Regulation of the actin-activated MgATPase activity of<i>Acanthamoeba</i>myosin II by phosphorylation of serine 639 in motor domain loop 2

Liu, Xiong; Lee, Duck‐Yeon; Cai, Sanjun; Yu, Shuhua; Shu, Shi; Levine, Rodney L.; Korn, Edward D.

doi:10.1073/pnas.1219713110

Cited by 8 publications

(17 citation statements)

References 52 publications

(35 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Protein Preparations-Recombinant WT and S639D AM2 S1s comprising heavy chain residues 1-900 and the two AM2 light chains were overproduced in Sf9 (Spodoptera frugiperda) insect cells and purified to homogeneity via FLAG affinity chromatography as described previously (6). F-actin was prepared from rabbit skeletal muscle according to Lehrer and Kerwar (8) and pyrene-labeled as described by Criddle et al (9).…”

Section: Methodsmentioning

confidence: 99%

“…Kinetic Measurements-Steady-state ATPase assays were conducted at 30°C with the radiometric ATPase assay, as described previously, in buffer containing 20 mM MOPS, pH 7.0, 25 mM KCl, 5 mM MgCl 2, 1 mM [␥- 32 P]ATP, and 0.12 M S1 (6). Stopped-flow assays were conducted at 20°C in a SF-61DX2 stopped-flow spectrophotometer (Hi-Tech Scientific) equipped with a 75-watt mercury-xenon arc lamp in buffer containing 25 mM MOPS, pH 7.0, 100 mM KCl, and 5 mM MgCl 2 unless stated otherwise.…”

Section: Methodsmentioning

confidence: 99%

“…In contrast to eukaryotic muscle and nonmuscle myosin-2s, filament assembly and actin-activated ATPase activity of AM2 are regulated by phosphorylation of heavy chain serines (6). Phosphorylation of four serines within the non-helical tailpiece regulates filament assembly and the structure of the bipolar minifilaments (7), whereas phosphorylation of Ser-639 within the motor domain down-regulates actinactivated ATPase activity of AM2 (6,7). Furthermore, phosphorylation of Ser-639 also inhibits the activity of recombinant single-headed subfragment-1 (S1), and the recombinant phosphomimetic S1 mutant, S639D, has similarly low actinactivated ATPase activity (6).…”

Section: Cytoplasmic Myosin-2 (Am2)mentioning

confidence: 99%

“…In vivo, myosin filaments undergo dynamic assembly/disassembly transitioning from octameric minifilaments to thick filaments and vice versa in a spatiotemporal manner (5). In contrast to eukaryotic muscle and nonmuscle myosin-2s, filament assembly and actin-activated ATPase activity of AM2 are regulated by phosphorylation of heavy chain serines (6). Phosphorylation of four serines within the non-helical tailpiece regulates filament assembly and the structure of the bipolar minifilaments (7), whereas phosphorylation of Ser-639 within the motor domain down-regulates actinactivated ATPase activity of AM2 (6,7).…”

Section: Cytoplasmic Myosin-2 (Am2)mentioning

confidence: 99%

See 3 more Smart Citations

Kinetic Characterization of the ATPase and Actin-activated ATPase Activities of Acanthamoeba castellanii Myosin-2

Heissler

Liu

Korn

et al. 2013

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Background: Actin-activated ATPase activity of Acanthamoeba myosin-2 is inhibited by phosphorylation of Ser-639. Results: Kinetic constants for each step of the ATPase cycle were determined for wild-type myosin and the phosphomimetic mutant S639D. Conclusion: Ser-639 phosphorylation reduces actin-activated phosphate-release and hence the steady-state ATPase activity of the myosin. Significance: This is the first example of regulation of a myosin by covalent modification of loop-2.

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Cytoplasmic Myosin-2 (Am2)mentioning

confidence: 99%

Section: Cytoplasmic Myosin-2 (Am2)mentioning

confidence: 99%

See 2 more Smart Citations

Kinetic Characterization of the ATPase and Actin-activated ATPase Activities of Acanthamoeba castellanii Myosin-2

Heissler

Liu

Korn

et al. 2013

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…We showed further that the actin-activated MgATPase activity of AMII is down-regulated by phosphorylation of Ser639 and not by phosphorylation of the serines in the nonhelical tailpiece. Moreover, we found that nonfilamentous AMII derivatives, heavy meromyosin and subfragment-1, also have actin-activated MgATPase activity that is downregulated by phosphorylation of Ser639 (1).…”

mentioning

confidence: 85%

Regulation of the filament structure and assembly ofAcanthamoebamyosin II by phosphorylation of serines in the heavy-chain nonhelical tailpiece

