Inhibition of Acanthamoeba myosin I heavy chain kinase by Ca(2+)-calmodulin (original) (raw)
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Journal of Biological Chemistry
The three isoforms of Acanthamoeba myosin I (nonfilamentous myosin with only a single heavy chain) express actin-activated M@+-ATPase activity only when phosphorylated at a single site by myosin I heavy chain kinase. The kinase is activated by autophosphorylation that is greatly stimulated by acidic phospholipids. Substantial fractions of the three myosins I and the kinase are associated in s i t u with membranes, and all four enzymes bind to purified membranes in vitro. W e now report that when kinase and myosin I are incubated together with phosphatidylserine vesicles not only does the kinase autophosphorylate more rapidly than soluble kinase in the absence of phosphatidylserine but that, probably as a result, the kinase phosphorylates myosin I more rapidly than soluble kinase phosphorylates soluble myosin I. Similarly, plasma membrane-bound kinase phosphorylates membrane-bound myosin I and activates its actin-activated Mg2"ATPase activity more rapidly than soluble kinase phosphorylates and activates soluble myosin I in the absence of membranes. However, the enhanced activity of membrane-bound kinase (which is comparable to the activity of kinase in the presence of phosphatidylserine) is not due to autophosphorylation of the membrane-bound kinase, which is very much slower than for kinase activated by phosphatidylserine vesicles.
The Journal of Cell Biology, 1991
The actin-activated Mg"-ATPase activities of Acanthamoeba myosins I are known to be maximally expressed only when a single threonine (myosin IA) or serine (myosins IB and IC) is phosphorylated by myosin I heavy chain kinase. The purified kinase is highly activated by autophosphorylation and the rate of autophosphorylation is greatly enhanced by the presence of acidic phospholipids . In this paper, we show by immunofluorescence and immunoelectron microscopy of permeabilized cells that myosin I heavy chain kinase is highly concentrated, but not exclusively, at the plasma membrane. Judged by their electrophoretic mobilities, kinase associated with purified plasma membranes may differ from the cytoplasmic kinase, possibly in the extent of its phosphorylation . Purified kinase binds to
Journal of Biological Chemistry, 1996
Acanthamoeba myosin I heavy chain (MIHC) kinase is a monomeric 97-kDa protein that is activated by binding to acidic phospholipids or by autophosphorylation. Activation by phospholipids is inhibited by Ca 2؉ -calmodulin. In the accompanying paper (Brzeska, H., Martin, B., and Korn, E. D. (1996) J. Biol. Chem. 271, 27049 -27055), we identified the catalytic domain as the COOH-terminal 35 kDa produced by trypsin digestion of phosphorylated MIHC kinase. In this paper, we report the cloning and sequencing of the corresponding cDNA and expression of fully active catalytic domain. The expressed catalytic domain has substrate specificity similar to that of native kinase and resistance to trypsin similar to that of fully phosphorylated MIHC kinase. MIHC kinase catalytic domain has only 25% sequence identity to the catalytic domain of protein kinase A and similarly low sequence identity to the catalytic domains of protein kinase C-and calmodulin-dependent kinases, but 50% sequence identity and 70% similarity to the p21-activated kinase (PAK) and STE20 family of kinases. This suggests that MIHC kinase is (at least) evolutionarily related to the PAK family, whose activities are regulated by small GTP-binding proteins. The homology includes the presence of a potential MIHC kinase autophosphorylation site as well as conserved Tyr and Ser/Thr residues in the region corresponding to the P؉1 loop of protein kinase A. A synthetic peptide corresponding to this region of MIHC kinase is phosphorylated by both the expressed catalytic domain and native MIHC kinase.
Purification and characterization of a third isoform of myosin I from Acanthamoeba castellanii
Journal of Biological Chemistry
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 11. However, myosin IC has most of the distinctive properties of a myosin I. It is a globular protein of native M, -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 11. Myosin IC has high Ca2+-and (K+,EDTA)-ATPase activities. Its low Mg2+-ATPase activity is stimulated to a maximum rate of 20 s-l 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-actin, 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 Factin, which, together with the kinetics of its actinactivated Mg2+-ATPase activity, suggests that it, like myosins IA and IB, possesses two independent actinbinding domains.
