A Conserved Negatively Charged Amino Acid Modulates Function in Human Nonmuscle Myosin IIA

Wang, F; Ev, Harvey; Ma, Chi; D, Wei; Sellers,

doi:10.1021/bi000133x

Cited by 65 publications

(69 citation statements)

References 39 publications

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“…In related experiments, substitution of Ala for Asp at the TEDS site of human nonmuscle myosin IIA heavy meromyosin reduced activity to 10% of wild type (32). Although the 1-2% of wild-type activity that we found for the S199T, E428V, and E445K mutants was not observed for the myosin II mutants on which the MYOA mutants were based, this level of activity might have been undetectable because the myosin II wild types have only about 3-10% of the actin-dependent MgATPase activity of wild-type MYOA.…”

Section: Discussionmentioning

confidence: 95%

Myosin I mutants with only 1% of wild-type actin-activated MgATPase activity retain essentialin vivofunction(s)

Liu

Osherov

Yamashita

et al. 2001

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

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The single class I myosin (MYOA) of Aspergillus nidulans is essential for hyphal growth. It is generally assumed that the functions of all myosins depend on their actin-activated MgATPase activity. Here we show that MYOA mutants with no more than 1% of the actin-activated MgATPase activity of wild-type MYOA in vitro and no detectable in vitro motility activity can support fungal cell growth, albeit with a delay in germination time and a reduction in hyphal elongation. From these and other data, we conclude that the essential role(s) of myosin I in A. nidulans is probably structural, requiring little, if any, actin-activated MgATPase or motor activity, which have long been considered the defining characteristics of the myosin family.

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Section: Discussionmentioning

confidence: 95%

Myosin I mutants with only 1% of wild-type actin-activated MgATPase activity retain essentialin vivofunction(s)

Liu

Osherov

Yamashita

et al. 2001

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

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“…The enzymatic properties of a nonmuscle myosin II HMMlike fragment expressed in the baculovirus/Sf9 system have been previously characterized and shown to be an excellent model for the tissue-purified protein (10). In this report, two separate point mutations in the myosin heavy chain (N93K and R702C) corresponding to mutations recently reported in some families with May-Hegglin anomaly and Fechtner syndrome were separately engineered into this background (4).…”

Section: Resultsmentioning

confidence: 99%

“…The in vitro motility of the HMM fragments were measured as described (10,17). The conditions were 80 mM KCl, 5 mM MgCl 2 , 1 mM ATP, 0.1 mM EGTA, 1 mM dithiothreitol, 20 mM MOPS (pH 7.4), 200 nM tropomyosin, 0.7% methylcellulose, 2.5 mg/ml glucose, 0.1 mg/ml glucose oxidase, and 2 mg/ml catalase at 30°C.…”

Section: Construction Of Baculovirus Transfermentioning

confidence: 99%

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Mutations in Human Nonmuscle Myosin IIA Found in Patients with May-Hegglin Anomaly and Fechtner Syndrome Result in Impaired Enzymatic Function

Wang

Sellers

2002

Journal of Biological Chemistry

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A family of autosomal-dominant diseases including May-Hegglin anomaly, Fechtner syndrome, Sebastian syndrome, Alport syndrome, and Epstein syndrome are commonly characterized by giant platelets and thrombocytopenia. In addition, there may be leukocyte inclusions, deafness, cataracts, and nephritis, depending on the syndrome. Mutations in the human nonmuscle myosin IIA heavy chain gene (MYH9) have been linked to these diseases. Two of the recently described mutations, N93K and R702C, are conserved in smooth and nonmuscle myosins from vertebrates and lie in the head domain of myosin. Interestingly, the two mutations lie within close proximity in the three-dimensional structure of myosin. These two mutations were engineered into a heavy meromyosin-like recombinant fragment of nonmuscle myosin IIA, which was expressed in baculovirus along with the appropriate light chains. The R702C mutant displays 25% of the maximal MgATPase activity of wild type heavy meromyosin and moves actin filaments at half the wild type rate. The effects of the N93K mutation are more dramatic. This heavy meromyosin has only 4% of the maximal MgATPase activity of wild type and does not translocate actin filaments in an in vitro motility assay. Biochemical characterization of the mutant is consistent with this mutant being unable to fully adopt the "on" conformation.