Liu

Hong

Shu

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

Acanthamoeba myosin II (AMII) has two heavy chains ending in a 27-residue nonhelical tailpiece and two pairs of light chains. In a companion article, we show that five, and only five, serine residues can be phosphorylated both in vitro and in vivo: Ser639 in surface loop 2 of the motor domain and serines 1489, 1494, 1499, and 1504 in the nonhelical tailpiece of the heavy chains. In that paper, we show that phosphorylation of Ser639 down-regulates the actin-activated MgATPase activity of AMII and that phosphorylation of the serines in the nonhelical tailpiece has no effect on enzymatic activity. Here we show that bipolar tetrameric, hexameric, and octameric minifilaments of AMII with the nonhelical tailpiece serines either phosphorylated or mutated to glutamate have longer bare zones and more tightly clustered heads than minifilaments of unphosphorylated AMII, irrespective of the phosphorylation state of Ser639. Although antiparallel dimers of phosphorylated and unphosphorylated myosins are indistinguishable, phosphorylation inhibits dimerization and filament assembly. Therefore, the different structures of tetramers, hexamers, and octamers of phosphorylated and unphosphorylated AMII must be caused by differences in the longitudinal stagger of phosphorylated and unphosphorylated bipolar dimers and tetramers. Thus, although the actin-activated MgATPase activity of AMII is regulated by phosphorylation of Ser639 in loop 2 of the motor domain, the structure of AMII minifilaments is regulated by phosphorylation of one or more of four serines in the nonhelical tailpiece of the heavy chain.A s summarized in the accompanying paper (1), Acanthamoeba myosin II (AMII) is a typical class II myosin with two heavy chains and two pairs of light chains (2, 3). The coiled-coil helical tails of the heavy chains terminate with a 27-residue nonhelical tailpiece, 1483PSSRGGSTRGASARGASVRAGSARAEE1509, which has a pattern of four contiguous repeats of XXSXR (4). Previous work had shown that phosphorylation of two or more of these serines (residues 1489, 1494, 1499, and 1504) correlates with, and was assumed to be responsible for, inactivation of AMII's actin-activated MgATPase activity (5, 6). Also, it had been concluded that only filamentous AMII has actin-activated MgATPase activity (7, 8), and it was inferred that the ATPase activity is regulated by a change in the conformation of the bipolar minifilaments (9-11). However, detailed studies by the Pollard laboratory found no significant differences in either the polymerization properties or electron microscopic images of minifilaments of phosphorylated and dephosphorylated myosins (12-16).The earlier studies were carried out with purified endogenous myosin. To avoid possible complications in enzymatic and structural studies caused by the partial phosphorylation of purified endogenous myosin and incomplete dephosphorylation by phosphatase, we initiated studies on the enzymatic activity and structure of expressed recombinant wild-type, truncated, and mutant myosins before and after phos...

show abstract

Mechanism of oxidative inactivation of human presequence protease by hydrogen peroxide

Chen

Teixeira

Glaser

et al. 2014

Free Radical Biology and Medicine

Self Cite

View full text Add to dashboard Cite

The mitochondrial presequence protease (PreP) is a member of the pitrilysin class of metalloproteases. It degrades the mitochondrial targeting presequences of mitochondria-localized proteins as well as unstructured peptides such as amyloid-β-peptide. The specific activity of PreP is reduced in Alzheimer patients and animal models of Alzheimer disease. The loss of activity can be mimicked in vitro by exposure to oxidizing conditions, and indirect evidence suggested that inactivation was due to methionine oxidation. We performed peptide mapping analyses to elucidate the mechanism of inactivation. None of the 24 methionine residues in recombinant human PreP was oxidized. We present evidence that inactivation was due to oxidation of cysteine residues and consequent oligomerization through intermolecular disulfide bonds. The most susceptible cysteine residues to oxidation are Cys34, Cys112, and Cys119. Most, but not all, of the activity loss is restored by the reducing agent, dithiothreitol. These findings elucidate a redox mechanism for regulation of PreP and also provide a rational basis for therapeutic intervention in conditions characterized by excessive oxidation of PreP.

show abstract

Regulation of the actin-activated MgATPase activity ofAcanthamoebamyosin II by phosphorylation of serine 639 in motor domain loop 2

Cited by 8 publications

References 52 publications

Kinetic Characterization of the ATPase and Actin-activated ATPase Activities of Acanthamoeba castellanii Myosin-2

Kinetic Characterization of the ATPase and Actin-activated ATPase Activities of Acanthamoeba castellanii Myosin-2

Regulation of the filament structure and assembly ofAcanthamoebamyosin II by phosphorylation of serines in the heavy-chain nonhelical tailpiece

Mechanism of oxidative inactivation of human presequence protease by hydrogen peroxide

Contact Info

Product

Resources

About