH-3 domain into GPA-1 and GPA-2. To define the functions of the tail domains more precisely, we determined the properties of expressed wild type and six mutant myosins, an SH3 deletion mutant and five mutants truncated at the C terminus of the SH3, GPA-2, TH-1, neck and head domains, respectively. We found that both the TH-1 and GPA-2 domains bind F-actin in the presence of ATP. Only the mutants that retained an actin-binding site in the tail exhibited triphasic actin-dependent MgATPase activity, in agreement with the F-actin-cross-linking model, but truncation reduced the MgATPase activity at both low and high actin concentrations. Deletion of the SH3 domain had no effect. Also, none of the tail domains, including the SH3 domain, affected either the K m orV max for the phosphorylation of Ser-329 by myosin I heavy chain kinase. Acanthamoeba myosin IC has a single 129-kDa heavy chain and a single 17-kDa light chain. The heavy chain comprises a 75-kDa catalytic head domain with an ATP-sensitive F-actin-binding site, a 3-kDa neck domain, which binds a single 17-kDa light chain, and a 50-kDa tail domain, which binds F-actin in the presence or absence of ATP. The actin-activated MgATPase activity of myosin IC exhibits triphasic actin dependence, apparently as a consequence of the two actin-binding sites, and is regulated by phosphorylation of Ser-329 in the head. The 50-kDa tail consists of a basic domain, a glycine/proline/alanine-rich (GPA) domain, and aSrc homology 3 (SH3) domain, often referred to as tail homology (TH)-1, -2, and -3 domains, respectively. The SH3 domain divides the TH-3 domain into GPA-1 and GPA-2. To define the functions of the tail domains more precisely, we determined the properties of expressed wild type and six mutant myosins, an SH3 deletion mutant and five mutants truncated at the C terminus of the SH3, GPA-2, TH-1, neck and head domains, respectively. We found that both the TH-1 and GPA-2 domains bind F-actin in the presence of ATP. Only the mutants that retained an actin-binding site in the tail exhibited triphasic actin-dependent MgATPase activity, in agreement with the F-actin-cross-linking model, but truncation reduced the MgATPase activity at both low and high actin concentrations. Deletion of the SH3 domain had no effect. Also, none of the tail domains, including the SH3 domain, affected either the K m orV max for the phosphorylation of Ser-329 by myosin I heavy chain kinase. tail homology glycine, proline and alanine-rich myosin I heavy chain kinase p21-activated kinase Src homology 3 polymerase chain reaction base pairs dithiothreitol bovine serum albumin The myosin superfamily includes more than 120 different isoforms falling into 15 classes based on the sequences of their catalytic domains and also differing in the structure of their tails (1Sellers J.R. Myosins. Oxford University Press, Oxford1999Google Scholar). Other than the conventional class II myosins, class I myosins are the most numerous, most widely distributed, and most extensively studied. At this writing, the complete DNA sequences of the heavy chains of 30 class I myosins from 13 species including yeast, protozoa, invertebrates, and vertebrates have been determined. The structure, enzymatic properties, and possible functions of many of the class I myosins have been investigated, none more extensively than the threeAcanthamoeba myosin Is (IA, IB, and IC), which were the first unconventional (nonclass II) myosins to be discovered (2Pollard T.D. Korn E.D. J. Biol. Chem. 1973; 248: 4682-4690Abstract Full Text PDF PubMed Google Scholar). All class I myosins have a single, relatively short (for myosins) heavy chain, one or more light chains, and, unlike class-II myosins, do not polymerize into filaments. By sequence analysis (3Jung G. Korn E.D. Hammer J.A. