Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

---

Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Phosphorylation of a single threonine (myosin IA) or serine (myosins IB and IC) in the heavy chains of the Acanthamoeba myosin I isozymes is required for expression of their actin-activated Mg2(+)-ATPase activities. We now report that the synthetic peptide Gly-Arg-Gly-Arg-Ser-Ser-Val-Tyr-Ser, which corresponds to the phosphorylated region of Acanthamoeba myosin IC, is a good substrate for myosin I heavy chain kinase: Km = 54 microM, and Vmax = 15 mumols/min.mg. The same serine is phosphorylated as in the native substrate (residue 6 in the above sequence), and kinase activity with the synthetic peptide as substrate is also stimulated by phosphatidylserine-enhanced autophosphorylation of the kinase. These results indicate that all of the essential sequence determinants of kinase specificity are contained within this 9-residue peptide. With the peptide as substrate, we found that another acidic phospholipid, phosphatidylinositol, also enhances autophosphorylation of the kinase whereas the neutral phospholipids phosphatidylcholine and phosphatidylethanolamine do not. By comparing the Km and Vmax values for a series of synthetic peptide substrates, we established that 1 basic amino acid is essential on the NH2-terminal side of the phosphorylation site, and two are preferable, and that a tyrosine is essential 2 residues away on the COOH-terminal side. There is a slight preference for arginines over lysines. All of these local sequence specificity determinants are present in the three native substrates, Acanthamoeba myosins IA, IB, and IC, and in two Dictyostelium myosin I isozymes that are putative substrates for the kinase. Similar sequences do not occur in the myosins I from intestinal brush border, which is not a substrate for the Acanthamoeba kinase.
...
PMID:Substrate specificity of Acanthamoeba myosin I heavy chain kinase as determined with synthetic peptides. 216 81

A third isoform of myosin I has been isolated from Acanthamoeba and designated myosin IC. Peptide maps and immunoassays indicate that myosin IC is not a modified form of myosin IA, IB, or II. However, myosin IC has most of the distinctive properties of a myosin I. It is a globular protein of native Mr approximately 162,000, apparently composed of a single 130-kDa heavy chain and a pair of 14-kDa light chains. It is soluble in MgATP at low ionic strength, conditions favoring filament assembly by myosin II. Myosin IC has high Ca2+- and (K+,EDTA)-ATPase activities. Its low Mg2+-ATPase activity is stimulated to a maximum rate of 20 s-1 by the addition of F-actin if its heavy chain has been phosphorylated by myosin I heavy chain kinase. The dependence of the Mg2+-ATPase activity of myosin IC on F-actin concentration is triphasic; and, at fixed concentrations of F-action, this activity increases cooperatively as the concentration of myosin IC is increased. These unusual kinetics were first demonstrated for myosins IA and IB and shown to be due to the presence of two actin-binding sites on each heavy chain which enable those myosins I to cross-link actin filaments. Myosin IC is also capable of cross-linking F-actin, which, together with the kinetics of its actin-activated Mg2+-ATPase activity, suggests that it, like myosins IA and IB, possesses two independent actin-binding domains.
...
PMID:Purification and characterization of a third isoform of myosin I from Acanthamoeba castellanii. 253 Feb 29

The motor function of vertebrate unconventional myosins is not well understood. In this study, we initiated the baculovirus expression system to characterize a novel myosin I from bovine adrenal gland that we had previously cloned [Zhu, T., & Ikebe, M. (1994) FEBS Lett. 339, 31-36], which is classified as myosin I beta. The expressed myosin I beta was well extracted when calmodulin was coexpressed in Sf9 cells. The recombinant myosin I beta cosedimented with actin in an ATP dependent manner. The purified myosin I beta was composed of one heavy chain and three calmodulins. The electron microscopic image of myosin I beta confirmed its single-headed structure with a short tail, which is similar to that of brush border myosin I (BBMI). Myosin I beta showed high K+,EDTA--ATPase activity (approximately 0.14 mumol/min/mg) and Ca(2+)-ATPase activity (approximately 0.32 mumol/min/mg), and the KCl/pH dependence of these activities was different from that of conventional myosin. Mg(2+)-ATPase activity of myosin I beta alone was increased above pCa 6, while the actin dependent activity was not affected by Ca2+. Actin sliding velocity of myosin I beta in the absence of Ca2+ was 0.3-0.5 microns/s at 25 degrees C, which is much greater than that of BBMI (< 0.05 microns/s). The actin sliding activity was abolished above pCa 6, and the sliding activity was restored when exogenous calmodulin was added in the absence of Ca2+. Within similar Ca2+ concentrations, one of the three calmodulins was dissociated from myosin I beta. The results suggest that Ca2+ dependent association of calmodulin may function as a regulatory mechanism of myosin I beta motor activity and that the motor activity of mammalian myosin I is largely different among distinct myosin I isoforms.
...
PMID:Functional expression of mammalian myosin I beta: analysis of its motor activity. 855 22

