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• W2060840701 abstract "This study examines the steady state activity and in vitro motility of single-headed (S1) and double-headed (HMM) myosin VI constructs within the context of two putative modes of regulation. Phosphorylation of threonine 406 does not alter either the rate of actin filament sliding or the maximal actin-activated ATPase rate of S1 or HMM constructs. Thus, we do not observe any regulation of myosin VI by phosphorylation within the motor domain. Interestingly, in the absence of calcium, the myosin VI HMM construct moves in an in vitro motility assay at a velocity that is twice that of S1 constructs, which may be indicative of movement that is not based on a “lever arm” mechanism. Increasing calcium above 10 μm slows both the rate of ADP release from S1 and HMM actomyosin VI and the rates of in vitro motility. Furthermore, high calcium concentrations appear to uncouple the two heads of myosin VI. Thus, phosphorylation and calcium are not on/off switches for myosin VI enzymatic activity, although calcium may alter the degree of processive movement for myosin VI-mediated cargo transport. Lastly, calmodulin mutants reveal that the calcium effect is dependent on calcium binding to the N-terminal lobe of calmodulin. This study examines the steady state activity and in vitro motility of single-headed (S1) and double-headed (HMM) myosin VI constructs within the context of two putative modes of regulation. Phosphorylation of threonine 406 does not alter either the rate of actin filament sliding or the maximal actin-activated ATPase rate of S1 or HMM constructs. Thus, we do not observe any regulation of myosin VI by phosphorylation within the motor domain. Interestingly, in the absence of calcium, the myosin VI HMM construct moves in an in vitro motility assay at a velocity that is twice that of S1 constructs, which may be indicative of movement that is not based on a “lever arm” mechanism. Increasing calcium above 10 μm slows both the rate of ADP release from S1 and HMM actomyosin VI and the rates of in vitro motility. Furthermore, high calcium concentrations appear to uncouple the two heads of myosin VI. Thus, phosphorylation and calcium are not on/off switches for myosin VI enzymatic activity, although calcium may alter the degree of processive movement for myosin VI-mediated cargo transport. Lastly, calmodulin mutants reveal that the calcium effect is dependent on calcium binding to the N-terminal lobe of calmodulin. Myosin VI was the first myosin demonstrated to move toward the pointed (–) end of an actin filament (1Wells A.L. Lin A.W. Chen L.-Q. Safer D. Cain S.M. Hasson T. Carragher B.O. Milligan R.A. Sweeney H.L. Nature. 1999; 401: 505-508Google Scholar). This discovery was based on the hypothesis that reverse direction would require a redesign of the domain of myosin that couples changes in the state of the nucleotide binding pocket and actin-myosin interface to movements of the myosin light chain binding domain (the myosin “lever arm”). Indeed, cryoelectron microscopy revealed that upon ADP release, the effective myosin VI lever arm rotates in the opposite direction (i.e. toward the pointed end of the actin filament) compared with other characterized myosins (1Wells A.L. Lin A.W. Chen L.-Q. Safer D. Cain S.M. Hasson T. Carragher B.O. Milligan R.A. Sweeney H.L. Nature. 1999; 401: 505-508Google Scholar). The light chain-binding region of each head of the myosin VI dimer is thought to consist of a single calmodulin bound to the heavy chain “IQ” motif. This light chain binding region is generally believed to act as a “lever arm” that amplifies nucleotide state-dependent structural changes within the core of the myosin motor domain (for review, see Refs. 2Holmes K.C. Geeves M.A. Annu. Rev. Biochem. 1999; 68: 687-728Google Scholar and 3Houdusse A. Sweeney H.L. Curr. Opin. Struct. Biol. 2001; 11: 182-194Google Scholar). For both myosin II and myosin V, the length of the “lever arm” has been shown to correspond to the step size associated with a single ATPase cycle (4Uyeda T.Q. Abramson P.D. Spudich J.