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 6720-6724Crossref PubMed Scopus (77) Google Scholar, 4Jung G. Schmitt C.J. Hammer III, J.A. Gene (Amst.). 1989; 82: 269-280Crossref PubMed Scopus (43) Google Scholar, 5Lee W.-L. Ostap E.M. Zot H.G. Pollard T.D. J. Biol. Chem. 1999; 274: 35159-35171Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar), the masses of the heavy chains of Acanthamoeba myosin IA, IB, and IC are 134, 125, and 129 kDa, respectively, consisting of an N-terminal head (catalytic, motor) domain of ∼75 kDa, a short neck domain that binds one (myosin IB and myosin IC (6Wang Z.Y. Wang F. Sellers J.R. Korn E.D. Hammer J.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15200-15205Crossref PubMed Scopus (36) Google Scholar)) or possibly as many as three (myosin IA (5Lee W.-L. Ostap E.M. Zot H.G. Pollard T.D. J. Biol. Chem. 1999; 274: 35159-35171Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar)) light chains and a nonhelical C-terminal tail domain of ∼50 kDa. Only the single light chain of myosin IC has been cloned and sequenced (7Wang Z.Y. Sakai J. Matsudaira P.T. Baines I.C. Sellers J.R. Hammer J.A. Korn E.D. J. Muscle Res. Cell Motil. 1997; 18: 395-398Crossref PubMed Scopus (28) Google Scholar); it is a calmodulin-like protein with a mass of ∼17 kDa. The tail domains have been subdivided by sequence into three regions: a basic domain (TH-1),1 a Gly/Pro/Ala-rich (GPA) domain (TH-2), and a Src homology 3 domain (TH-3). These three regions occur sequentially in Acanthamoebamyosins IA (5Lee W.-L. Ostap E.M. Zot H.G. Pollard T.D. J. Biol. Chem. 1999; 274: 35159-35171Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar) and IB (4Jung G. Schmitt C.J. Hammer III, J.A. Gene (Amst.). 1989; 82: 269-280Crossref PubMed Scopus (43) Google Scholar), but the TH-3 region of myosin IC splits its C-terminal TH-2 domain in two (3Jung G. Korn E.D. Hammer J.A. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 6720-6724Crossref PubMed Scopus (77) Google Scholar). It seems highly likely that specific interactions of these three tail domains play important roles in determining the different localizations and different functions of the three isozymes (8Baines I.C. Brzeska H. Korn E.D. J. Cell Bio l. 1992; 119: 1193-1203Crossref PubMed Scopus (84) Google Scholar, 9Baines I.C. Corigliano-Murphy A. Korn E.D. J. Cell Bio l. 1995; 130: 591-603Crossref PubMed Scopus (52) Google Scholar, 10Doberstein S.K. Baines I.C. Wiegand G. Korn E.D. Pollard T.D. Nature. 1993; 365: 841-843Crossref PubMed Scopus (103) Google Scholar). The bacterially expressed TH-1 domain of Acanthamoeba myosin IC binds acidic phospholipids (11Doberstein S.K. Pollard T.D. J. Cell Biol. 1992; 117: 1241-1249Crossref PubMed Scopus (102) Google Scholar) and, therefore, TH-1 is almost certainly responsible for the ability of the native myosins to bind to acidic lipids (12Adams R.J. Pollard T.D. Nature. 