Archives of Biochemistry and Biophysics, 2000
Acanthamoeba myosin II is regulated in an unique way by phosphorylation of three serine residues located within nonhelical tailpiece of the rod domain. Phosphorylation inhibits functions associated with the NH 2-terminal motor domain, i.e., actin-activated activity and ability to move actin filaments. Number of data indicate functional communication between these distant domains. In this work, effect of modification of arginine residues with phenylglyoxal on the Ca 2؉-ATPase activity and susceptibility to endoproteinase ArgC cleavage of monomeric phospho-and dephosphomyosin II has been investigated. Upon the phenylglyoxal treatment the activity of dephosphomyosin II was decreasing faster that the activity of phosphomyosin. The modification also affected the proteolytic fragmentation of phospho-and dephosphomyosin II: the cleavage of heavy chain was further inhibited for phosphomyosin and enhanced for dephosphomyosin with a concomitant exposure of an additional cleavage site within the head domain. No difference in the quantity of modified arginines was observed. These results indicate a difference between the conformation of active sites of phospho-and dephosphomyosin II.
Proceedings of the National Academy of Sciences, 1998
The three single-headed monomeric myosin I isozymes of Acanthamoeba castellanii (AMIs)—AMIA, AMIB, and AMIC—are among the best-studied of all myosins. We have used AMIC to study structural correlates of myosin’s actin-activated ATPase. This activity is normally controlled by phosphorylation of Ser-329, but AMIC may be switched into constitutively active or inactive states by substituting this residue with Glu or Ala, respectively. To determine whether activation status is reflected in structural differences in the mode of attachment of myosin to actin, these mutant myosins were bound to actin filaments in the absence of nucleotide (rigor state) and visualized at 24-Å resolution by using cryoelectron microscopy and image reconstruction. No such difference was observed. Consequently, we suggest that regulation may be affected not by altering the static (time-averaged) structure of AMIC but by modulating its dynamic properties, i.e., molecular breathing. The tail domain of vertebrate i...
Proceedings of the National Academy of Sciences, 1998
Phosphorylation of Ser-627 is both necessary and sufficient for full activity of the expressed 35-kDa catalytic domain of myosin I heavy chain kinase (MIHCK). Ser-627 lies in the variable loop between highly conserved residues DFG and APE at a position at which a phosphorylated Ser͞Thr also occurs in many other Ser͞Thr protein kinases. The variable loop of MIHCK contains two other hydroxyamino acids: Thr-631, which is conserved in almost all Ser͞Thr kinases, and Thr-632, which is not conserved. We determined the effects on the kinase activity of the expressed catalytic domain of mutating Ser-627, Thr-631, and Thr-632 individually to Ala, Asp, and Glu. The S627A mutant was substantially less active than wild type (wt), with a lower k cat and higher K m for both peptide substrate and ATP, but was more active than unphosphorylated wt. The S627D and S627E mutants were also less active than phosphorylated wt, i.e., acidic amino acids cannot substitute for phospho-Ser-627. The activity of the T631A mutant was as low as that of the S627A mutant, whereas the T632A mutant was as active as phosphorylated wt, indicating that highly conserved Thr-631, although not phosphorylated, is essential for catalytic activity. Asp and Glu substitutions for Thr-631 and Thr-632 were inhibitory to various degrees. Molecular modeling indicated that Thr-631 can hydrogen bond with conserved residue Asp-591 in the catalytic loop and that similar interactions are possible for other kinases whose activities also are regulated by phosphorylation in the variable loop. Thus, this conserved Thr residue may be essential for the activities of other Ser͞Thr protein kinases as well as for the activity of MIHCK. Acanthamoeba myosin I heavy chain kinase (MIHCK) activates amoeba myosin I molecules by phosphorylating a single Ser or Thr residue within the subfragment 1 portion of their single heavy chains (refs. 1 and 2; for review, see ref. 3). Acanthamoeba MIHCK also phosphorylates the light chain of, and activates, smooth muscle myosin II (3, 4). MIHCK is a member of the p21-activated protein kinase (PAK) family (5). All PAKs have highly homologous catalytic domains (6-8) and most, including MIHCK (9), have been shown to be activated by the small GTP-binding proteins Rac and Cdc42. MIHCK and typical mammalian and yeast Paks have similar substrate specificities in vitro including the ability to phosphorylate and activate Acanthamoeba and Dictyostelium myosin I molecules (10, 11). MIHCK is a 79.3-kDa protein with a 35-kDa C-terminal catalytic domain (5, 12). The activity of native MIHCK is enhanced ϳ50-fold by phospholipid (and plasma membrane)-MATERIALS AND METHODS DNA Preparation. PCR (Pfu polymerase, Perkin-Elmer) were used to prepare the DNA coding for each of the mutants of the MIHCK catalytic domain, as described for the wild-type (wt) catalytic domain (5). The internal primer for the Ser mutants consisted of bases 514-543 and the primer for both Thr mutants was bases 527-556, with appropriate changes. The The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ''advertisement'' in accordance with 18 U.S.C. §1734 solely to indicate this fact.