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4459-4464Google Scholar, 5Purcell T.J. Morris C. Spudich J.A. Sweeney H.L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14159-14164Google Scholar). Recent kinetic and single molecule data demonstrate that myosin VI has a high duty ratio (i.e. remains strongly bound to actin for >90% of its actomyosin ATPase cycle) and is processive (6De La Cruz E.M. Ostap E.M. Sweeney H.L. J. Biol. Chem. 1999; 276: 32373-32381Google Scholar, 7Rock R.S. Rice S.E. Wells A.L. Purcell T.J. Spudich J.A. Sweeney H.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13655-13659Google Scholar, 8Nishikawa S. Homma K. Komori Y. Iwaki M. Wazawa T. Iwane A.H. Saito J. Ikebe R. Katayama E. Yanagida T. Ikebe M. Biochem. Biophys. Res. Commun. 2002; 290: 311-317Google Scholar). Cryoelectron microscopy studies of myosin VI bound to actin showed a very compact effective “lever arm” that appears too small to account for the large step size of myosin VI (1Wells A.L. Lin A.W. Chen L.-Q. Safer D. Cain S.M. Hasson T. Carragher B.O. Milligan R.A. Sweeney H.L. Nature. 1999; 401: 505-508Google Scholar, 7Rock R.S. Rice S.E. Wells A.L. Purcell T.J. Spudich J.A. Sweeney H.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13655-13659Google Scholar). Furthermore, the single molecule data reveal that myosin VI has a broad distribution of step sizes centered on 30 nm (7Rock R.S. Rice S.E. Wells A.L. Purcell T.J. Spudich J.A. Sweeney H.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13655-13659Google Scholar), which also is not consistent with a lever arm mechanism. Thus, it is unclear what role the region containing the IQ motif of myosin VI is playing in motility. To provide further insight into the mechanism of movement, we examined the motility of myosin VI S1 and HMM constructs. The expectation based on myosin V is that for a lever arm mechanism, the asymmetry of the lever arm swing (9Veigel C. Wang F. Bartoo M.L. Sellers J.R. Molloy J.E. Nat. Cell Biol. 2002; 4: 59-65Google Scholar) will allow an ensemble of the S1 constructs to move actin filaments at a high percentage of the velocity of the HMM construct. Whereas the light chain-binding region of myosin VI may not function as a conventional lever arm, the fact that it contains calmodulin could allow some form of regulation by calcium. As shown for myosin V, there could be altered motor function of myosin VI as a function of calcium concentration (10Homma K. Saito J. Ikebe R. Ikebe M. J. Biol. Chem. 2000; 275: 34766-34771Google Scholar, 11Trybus K.M. Krementsova E. Freyzon Y. J. Biol. Chem. 1999; 274: 27448-27456Google Scholar). A recent report suggested that calcium binding to myosin VI serves as an “on/off” switch for myosin VI motility (12Yoshimura M. Homma K. Saito J. Inoue A. Ikebe R. Ikebe M. J. Biol. Chem. 2001; 276: 39600-39607Google Scholar). Herein, we perform a more detailed investigation of the nature of calcium regulation of myosin VI using S1 and HMM myosin VI constructs. Myosin VI, like a number of class I myosins, has a putative phosphorylation site in one of its actin binding loops (the “HCM” loop) (13Bement W.M. Mooseker M.S. Cell Motil. Cytoskeleton. 1995; 31: 87-92Google Scholar). There is evidence that Thr406 is a PAK phosphorylation site (12Yoshimura M. Homma K. Saito J. Inoue A. Ikebe R. Ikebe M. J. Biol. Chem. 2001; 276: 39600-39607Google Scholar, 14Buss F. Kendrick-Jones J. Lionne C. Knight A.E. Cote G.P. Luzio P.J. J. Cell Biol. 1998; 143: 1535-1545Google Scholar), and recently Yoshimura et al. (12Yoshimura M. Homma K. Saito J. Inoue A. Ikebe R. Ikebe M. J. Biol. Chem. 2001; 276: 39600-39607Google Scholar) raised the possibility that phosphorylation of the loop serves as an “on/off” switch for motility. However, this mechanism is inconsistent with a recent kinetic study of myosin VI using mutants T406E and T406A, designed to mimic the phosphorylated and dephosphorylated loop, respectively (6De La Cruz E.M. Ostap E.M. Sweeney H.L. J. Biol. Chem. 1999; 276: 32373-32381Google Scholar). Those results suggested that phosphorylation does not alter the rate-limiting step in the actomyosin VI ATPase cycle (ADP release); rather, phosphorylation increases the rate of phosphate release, thereby increasing the duty cycle. Since ADP release also limits in vitro motility, the expectation, based on the work of De La Cruz et al. (6De La Cruz E.M. Ostap E.M. Sweeney H.L. J. Biol. Chem. 1999; 276: 32373-32381Google Scholar), was that phosphorylation would not be an on/off switch for either ATPase