1989; 340: 505-508Crossref PubMed Scopus (5) Google Scholar) and amoeba plasma membranes (13Miyata H. Bowers B. Korn E.D. J. Cell Biol. 1989; 109: 1519-1528Crossref PubMed Scopus (79) Google Scholar). Experiments with bacterially expressed TH-2 plus TH-3 domains of Acanthamoebamyosin IC (11Doberstein S.K. Pollard T.D. J. Cell Biol. 1992; 117: 1241-1249Crossref PubMed Scopus (102) Google Scholar), with bacterially expressed TH-2 region ofDictyostelium myosin IB (14Jung G. Hammer III, J.A. FEBS Lett. 1994; 342: 197-202Crossref PubMed Scopus (58) Google Scholar) and with a C-terminal 30-kDa proteolytic fragment of Acanthamoeba myosin IA (15Lynch T., J. Albanesi J.P. Korn E.D. Robinson E.A. Bowers B. Fujisaki H. J. Biol. Chem. 1986; 261: 17156-17162Abstract Full Text PDF PubMed Google Scholar), had indicated that the TH-2 region may be principally responsible for the ability of the amoeba myosin Is to bind to F-actin in the presence of ATP (15Lynch T., J. Albanesi J.P. Korn E.D. Robinson E.A. Bowers B. Fujisaki H. J. Biol. Chem. 1986; 261: 17156-17162Abstract Full Text PDF PubMed Google Scholar, 16Fujisaki H. Albanesi J.P. Korn E.D. J. Biol. Chem. 1985; 260: 11182-11189Abstract Full Text PDF Google Scholar, 17Albanesi J.P. Coué M. Fujisaki H. Korn E.D. J. Biol. Chem. 1985; 260: 13276-13280Abstract Full Text PDF PubMed Google Scholar). As we will discuss later (see “Discussion”), the TH-3 domain is likely to be involved in the localization of myosin Is and the organization of the actin cytoskeleton (18Barylko B. Binns D.D. Albanesi J.P. Biochim. Biophys. Acta. 2000; 1496: 23-25Crossref PubMed Scopus (48) Google Scholar). The three Acanthamoeba myosin Is have similar catalytic activities. All have high ATPase activity in the presence of EDTA and either NH4+ or K+ and low activity in the presence of Mg2+ that is substantially activated by F-actin, a diagnostic characteristic of the myosin superfamily. However, the MgATPase activity of these amoeba myosin Is has an unusual triphasic dependence on the F-actin concentration (17Albanesi J.P. Coué M. Fujisaki H. Korn E.D. J. Biol. Chem. 1985; 260: 13276-13280Abstract Full Text PDF PubMed Google Scholar, 19Brzeska H. Lynch T.J. Korn E.D. J. Biol. Chem. 1988; 263: 427-435Abstract Full Text PDF PubMed Google Scholar, 20Lynch T.J. Brzeska H. Miyata H. Korn E.D. J. Biol. Chem. 1989; 264: 19333-19339Abstract Full Text PDF PubMed Google Scholar),i.e. substantial activation at low F-actin concentrations peaking at about 2 μm, followed by a decrease in the activity and then normal hyperbolic activation that begins to plateau at about 80 μm F-actin. A variety of experimental data and computer modeling strongly support the conclusion that this triphasic actin dependence is caused by cooperative cross-linking of actin filaments by myosin I at high ratios of myosin I to actin (17Albanesi J.P. Coué M. Fujisaki H. Korn E.D. J. Biol. Chem. 1985; 260: 13276-13280Abstract Full Text PDF PubMed Google Scholar,19Brzeska H. Lynch T.J. Korn E.D. J. Biol. Chem. 1988; 263: 427-435Abstract Full Text PDF PubMed Google Scholar, 21Albanesi J.P. Fujisaki H. Korn E.D. J. Biol. Chem. 1985; 260: 11174-11179Abstract Full Text PDF PubMed Google Scholar, 22Pantaloni D. J. Biol. Chem. 1985; 260: 11180-11182Abstract Full Text PDF PubMed Google Scholar). The ability of these myosins to cross-link actin filaments can be explained by the presence of two actin-binding sites, the ATP-sensitive actin-binding site in the catalytic domain that is common to all myosins of all classes and the ATP-insensitive actin-binding site(s) in the tail that occurs in some (but not all) class I myosins including, in addition to the three Acanthamoeba myosin Is, two of the five Dictyostelium myosin Is (23Lee S.F. Côté G.P. J. Biol. Chem. 1993; 268: 20923-20929Abstract Full Text PDF PubMed Google Scholar, 24Rosenfeld S.S. Rener B. Biochemistry. 