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“…The proteins containing only one motor domain were purified from the cell extract by FLAG affinity chromatography followed by actin selection to isolate the proteins containing motor domains. A detailed procedure is described in Wang et al (23).…”

Section: Methodsmentioning

confidence: 99%

Phosphorylation-dependent Regulation Is Absent in a Nonmuscle Heavy Meromyosin Construct with One Complete Head and One Head Lacking the Motor Domain

Cremo¹,

Wang²,

Facemyer³

et al. 2001

Journal of Biological Chemistry

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To understand the domain requirements of phosphorylation-dependent regulation, we prepared three recombinant constructs of nonmuscle heavy meromyosin IIB containing 1) two complete heads, 2) one complete head and one head lacking the motor domain, and 3) one complete head and one head lacking both motor and regulatory domains. Steady-state ATPase measurements showed that phosphorylation did not alter the affinity for actin by more than a factor of 2 for any construct. Phosphorylation increased V max by a factor of 10 for construct 1 and 1.5-3 for construct 2 but had no effect for construct 3. Single turnover measurements, a better measure of slow rates inherent to unphosphorylated regulated myosins, showed that the single-headed construct 2, like construct 3 retains less than 1% of the regulatory properties of the double-headed construct 1 (300-fold activation). Therefore, a complete head cannot The conventional class II myosins are hexameric proteins consisting of two heavy chains and two pairs of light chains. The carboxyl-terminal half of the two heavy chains dimerize via an ␣-helical coiled-coil to form a long rod through which myosin can self-associate to form filaments. The amino-terminal half of the two heavy chains forms the two separate motor (catalytic) domains, which bind actin and hydrolyze ATP. Each is connected to the rod via a neck region that contains the binding site for the two light chains. The class of light chains that binds nearest to the motor domain is termed the essential light chain (ELC) 1 whereas the class that binds nearest to the coiled-coil rod is termed the regulatory light chain (RLC). One ELC and one RLC together with the portion of the heavy chain to which they bind are commonly referred to as the "regulatory domain." The motor domain and the regulatory domain together form the soluble myosin "head" termed subfragment-1 (S1). Truncation of the coiled-coil rod of myosin produces a soluble two-headed structure termed heavy meromyosin (HMM).The actin-activated MgATPase activity and motor properties of smooth muscle and nonmuscle myosins are regulated by phosphorylation of the RLC (1-3). The unphosphorylated forms of these regulated myosins have low ATPase activity and are unable to move actin filaments, whereas the phosphorylated forms are activated in both respects. S1 is active in both unphosphorylated and phosphorylated states (4 -6), whereas HMM is well regulated by phosphorylation (4, 7). Singleheaded myosin, which lacks the motor domain and regulatory domain of one head, is not regulated by phosphorylation (8, 9). Similarly, recombinantly expressed fragments that were sufficiently truncated from the carboxyl terminus to destabilize the coiled-coil, and thus the two-headed structure, were also not regulated by phosphorylation (10 -12). These data suggest that the presence of two heads is critical for down-regulation, suggesting that head-head interaction is an important feature of the unphosphorylated state. However, these studies do not elucidate which domain interactions ar...

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A Conserved Negatively Charged Amino Acid Modulates Function in Human Nonmuscle Myosin IIA

Cited by 65 publications

References 39 publications

Myosin I mutants with only 1% of wild-type actin-activated MgATPase activity retain essentialin vivofunction(s)

Myosin I mutants with only 1% of wild-type actin-activated MgATPase activity retain essentialin vivofunction(s)

Mutations in Human Nonmuscle Myosin IIA Found in Patients with May-Hegglin Anomaly and Fechtner Syndrome Result in Impaired Enzymatic Function

Phosphorylation-dependent Regulation Is Absent in a Nonmuscle Heavy Meromyosin Construct with One Complete Head and One Head Lacking the Motor Domain

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