activity or motility, contrary to the conclusions of Yoshimura et al. (12Yoshimura M. Homma K. Saito J. Inoue A. Ikebe R. Ikebe M. J. Biol. Chem. 2001; 276: 39600-39607Google Scholar). To resolve the apparent contradictions pertaining to the mechanism and regulation of movement of myosin VI on actin filaments, we examined the in vitro motility and solution kinetics of both single- and double-headed myosin VI with different levels of phosphorylation and at different calcium concentrations. Additionally, we examined the site of calcium action using calmodulin mutants with either N- or C-terminal calcium binding eliminated. Myosin VI Expression and Purification—To create single-headed, S1-like myosin VI constructs (S1), porcine myosin VI wild-type cDNA (15Hasson T. Mooseker M.S. J. Cell Biol. 1994; 127: 425-440Google Scholar) was truncated at Gly840. A FLAG tag (encoding GDYKDDDDK) was inserted at the C terminus to facilitate purification (16Hopp T.P. Prickett K.S. Price V. Libby R.T. March C.J. Cerretti P. Urdal D.L. Conlon P.J. Biotechnology. 1988; 6: 1205-1210Google Scholar), and a Myc tag (encoding EQKLISEEDL) was inserted preceding the FLAG tag for use in in vitro motility assays (17Kolodziej P.A. Young R.A. Methods Enzymol. 1991; 194: 508-519Google Scholar). The recombinant heavy chain protein contains the motor domain and the single calmodulin/light chain-binding site (IQ motif). The double-headed, HMM-like construct was truncated at Arg994 to include 20 native heptad repeats of predicted coiled-coil with a C-terminal leucine zipper (GCN4) to ensure dimerization and improve protein yield (18Trybus K.M. Freyzon Y. Faust L.Z. Sweeney H.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 48-52Google Scholar) followed by the Myc and FLAG tag sequences. For comparative motility studies, we used two myosin V constructs, single-headed, 1-IQ (S1-like) and double-headed, 6-IQ (HMM) proteins. The double-headed construct was produced as previously described (7Rock R.S. Rice S.E. Wells A.L. Purcell T.J. Spudich J.A. Sweeney H.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13655-13659Google Scholar). Construction of the single-headed construct involved truncation of the myosin V heavy chain at Lys909. As for myosin VI, the FLAG and Myc epitope were added for purification and motility, respectively. Expression of the recombinant single- and double-headed fragments of myosin VI was accomplished via infection of SF9 insect cells with a viral expression vector (baculovirus) capable of driving high level expression of foreign proteins. The SF9 cells were co-infected with recombinant virus expressing the myosin VI heavy chain and a recombinant virus for calmodulin expression. Details of the protein expression and purification have been published (1Wells A.L. Lin A.W. Chen L.-Q. Safer D. Cain S.M. Hasson T. Carragher B.O. Milligan R.A. Sweeney H.L. Nature. 1999; 401: 505-508Google Scholar, 19Sweeney H.L. Rosenfeld S. Brown F. Faust L. Smith J. Stein L. Sellers J. J. Biol. Chem. 1998; 273: 6262-6270Google Scholar). Greater than five separate protein preparations were used in the experiments described in this study to account for any preparation-specific effects. Calmodulin Mutants—To generate calmodulin with either the N- or C-terminal calcium binding sites eliminated, the cDNA of chicken calmodulin was mutated to make E104A,E140A for the C-terminal mutant and E31A,E67A for the N-terminal mutant. The mutant cDNA constructs were then inserted into a viral expression vector for co-transfection with the myosin VI constructs or into a bacteria expression vector to generate exogenous calmodulin for use during the kinetic and motility experiments. Protein and Reagents—mantADP 1The abbreviation used is: mantADP, 2′(3′)-O-(N-methylanthraniloyl)-ADP. was synthesized as previously described (20Hiratsuka T. Biochim. Biophys. Acta. 1983; 742: 496-508Google Scholar). Actin was prepared from acetone powder as described (21Pardee J.D. Spudich J.A. Methods Enzymol. 