1994; 33: 2322-2328Crossref PubMed Scopus (39) Google Scholar). The actin-activated MgATPase activities of the myosin Is fromAcanthamoeba (25Hammer III, J.A. Albanesi J.P. Korn E.D. J. Biol. Chem. 1983; 258: 10168-10175Abstract Full Text PDF PubMed Google Scholar), Dictyostelium (26Côté 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), andAspergillus (27Brzeska H. Liu X. Deresso G. Korn E.D. May G.S. Yamashita R. Mol. Biol. Cell. 1999; 10: 161aCrossref PubMed Scopus (18) Google Scholar) are activated by phosphorylation of a Thr or Ser at a position (Ser-329 in Acanthamoeba myosin IC (28Brzeska H. Lynch T.J. Martin B. Korn E.D. J. Biol. Chem. 1989; 264: 19340-19348Abstract Full Text PDF PubMed Google Scholar)) in a conserved actin-binding surface loop (29Rayment I. Rypniewski R.W. Schmidt-Bäse K. Smith R. Tomchick D.R. Benning M.M. Winkelmann D.A. Wesenberg G. Holden H.M. Science. 1993; 133: 50-65Crossref Scopus (1850) Google Scholar) where almost all other myosins have a Glu or Asp residue (1Sellers J.R. Myosins. Oxford University Press, Oxford1999Google Scholar, 30Bement W. Mooseker M.S. Cell. Motil. Cytoske l. 1995; 31: 87-92Crossref PubMed Scopus (145) Google Scholar, 31Brzeska H. Korn E.D. J. Biol. Chem. 1996; 271: 16983-16986Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). TheAcanthamoeba (32Brzeska H. Szczepanowska J. Hoey J. Korn E.D. J. Biol. Che m. 1996; 271: 27056-27062Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) and Dictyostelium (33Lee S.F. Egelhoff T.T. Mahasneh A. Côté G.P. J. Biol. Chem. 1996; 271: 27044-27048Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar) myosin I heavy chain kinases (MIHCK) that phosphorylate the Ser and Thr residues are members of the p21-activated kinase (Pak) family. Like other Paks, the 97-kDa MIHCK is activated by Rac and Cdc42 (34Brzeska H. Young R. Knaus U. Korn E.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 394-399Crossref PubMed Scopus (36) Google Scholar) and lipids (35Brzeska H. Lynch T.J. Korn E.D. J. Biol. Chem. 1990; 265: 3591-3594Abstract Full Text PDF PubMed Google Scholar). Only the 35-kDa C-terminal catalytic domain and a short p21-binding domain near the N terminus of MIHCK have sequence similarity to other Paks (34Brzeska H. Young R. Knaus U. Korn E.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 394-399Crossref PubMed Scopus (36) Google Scholar). Interestingly, the central region of MIHCK is very rich in Pro residues, including multiple PXXP repeats characteristic of SH3-binding domains in other proteins (34Brzeska H. Young R. Knaus U. Korn E.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 394-399Crossref PubMed Scopus (36) Google Scholar). Therefore, it seemed possible that the TH-3 (SH3) tail subdomain of theAcanthamoeba myosin Is might be a site for interaction with MIHCK. From this brief introduction, it is apparent that the three tail subdomains have important roles in the activity and function of theAcanthamoeba myosin Is. However, the properties of the tail domains have been characterized mostly by studying the properties of bacterially expressed peptides. In the work described in this paper, we compared the properties of expressed wild type Acanthamoebamyosin IC and truncated and deletion tail mutants to define more quantitatively the actin-binding region in the tail, to confirm that the triphasic actin dependence of the MgATPase activity is caused by the second actin-binding site in the tail, and to evaluate the possibility that the SH3 domain of Acanthamoeba myosin IC may interact with the Pro-rich region of MIHCK. After these studies were completed, Lee et al. (5Lee W.