Deflection of the mechanically sensitive hair bundle atop a hair cell opens transduction channels, some of which subsequently reclose during a Ca2+-dependent adaptation process. Myosin I in the hair bundle is thought to mediate this adaptation; in the bullfrog's hair cell, the relevant isozyme may be the 119-kDa amphibian myosin I beta. Because this molecule resembles other forms of myosin I, we hypothesized that calmodulin, a cytoplasmic receptor for Ca2+, regulates the ATPase activity of myosin. We identified an approximately 120-kDa calmodulin-binding protein that shares with hair-bundle myosin I the properties of being photolabeled by vanadate-trapped uridine nucleotides and immunoreactive with a monoclonal antibody raised against mammalian myosin I beta. To investigate the possibility that calmodulin mediates Ca2+-dependent adaptation, we inhibited calmodulin action and measured the results with two distinct assays. Calmodulin antagonists increased photolabeling of hair-bundle myosin I by nucleotides. In addition, when introduced into hair cells through recording electrodes, calmodulin antagonists abolished adaptation to sustained mechanical stimuli. Our evidence indicates that calmodulin binds to and controls the activity of hair-bundle myosin I, the putative adaptation motor.
...
PMID:Calmodulin controls adaptation of mechanoelectrical transduction by hair cells of the bullfrog's sacculus. 870 Sep 9

The actin-activated ATPase activity of Acanthamoeba myosin IC is stimulated 15- to 20-fold by phosphorylation of Ser-329 in the heavy chain. In most myosins, either glutamate or aspartate occupies this position, which lies within a surface loop that forms part of the actomyosin interface. To investigate the apparent need for a negative charge at this site, we mutated Ser-329 to alanine, asparagine, aspartate, or glutamate and coexpressed the Flag-tagged wild-type or mutant heavy chain and light chain in baculovirus-infected insect cells. Recombinant wild-type myosin IC was indistinguishable from myosin IC purified from Acanthamoeba as determined by (i) the dependence of its actin-activated ATPase activity on heavy-chain phosphorylation, (ii) the unusual triphasic dependence of its ATPase activity on the concentration of F-actin, (iii) its Km for ATP, and (iv) its ability to translocate actin filaments. The Ala and Asn mutants had the same low actin-activated ATPase activity as unphosphorylated wild-type myosin IC. The Glu mutant, like the phosphorylated wild-type protein, was 16-fold more active than unphosphorylated wild type, and the Asp mutant was 8-fold more active. The wild-type and mutant proteins had the same Km for ATP. Unphosphorylated wild-type protein and the Ala and Asn mutants were unable to translocate actin filaments, whereas the Glu mutant translocated filaments at the same velocity, and the Asp mutant at 50% the velocity, as phosphorylated wild-type proteins. These results demonstrate that an acidic amino acid can supply the negative charge in the surface loop required for the actin-dependent activities of Acanthamoeba myosin IC in vitro and indicate that the length of the side chain that delivers this charge is important.
...
PMID:Analysis of the regulatory phosphorylation site in Acanthamoeba myosin IC by using site-directed mutagenesis. 986 Sep 46

Acanthamoeba myosin IC (AMIC) is a single-headed myosin comprised of one heavy chain (129 kDa) and one light chain (17 kDa). The heavy chain has head, neck (light chain-binding), and tail domains. The tail consists of four subdomains: a basic region (BR) (23 kDa) and two Gly/Pro/Ala-rich (GPA) regions, GPA1 (6 kDa) and GPA2 (15 kDa), flanking an Src homology 3 region (6 kDa). Although the AMIC head is similar in sequence, structure, and function (ATPase motor) to other myosin heads, the organization of the tail has been less clear as has its function beyond an assumed role in binding interaction partners, e.g., the BR has a membrane affinity and the GPA components bind F-actin in an ATP-independent manner. To investigate the spatial arrangement of subdomains in the tail, we have used cryo-electron microscopy and image reconstruction to compare actin filaments decorated with WT AMIC and tail-truncated mutants of various lengths. The BR forms an oval-shaped feature, approximately 40 A long, that diverges obliquely from the head, extending azimuthally around the actin filament and toward its barbed end. GPA2 and GPA1 are located together on the inner (actin-proximal) side of the tail, close enough to act in concert in binding the same or another actin filament. The outer face of the BR is strategically exposed for membrane or vesicle binding.
...
PMID:Subdomain organization of the Acanthamoeba myosin IC tail from cryo-electron microscopy. 1530 34

Class I myosins have a single heavy chain comprising an N-terminal motor domain with actin-activated ATPase activity and a C-terminal globular tail with a basic region that binds to acidic phospholipids. These myosins contribute to the formation of actin-rich protrusions such as pseudopodia, but regulation of the dynamic localization to these structures is not understood. Previously, we found that Acanthamoeba myosin IC binds to acidic phospholipids in vitro through a short sequence of basic and hydrophobic amino acids, BH site, based on the charge density of the phospholipids. The tail of Dictyostelium myosin IB (DMIB) also contains a BH site. We now report that the BH site is essential for DMIB binding to the plasma membrane and describe the molecular basis of the dynamic relocalization of DMIB in live cells. Endogenous DMIB is localized uniformly on the plasma membrane of resting cells, at active protrusions and cell-cell contacts of randomly moving cells, and at the front of motile polarized cells. The BH site is required for association of DMIB with the plasma membrane at all stages where it colocalizes with phosphoinositide bisphosphate/phosphoinositide trisphosphate (PIP(2)/PIP(3)). The charge-based specificity of the BH site allows for in vivo specificity of DMIB for PIP(2)/PIP(3) similar to the PH domain-based specificity of other class I myosins. However, DMIB-head is required for relocalization of DMIB to the front of migrating cells. Motor activity is not essential, but the actin binding site in the head is important. Thus, dynamic relocalization of DMIB is determined principally by the local PIP(2)/PIP(3) concentration in the plasma membrane and cytoplasmic F-actin.
...
PMID:Molecular basis of dynamic relocalization of Dictyostelium myosin IB. 2236 11

---