1982; 85: 164-181Google Scholar) and gel-filtered. Phalloidin (Sigma) was added to stabilize the F-actin following polymerization and dialysis into the appropriate buffer. Apyrase at 0.02 units/ml (Sigma) was used to remove contaminating ADP. The expressed proteins were run on SDS-polyacrylamide gels to determine purity of the preparation, and the protein concentrations were measured colorimetrically using the Bio-Rad protein assay. The solutions used in both the kinetic measurements and the in vitro motility assay were at an ionic strength of 78 mm and contained 0.37 mm free Mg2+. They were composed of 10 mm imidazole, 46 mm KCl, 2.4 mm MgCl2, and 2 mm total EGTA (pH 7.3). The calcium concentration was varied by adjusting the relative amounts of K+-EGTA and Ca2+-K+-EGTA or by the addition of CaCl2 for the pCa 3.0 and pCa 4.0 solutions while maintaining total EGTA at 2 mm. The addition of Na2ATP to the different pCa solutions was adjusted to maintain a final [Mg-ATP] of 2 mm. All experimental solutions contained 6 μm excess calmodulin unless otherwise noted. Phosphorylation and Dephosphorylation of Myosin VI Thr406— Phosphorylation of myosin VI was accomplished using a constitutively active recombinant PAK3 protein (provided by G. Coté, Queen's University) following procedures described previously (12Yoshimura M. Homma K. Saito J. Inoue A. Ikebe R. Ikebe M. J. Biol. Chem. 2001; 276: 39600-39607Google Scholar). Dephosphorylation of myosin VI was performed by incubation with λ-phosphatase (New England Biolabs). Briefly, the myosin VI was treated with 250–500 units of λ-phosphatase at 30 °C for 30 min in the presence of 2 mm MnCl2. Untreated samples were incubated in the same reaction buffer without the addition of λ-phosphatase. For blot analysis, anti-phosphothreonine antibodies were purchased from Zymed Laboratories (San Francisco, CA) and used at a 1:250 dilution following standard electrophoretic and blotting techniques. Image analysis was performed using Kodak image software (Eastman Kodak Co.). The band intensities were determined relative to the PAK3-treated sample on each blot. Six separate blots were analyzed using five different protein preparations (both S1 and HMM), and the results were pooled to determine the average intensities of the untreated and λ-phosphatase-treated myosin VI constructs. Stopped-flow Experiments and Steady State ATPase Measurements— Transient kinetic measurements were performed as previously described (6De La Cruz E.M. Ostap E.M. Sweeney H.L. J. Biol. Chem. 1999; 276: 32373-32381Google Scholar) at either 25 or 30 °C with an Applied Photophysics SX.18MV stopped-flow device (Surry, UK) in either single mix or sequential mix mode. For the stopped-flow experiments, all of the reagent concentrations are described prior to 1:1 mixing for measurement. mantADP was directly excited at 365 nm, and the fluorescence was measured using a 400-nm long pass filter. N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)-coumarin-3-carboxamide (MDCC)-labeled phosphate-binding protein (22Brune M. Hunter J.L. Corrie J.E. Webb M.R. Biochemistry. 1994; 33: 8262-8271Google Scholar) was excited at 425 nm, and the fluorescence was monitored using a 455-nm colored glass filter. mantADP Release—ADP release from actomyosin VI was determined by preincubating either 1 μm (S1) or 0.5 μm (HMM) in the appropriate pCa solution containing 50 μm mantADP and 5 μm F-actin. The actomyosin VI-mantADP complex was mixed 1:1 with 4 mm Mg-ADP. A single exponential was used to fit the time course of the fluorescence decrease. The experiments were performed at 30 °C. Transient PiRelease—Phosphate release was measured using the stopped-flow apparatus in sequential mix mode. Myosin VI S1 (4 μm; starting concentration) was mixed with Mg-ATP (400 μm), aged for 300 ms to populate the actomyosin-ADP-Pi state, and then mixed with F-actin. The initial burst was fit by a single exponential with a slope. 2 mm ADP was added to the F-actin solution to limit further ATP binding. N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC)-labeled phosphate binding protein (PBP) was added to the initial myosin and actin solutions to produce a final PBP concentration of 4 μm. All solutions and the stopped-flow were preincubated with a Pi mop solution containing 0.5 units/ml purine nucleoside phosphorylase and 250 μm 7-methylguanosine. The experiments were performed at 25 °C. Steady State ATPase—The Mg-ATPase activity of the myosin constructs was determined using the NADH-coupled assay in the stopped-flow or spectrophotometer by monitoring the absorbance change at 340 nm. The ATPase solution was composed of the described pCa