-L. Ostap E.M. Zot H.G. Pollard T.D. J. Biol. Chem. 1999; 274: 35159-35171Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar) reported interesting results, which we will discuss later in this paper, on the actin-binding properties of bacterially expressed peptides corresponding to the TH-1 and TH-2 plus TH-3 regions of Acanthamoeba myosin IA. Standard methods were used for all DNA manipulations (36Ausubel F.M. Brent R. Kington R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Green Publishing Associates, Inc., New York1993Google Scholar). The myosin I heavy chain cDNA (accession number AF051353) cloned into pBluescript plasmid (3Jung G. Korn E.D. Hammer J.A. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 6720-6724Crossref PubMed Scopus (77) Google Scholar,6Wang Z.Y. Wang F. Sellers J.R. Korn E.D. Hammer J.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15200-15205Crossref PubMed Scopus (36) Google Scholar) served as the template for the PCR reactions described below. Fig. 1shows the mutants constructed in this study. The plasmid containing the TH-3 deletion mutant (ΔSH3) was constructed utilizing a unique SanDI site located upstream of the SH3 domain of myosin IC and an XbaI site in the vector located downstream of the myosin IC stop codon. Two primers were synthesized for PCR. The sense primer (primer A), 5′-CGTCCTACGTCGAGGGTCCCCCGCGCGCCG-3′ (bp 3134 and 3163 of myosin IC heavy chain) introduced a new SanDI restriction site (underlined) immediately downstream of the SH3 domain. The antisense primer (primer B) was 5′-GCTCACATGTTCTTTCCTGCGTTATCCCCTGATTC-3′ (bp 1128–1162 of pBluescript II KS+ vector). The original myosin IC plasmid was digested withSanDI and XbaI, and the PCR product was cloned between these sites resulting in a deletion of 198 bp, which encode the amino acids between Pro-986 and Ile-1051 in the myosin IC heavy chain (bp 2956–3153). An intermediate vector with a unique MluI site immediately following the myosin IC stop codon was constructed to generate all the truncation mutants. The sense primer (primer C), 5′-GTCCTCCTCCCCCGGGTCCC TA ACGCGTCCGGACCAATGC-3′ (bp 3539–3579 of myosin IC), was designed to introduce aSanDI restriction site (first underlined sequence) upstream of the stop codon (in bold) and an MluI site (second underlined sequence) downstream of the stop codon. The PCR was run between sense primer C and antisense primer B, and the product was cloned into the original plasmid digested with SanDI andXbaI. This produced a vector containing myosin IC heavy chain cDNA truncated at the SanDI site with anMluI site downstream of the stop codon. The T-2, T-3, T-4, T-5, and T-6 truncants were generated by PCR utilizing a unique ScaI site located within the light chain-binding region of the myosin IC heavy chain and anMluI site of the intermediate vector. The sense primer (primer D), 5′-CCGCGTCGTGTGCCCCAAGACCTGGTCCG-3′ (bp 1881–1909) was used to generate all the truncants. The antisense primers for each truncation contained a stop codon (in bold) that was introduced immediately after the truncation point and an MluI restriction site (underlined) downstream of the stop codon. The antisense primers were: for T-2, 5′-CAGGCGACGACGCACGCGT TAGTTGACGAGCGCGTCATTG-3) (bp 2142–2181); for T-3, 5′-ACGCTGATCAGCACGCGT TAGTCCTGCAGCGGGCTGAGGG-3′ (bp 2471–2511); for T-4, 5′-GCCGCCGCCTCCCACGCGT AAGAGGATCTGGTCCTTGTAGG-3′ (bp 2977–3012); for T-5, 5′-GTCATACAGCGCACGCACGCGT AAGGGTCCGGGCGC-3′ (bp 3141–3169); for T-6, 5′-GCGCAGCGACGCGTTAGATGAGTTCGACG-3′ (bp 3149–3169). The wild type myosin IC plasmid was used as template. Each of the PCR products was subcloned into the intermediate vector digested withMluI and partially digested with ScaI (because the vector DNA has a second ScaI site). T-1 was constructed utilizing the PstI site located within the head domain of the