solutions with 200 μm NADH (Sigma), 250 μm phosphoenol pyruvate (Sigma), 10 units/ml lactate dehydrogenase (Sigma), and 50 units/ml pyruvate kinase (Sigma) and included 0.025–0.1 μm myosin VI heads. The solutions also contained 0–30 μm F-actin and 6 μm calmodulin (unless otherwise noted). The reaction was initiated by the addition of 2 mm Mg-ATP. The assays were performed at 30 °C for direct comparison with the motility data. The transient kinetic data was initially analyzed using the software supplied with the stopped-flow apparatus. Further analysis and the generation of figures were completed using Sigmaplot 4.0 or Microsoft Excel. In Vitro Motility Assay—Single- and double-headed myosin VI (or myosin V) motility was measured at 30 °C using standard procedures (1Wells A.L. Lin A.W. Chen L.-Q. Safer D. Cain S.M. Hasson T. Carragher B.O. Milligan R.A. Sweeney H.L. Nature. 1999; 401: 505-508Google Scholar). The activating solutions were composed of the described pCa solutions supplemented with 0.3–0.5% methylcellulose and 2.5 mg/ml glucose, 0.1 mg/ml glucose oxidase, 0.02 mg/ml catalase, and 2 mm dithiothreitol to inhibit photobleaching. The single- and double-headed myosin VI constructs were attached to the nitrocellulose-coated glass coverslips via an antibody directed against the C-terminal Myc tag (23Sellers J.R. Cuda G. Wang F. Homsher E. Methods Cell Biol. 1993; 39: 23-49Google Scholar). The Myc antibodies were added to the flow cell, incubated for 2 min, and washed out, and 1 mg/ml bovine serum albumin was added to block nonspecific sites. The myosin VI was left to incubate for 2 min, followed by washing with solution containing bovine serum albumin. This was followed by the addition of 5 μm sheared unlabelled F-actin to block any noncycling myosin molecules (“dead heads”). Following two washes with 2 mm ATP to remove the excess sheared actin and two washes to remove the ATP, 20 nm rhodamine-phalloidin-labeled F-actin was added to the flow cell and incubated for 1 min, and the excess was washed out. The activating solution was added to the flow cell, and the sliding speed of the actin filaments was quantified using a fluorescence microscope equipped with a low light level camera. The raw data were recorded onto videotape and analyzed offline using a motion analysis system (Santa Rosa, CA), as described previously (23Sellers J.R. Cuda G. Wang F. Homsher E. Methods Cell Biol. 1993; 39: 23-49Google Scholar). Phosphorylation State of Expressed Wild-type Myosin VI—In Fig. 1A, the untreated, wild-type myosin VI S1 following expression and purification from SF9 cells is shown with myosin VI treated with either PAK3 or λ–phosphatase, probed on a Western blot with an anti-phosphothreonine antibody. The untreated myosin VI samples show relatively high levels of threonine phosphorylation, whereas incubation with λ-phosphatase (+λ) greatly reduced the reactivity to background levels. Similar background levels are observed with the T406E mutant, whether or not it has been exposed to λ-phosphatase or PAK3 treatment, suggesting the antibody is reacting only with the phosphorylated Thr406. Following treatment with PAK3, the intensity of the heavy chain reactivity is increased slightly (Fig. 1A; +PAK) when compared with the untreated myosin VI construct. Several separate preparations were analyzed to determine the level of myosin VI phosphorylation without any further treatment. The blots showed consistently high levels of phosphorylation at Thr406 in the untreated samples (Fig. 1B). Under our culture conditions (serum-containing media), the S1 and HMM myosin VI constructs expressed in SF9 cells are consistently isolated in a highly phosphorylated state that can be subsequently dephosphorylated by λ-phosphatase. Assuming that PAK3 treatment resulted in near 100% phosphorylation, the isolated myosin VI constructs are ∼80% phosphorylated. Steady State ATPase as a Function of Phosphorylation of Thr406—Fig. 2, A and B, depicts the actin-activated ATPase activity of untreated or λ-phosphatase-treated myosin VI S1 and HMM, respectively, as a function of actin concentration. As shown, neither the Vmax nor the KATPase are altered by the level of phosphorylation in either the single- or double-headed construct. PiRelease as a Function of Phosphorylation of Thr406—Previous work showed that the major kinetic difference due to mutations at Thr406 (T406E and T406A) was the rate of Pi release from actomyosin VI (4Uyeda T.Q. Abramson P.D. Spudich J.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4459-4464Google Scholar). The release rate was ∼3-fold greater for T406E (50 versus 18 s–1) at 40 μm actin. Accordingly, we measured the rate of Pi release from actomyosin VI with or without phosphorylation of Thr406. At 40 μm actin, the rate was 31.4 ± 1.4 s–1 for the phosphorylated S1 construct and 27.3 ± 2.7 s–1 for the dephosphorylated construct. In Vitro Motility as a Function of Phosphorylation of Thr406—We next addressed whether or not the rate of actin filament sliding is affected by phosphorylation of Thr406 (Table I). The myosin was incubated at 30 °C for 30 min either with or without added λ-phosphatase and then applied to the flow cell to determine the sliding speed. The velocity was unaffected by the level of myosin VI Thr406 phosphorylation. Note that the protocol used for λ-phosphatase treatment, 30 min at 30 °C, leads to a reduction in the in vitro motility rates observed with untreated proteins.Table IComparison of in vitro motility of wild type myosin V and VI constructsActin filament velocityS1, single-headedHMM, double-headednms-1Myosin V322 ± 32459 ± 45Myosin VI (untreated)131 ± 20307 ± 39Control (for λ-phosphatase)238 ± 64Treated (+ λ-phosphatase)222 ± 65 Open table in a new tab Actin-activated ATPase Activity as a Function of Calcium Concentration—To examine the effect of calcium on myosin VI function, actin-activated ATPase assays on single- and double-headed constructs were performed over a range of calcium concentrations from pCa 9.0 to pCa 4.0. The Vmax and KATPase values for the low and high calcium concentrations are provided in Table II with the ATPase rates, at 30 μm F-actin, plotted over the complete calcium range in Fig. 3. The HMM ATPase rates were similar at all [Ca2+]; however, a slight increase is observed at higher [Ca2+]. The ATPase rate of myosin VI S1 was significantly reduced (p < 0.05) at calcium concentrations higher than pCa 5.0 when compared with the rates obtained at pCa 9.0. There is no significant difference in the pCa 5.0 ATPase rate versus either pCa 4.0 or pCa 3.0 rates.Table IIEffect of [Ca2+] on myosin VI actin-activated ATPase activitypCa 9.0pCa 4.0KATPaseVmaxKATPaseVmaxμMhead-1s-1μMhead-1s-1S1 (single-headed)5.7 ± 0.75.3 ± 0.24.4 ± 0.33.7 ± 0.1HMM (double-headed)1.2 ± 0.22.8 ± 0.21.1 ± 0.23.2 ± 0.2 Open table in a new tab Control experiments were performed to determine whether excess calmodulin was necessary to maintain maximal activity of the myosin VI constructs. For these experiments, 0–30 μm wild-type chicken calmodulin, expressed in Escherichia coli and purified, was mixed with myosin VI S1. The actin-activated ATPase activity was measured in a pCa 4.0 or pCa 9.0 solution at 30 °C using the NADH-coupled assay, as described in the legend to Fig. 2 and under “Experimental Procedures,” in the presence of 20 μm F-actin. At both pCa 4.0 and pCa 9.0, the ATPase rates were similar regardless of the calmodulin concentration, in agreement with the results of Yoshimura et al. (12Yoshimura M. Homma K. Saito J. Inoue A. Ikebe R. Ikebe M. J. Biol. Chem. 2001; 276: 39600-39607Google Scholar). The activity ranged from 4.2 to 4.6 s–1 at pCa 9.0 and from 3.0 to 3.3 s–1 at pCa 4.0 (data not shown). Further, as shown previously by Yoshimura et al. (12Yoshimura M. Homma K. Saito J. Inoue A. Ikebe R. Ikebe M. J. Biol. Chem. 2001; 276: 39600-39607Google Scholar), control steady state measurements indicated that the phosphorylation state did not significantly change the calcium dependent effect observed when the calcium concentration was elevated from pCa 9.0 to pCa 4.0 (data not shown). In Vitro Motility as a Function of Calcium Concentration— The results of in vitro motility assays on single- and double-headed myosin VI over the calcium range pCa 9.0 to pCa 3.0 are shown in Fig. 4. In the absence of calcium, the HMM species moves actin filaments at slightly greater than twice the speed of the S1 constructs (307 versus 131 nms–1). This is a much greater difference than was seen in the case of single-headed versus double-headed myosin V (Table I). At high calcium concentrations, the speed of both constructs is reduced, but to