myosin IC heavy chain and the MluI site in the T-2 plasmid. Primers used for PCR were: sense primer 5′-CAACTTCGTGAAGCTGCAGCAGATCTTC-3′ (bp 1234–1267 of myosin IC heavy chain), which contains a PstI site (underlined); antisense primer 5′-GGTCTTGCGGAGGAATCTCACGCGT TAGTTGGCGTAGGAGAAC-3′ (bp 2061–2103 of myosin IC), which introduced a new stop codon (in bold) after the truncation point and an MluI site (underlined) immediately downstream of the stop codon. The PCR product was subcloned into pBluescript T-2 digested completely withMluI and partially with PstI (because the vector contains another PstI site). This produced truncated DNA encoding T-1 (Fig. 1). The sequences of all the mutant DNAs were confirmed. The wild type and mutant heavy chain DNAs were subcloned from pBluescript into the expression vector PVL1393 (PharMingen) by digestion with BamHI. A Flag epitope tag, MADYKDDDDYA, was placed at the N terminus of all of the heavy chains to provide a means for rapid purification of the wild type and mutant myosins. Sf-9 cells were cultured in suspension using Grace's medium supplemented with 10% fetal calf serum (Life Technologies, Inc.). Transfection was achieved by mixing 2–4 μg of plasmid DNA with 0.5 μg of BaculoGold vector DNA (PharMingen) according to the protocol provided by the manufacturer. Recombinant viruses were identified as occlusion-negative plaques. Viral stock was amplified according to the manufacturer's protocols and kept at 4 °C. The myosin IC light chain (6Wang Z.Y. Wang F. Sellers J.R. Korn E.D. Hammer J.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15200-15205Crossref PubMed Scopus (36) Google Scholar), the 35K catalytic domain (32Brzeska H. Szczepanowska J. Hoey J. Korn E.D. J. Biol. Che m. 1996; 271: 27056-27062Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), and full-length MIHCK 2C. Tan, E. D. Korn, and H. Brzeska, unpublished data. viral stocks were prepared previously in this laboratory. Wild type and mutant myosins were produced by co-infection of Sf-9 cells (2 × 106 cells/ml) with heavy chain and light chain viral stocks. All purification procedures were carried out at 4 °C. Wild type and mutant myosins were purified as described previously (6Wang Z.Y. Wang F. Sellers J.R. Korn E.D. Hammer J.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15200-15205Crossref PubMed Scopus (36) Google Scholar) with some modification. Briefly, Sf-9 cells were harvested 48 h after infection. About 2 g of cells were homogenized in 20 ml of extraction buffer (200 mm NaCl, 4 mmMgCl2, 2 mm ATP, 1 mm DTT, and 10 mm Tris, pH 7.5) containing 0.1 mmphenylmethylsulfonyl fluoride and 1 tablet of protease inhibitor mixture (Roche Molecular Biochemicals). The lysate was centrifuged at 45,000 rpm in a Beckman T170 rotor for 1 h, and the resultant supernatant was added to 1 ml of packed anti-FLAG antibody resin (Sigma) that had been washed with 0.1 m glycine (pH 3.5) and equilibrated with the extraction buffer. The resin was washed with 20 ml of extraction buffer, and myosin was eluted with 2.5 ml of extraction buffer containing 0.15 mg/ml FLAG peptide. The eluted fraction was concentrated about 3-fold by dialysis against 50% glycerol, 100 mm NaCl, 1 mm DTT, and 10 mm Tris, pH 7.5. After dialysis the sample was clarified by spinning at 15,000 rpm for 10 min at 4 °C, and the final product was kept in liquid nitrogen until use. The myosins were partially phosphorylated during expression in Sf-9 cells. To prepare unphosphorylated myosins for use as substrates for MIHCK, wild type myosin IC and the T-2 mutant were dephosphorylated while bound to the FLAG