a much greater extent for the HMM construct. Increasing the calcium concentration reduced the single-headed myosin VI motility from 131 to 80 nms–1, a reduction to 60% from pCa 9.0 speeds, whereas the myosin VI HMM motility was reduced to 30% of the pCa 9.0 motility, dropping from a speed of 307 to 103 nms–1. The greatest reduction in sliding speed is observed between pCa 6.0 and pCa 5.0 for the HMM construct, thus over a physiological calcium concentration range. At high calcium concentrations, the motility is similar for the single- and double-headed constructs. mantADP Release Rates as a Function of Calcium Concentration—In the absence of Ca2+, the rate of mantADP release from the actomyosin VI S1 complex was 5.2 ± 1.1 s–1; however, in the presence of 100 μm Ca2+, the rate was reduced to 3.7 ± 0.8 s–1 (Table III). Similar rates of dissociation of mantADP were measured in the presence and absence of calcium for the myosin VI HMM complexed with actin and mantADP. At high calcium concentration (pCa 4.0), the maximal steady state ATPase rates of the single- and double-headed constructs were both nearly identical to the ADP release rate (Tables II and III). However, at low calcium concentrations (pCa 9.0), the activity per head of the HMM species is approximately half that observed for the S1 species and half of the ADP release rate.Table IIIEffect of [Ca2+] on mantADP release rate of wild type myosin VIpCa 9.0pCa 4.0s-1MVI (S1, single-headed)5.2 ± 1.13.7 ± 0.8MVI (HMM, double-headed)5.4 ± 0.43.6 ± 0.5 Open table in a new tab Site of Action of Calcium—Whereas calcium binding to calmodulin probably mediates the effect on myosin VI activity, it was unclear which calmodulin binding sites are involved. Thus, the myosin VI S1 construct was expressed with a calmodulin mutant that either eliminated both C-terminal calcium binding sites (E104A,E140A) or both N-terminal calcium-binding sites (E31A,E67A). The isolated protein was assayed for the effect on actin-activated ATPase activity. As summarized in Fig. 5, the elimination of calcium binding to the C-terminal sites did not block the calcium effect on myosin VI. However, the removal of the N-terminal calcium-binding sites eliminated a calcium effect on the myosin VI ATPase activity. Myosin VI Phosphorylation at Thr406—We have shown that myosin VI, in our hands, is expressed and purified under conditions that maintain the myosin in a highly phosphorylated state (Fig. 1). Assuming that PAK3 phosphorylates nearly 100% of the myosin, we find ∼80–90% of both myosin VI heads are phosphorylated following purification. Blot analysis and densitometry of the T406E mutant suggests that other threonines within our constructs are not phosphorylated by PAK3 or dephosphorylated by λ-phosphatase (Fig. 1). Further controls using an anti-phosphoserine antibody show no changes in phosphorylation following PAK3 or λ-phosphatase treatment (data not shown). The data demonstrate that the kinetic parameters of the actin-activated ATPase for the single- or double-headed wild-type myosin VI are unchanged whether or not Thr406 is phosphorylated (Fig. 2). The kinetics in either case are intermediate to the values published for the T406E and T406A S1 mutants (6De La Cruz E.M. Ostap E.M. Sweeney H.L. J. Biol. Chem. 1999; 276: 32373-32381Google Scholar), which differed primarily in the KATPase and the actin-activated rate of Pi release. The lack of a change in the KATPase as a function of phosphorylation suggests that the rate of Pi release from actomyosin VI is unchanged. Indeed, in the present experiments, a comparison of the Pi release rate at 40 μm actin revealed no effect of phosphorylation. The rate measured in this study for the wild-type protein with or without phosphorylation of Thr406 (∼30 s–1) was intermediate to the values measured for the T406E and T406A mutants (6De La Cruz E." @default.
• W2060840701 created "2016-06-24" @default.
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• W2060840701 creator A5028495424 @default.
• W2060840701 creator A5033185368 @default.
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• W2060840701 date "2003-06-01" @default.
• W2060840701 modified "2023-09-29" @default.
• W2060840701 title "Calcium Functionally Uncouples the Heads of Myosin VI" @default.
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