affinity resin as an added step in the purification procedure. The resin with bound myosin was washed with ∼20 volumes of extraction buffer and then with one volume of phosphatase buffer (New England Biolabs, Beverly, MA). About 4000 units of lambda protein phosphatase (New England Biolabs) in 1 ml of phosphatase buffer (New England Biolabs) was added to ∼1 ml of the resin-bound myosin. Dephosphorylation was carried out at room temperature for 15 min with occasional resuspension of the resin by pipetting. The resin was then washed with the extraction buffer, and myosin was eluted and collected as described above. For expression of maximum actin-dependent MgATPase activity, the myosins must be fully phosphorylated on Ser-329. The wild type and mutant myosins were incubated with activated (autophosphorylated) 35K catalytic domain of MIHCK at a molar ratio of 1 to 8 in buffer containing 50 mm imidazole, pH 7.0, 2.5 mm ATP, 3.5 mm MgCl2, and 2 mm EGTA (and 50 mm NaCl and 25% glycerol derived from the myosin storage buffer) at 30 °C for 10 min. As determined by the incorporation of 32P from [γ-32P]ATP, phosphorylation was complete (1 mol/mol). The 35K catalytic domain and full-length MIHCK were purified using a nickel-nitrilotriacetic acid resin column as described (32Brzeska H. Szczepanowska J. Hoey J. Korn E.D. J. Biol. Che m. 1996; 271: 27056-27062Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Purified kinase was dialyzed against 50% glycerol, 10 mm Tris, pH 7.5, and 1 mm DTT and kept in liquid nitrogen until use. The kinase and 35K catalytic domain were essentially homogeneous as determined by SDS-polyacrylamide gel electrophoresis (data not shown). To obtain fully active, autophosphorylated kinases, the purified 35K catalytic domain and full-length MIHCK (both at 0.2 mg/ml) were first incubated for 30 min at 30 °C in 50 mm imidazole (pH 7.0) containing 2.5 mm ATP, 3.5 mmMgCl2, and 2 mm EGTA and BSA (0.2 mg/ml). Rabbit skeletal muscle actin was prepared from rabbit skeletal muscle acetone powder according to Spudich and Watt (37Spudich J.A. Watt S. J. Biol. Chem. 1971; 246: 4866-4871Abstract Full Text PDF PubMed Google Scholar).Acanthamoeba actin, which was kindly provided by Dr. Kirsten Remmert (National Heart, Lung, and Blood Institute), was prepared as described (38Gordon D.J. Yang Y.Z. Korn E.D. J. Biol. Chem. 1976; 251: 7474-7479Abstract Full Text PDF PubMed Google Scholar). The concentrations of myosins and kinases were determined by the Bradford method using BSA as the standard (39Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211946) Google Scholar). Actin concentrations were determined spectrophotometrically using an extinction coefficient of 0.62 cm2/ml at 290 nm. Assays were performed essentially as described (40Szczepanowska J.R., U. Herring C.J. Gruschus J.M Qin J. Korn E.D. Brzeska H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4146-4151Crossref PubMed Scopus (15) Google Scholar). The activated kinases (4.5 nm) were incubated at 30 °C for 20 s with various concentrations of dephosphorylated wild type and T-2 mutant myosins, as substrates, in 50 mmimidazole (pH 7.0) containing 2.5 mm[γ-32P]ATP (30,000 cpm/nmol), 3.5 mmMgCl2, and 2 mm EGTA and BSA (0.2 mg/ml). At all substrate concentrations, the rate of phosphorylation was linear with time for the period of incubation. The binding of wild type and mutantAcanthamoeba myosin IC to F-actin was assayed in solutions containing 10 mm Tris, pH 7.5, 3.5 mmMgCl2, 1 mm EGTA, 0.2 mg/ml BSA, with or without 2.5 mm ATP as indicated, and NaCl at the concentrations indicated in the figure legends. Myosins were mixed with various concentrations of F-actin and centrifuged for 30 min at 100,000 rpm in a Beckman TL centrifuge at 4 °C. The fraction of myosin unbound was determined by measuring the NH4/EDTA-ATPase activity remaining in the supernatant. Steady state ATPase activities were determined at 30 °C by measuring the radioactivity of Pireleased from [γ-32P]ATP as described (2Pollard T.D. Korn E.D. J. Biol. Chem. 1973; 248: 4682-4690Abstract Full Text PDF PubMed Google Scholar). The reaction mixtures for the assay of MgATPase activity contained 20 mmimidazole, pH 7.5, 4 mm MgCl2, 1 mmEGTA, 1 mm DTT, 3 mm [γ-32P]ATP (120 cpm/nmol) with or without F-actin as indicated. The reaction mixtures for the assay of NH4/EDTA-ATPase activity contained 25 mm Tris (pH 7.5), 400 mmNH4Cl, 35 mm EDTA, 1 mm DTT, and 3 mm [γ-32P]ATP (120 cpm/nmol). The reactions were started by the addition of myosin at 30 °C. SDS-polyacrylamide gel electrophoresis was carried out according to Laemmli (41Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205523) Google Scholar). The separating gel consisted of two layers; the upper half contained 7.5% acrylamide and the lower half contained 13% acrylamide. The 129-kDa heavy chain of myosin IC can be divided into a head domain, residues 1–693; a light chain-binding neck domain, residues 694–720; and four tail domains: the basic TH-1 domain, residues 721–940; the TH-3 (SH3) domain, residues 997–1051; and the two segments of the GPA TH-2 domain, GPA-1, residues 941–996, and GPA-2, residues 1052–1186, that are separated by the SH3 domain (Fig.1). As described under “Materials and Methods,” we constructed six truncated mutants and one deletion mutant (Fig. 1): T-1, the head only; T-2, the head and neck; T-3, the head, neck, and one-half of TH-1; T-4, the head, neck, and entire TH-1; T-5, the head, neck, TH-1, and GPA-1; T-6, the head, neck, GPA-1, and SH3; and ΔSH3, the entire heavy chain except for the deleted SH3 region. The wild type heavy chain and seven mutant heavy chain constructs were individually co-expressed with the light chain in Sf-9 cells. All of the heavy chains were expressed very well (see Fig.2, first lane, for expression of wild type heavy chain). All of the myosins were readily purified by the procedure described under “Materials and Methods” (Fig. 2), except for T-3, the mutant that was truncated within the TH-1 domain, which we were unable to solubilize and, therefore, could not study further. We compared the binding of the wild type and six mutant myosins to an excess of muscle F-actin in the presence or absence of ATP. As expected, because the head domain of all myosins has a high affinity, ATP-sensitive actin-binding site, all of the expressed myosins bound to actin essentially quantitatively in the absence of ATP (Fig. 3). The wild type and ΔSH3 myosins also bound to F-actin in the presence of ATP but T-1 and T-2 did not. T-4, T-5, and T-6 had intermediate behavior. Each bound extensively to F-actin in the presence of ATP but not as well as in the absence of ATP. These results are consistent with the recent report by Lee et al. (5Lee W.-L. Ostap E.M. Zot H.G. Pollard T.D. J. Biol. Chem. 1999; 274: 35159-35171Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar) that bacterially expressed peptides corresponding to the TH-1, TH-2, and TH-2/3 domains ofAcanthamoeba myosin IA bind to F-actin in the presence of ATP. Next, we quantified the relative affinities for F-actin of wild type myosin and the T-4, T-5, T-6, and ΔSH3 mutants in the presence of ATP (Fig. 4). The measuredKd values were: wild type, 22 nm; ΔSH3, 35 nm;