Hanna Brzeska - Academia.edu (original) (raw)

Papers by Hanna Brzeska

Research paper thumbnail of Effect of Mutating the Regulatory Phosphoserine and Conserved Threonine Onthe Activity of the Expressed Catalytic Domain of Acanthamoeba Myosin I Hea Vy Chain Kinase

Proceedings of the National Academy of Sciences of the United States of America, 1998

Phosphorylation of Ser-627 is both necessary and sufficient for full activity of the expressed 35... more 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.

Research paper thumbnail of Properties of Acanthamoeba Myosin I Heavy Chain Kinase Bound to Phospholipid Vesicles

J Biol Chem, 1995

The actin-activated Mg(2+)-ATPase and in vitro motility activities of the three Acanthamoeba myos... more The actin-activated Mg(2+)-ATPase and in vitro motility activities of the three Acanthamoeba myosin I isozymes depend upon phosphorylation of their single heavy chains by myosin I heavy chain kinase. Previously, the kinase had been shown to be activated by autophosphorylation, which is enhanced by acidic phospholipids, or simply by binding to purified plasma membranes in the absence of significant autophosphorylation. In this paper, we show that the rate of phosphorylation of myosin I by unphosphorylated kinase is approximately 20-fold faster when both the myosin I and the kinase are bound to acidic phospholipid vesicles than when both are soluble. This activation is not due to an increase in the local concentrations of vesicle-bound kinase and myosin I. Thus, acidic phospholipids, like membranes, can activate myosin I heavy chain kinase in the absence of significant autophosphorylation, i.e. membrane proteins are not required. Kinetic studies show that both binding of kinase to phospholipid vesicles and autophosphorylation of kinase in the absence of phospholipid increase the Vmax relative to soluble, unphosphorylated kinase with either an increase in the apparent Km (when myosin I is the substrate) or no significant change in Km (when a synthetic peptide is the substrate). Kinetic data showed that autophosphorylation of phospholipid-bound kinase is both intermolecular and intervesicular, and that phosphorylation of phospholipid-bound myosin I by phospholipid-bound kinase is also intervesicular even when the kinase and myosin are bound to the same vesicles. The relevance of these results to the activation of myosin I heavy chain kinase and phosphorylation of myosin I isozymes in situ are discussed.

Research paper thumbnail of Selective Localization of Myosin-I Proteins in Macropinosomes and Actin Waves

Cytoskeleton, 2016

Class I myosins are widely expressed with roles in endocytosis and cell migration in a variety of... more Class I myosins are widely expressed with roles in endocytosis and cell migration in a variety of cell types. Dictyostelium express multiple myosin Is, including three short-tailed (Myo1A, Myo1E, Myo1F) and three long-tailed (Myo1B, Myo1C, Myo1D). Here we report the molecular basis of the specific localizations of short-tailed Myo1A, Myo1E and Myo1F compared to our previously determined localization of long-tailed Myo1B. Myo1A and Myo1B have common and unique localizations consistent with the various features of their tail region; specifically the BH sites in their tails are required for their association with the plasma membrane and heads are sufficient for relocalization to the front of polarized cells. Myo1A does not localize to actin waves and macropinocytic protrusions, in agreement with the absence of a tail region which is required for these localizations of Myo1B. However, in spite of the overall similarity of their domain structures, the cellular distributions of Myo1E and Myo1F are quite different from Myo1A. Myo1E and Myo1F, but not Myo1A, are associated with macropinocytic cups and actin waves. The localizations of Myo1E and Myo1F in macropinocytic structures and actin waves differ from the localization of Myo1B. Myo1B colocalizes with F-actin in the actin waves and at the tips of mature macropinocytic cups whereas Myo1E and Myo1F are in the interior of actin waves and along the entire surface of macropinocytic cups. Our results point to different mechanisms of targeting of short- and long-tailed myosin Is, and are consistent with these myosins having both shared and divergent cellular functions. This article is protected by copyright. All rights reserved.

Research paper thumbnail of Cardiac troponin-C: a rapid and effective method of purification

Febs Letters, Feb 1, 1985

A new purification procedure for cardiac troponin-C is described which has several advantages ove... more A new purification procedure for cardiac troponin-C is described which has several advantages over previous methods. High purity of the final product was assessed by electrophoretic, enzymatic and spectroscopic methods.

Research paper thumbnail of Calmodulin-binding and Autoinhibitory Domains of Acanthamoeba Myosin I Heavy Chain Kinase, a p21-activated Kinase (PAK)

J Biol Chem, 2001

The sequence homology between Acanthamoeba myosin I heavy chain kinase (MIHCK) and other p21-acti... more The sequence homology between Acanthamoeba myosin I heavy chain kinase (MIHCK) and other p21-activated kinases (PAKs) is relatively low, including only the catalytic domain and a short PAK N-terminal motif (PAN), and even these regions are not highly homologous. In this paper, we report the expression in insect cells of full-length, fully regulated Acanthamoeba MI-HCK and further characterize the regulation of this PAK by Rac, calmodulin, and autoinhibition. We map the autoinhibitory region of MIHCK to its PAN region and show that the PAN region inhibits autophosphorylation and kinase activity of unphosphorylated fulllength MIHCK and its expressed catalytic domain but has very little effect on either when they are phosphorylated. These properties are similar to those reported for mammalian PAK1. Unlike PAK1, MIHCK is activated by Rac only in the presence of phospholipid. However, peptides containing the PAN region of MIHCK bind Rac in the absence of lipid, and Rac binding reverses the inhibition of the MIHCK catalytic domain by PAN peptides. Our data suggest that a region N-terminal to PAN is required for optimal binding of Rac. Also unlike mammalian PAK, phospholipid stimulation of Acanthamoeba MIHCK and Dictyostelium MIHCK) (which is also a PAK) is inhibited by Ca 2؉ -calmodulin. In contrast to Dictyostelium MIHCK, however, Ca 2؉ -calmodulin also inhibits Rac-induced activity of Acanthamoeba MIHCK. The basic region N-terminal to PAN is essential for calmodulin binding.

Research paper thumbnail of Studies on calmodulin structure using its tryptic fragments. In: Calcium-Binding Proteins: Structure and Function

Research paper thumbnail of Inhibition of Acanthamoeba myosin I heavy chain kinase by Ca(2+)-calmodulin

Journal of Biological Chemistry

vesicles and purified plasma membranes. Proteolytic removal of a 7-kDa NHz-terminal segment from ... more vesicles and purified plasma membranes. Proteolytic removal of a 7-kDa NHz-terminal segment from the 97-kDa kinase prevents binding of both calmodulin and phospholipid; therefore, we propose that they bind to the same or overlapping sites. These data provide a mechanism by which Ca2+ could inhibit the actin-activated Mg2+-ATPase activity of the myosin I isozymes in vivo and thus regulate myosin I-dependent motile activities.

Research paper thumbnail of The effect of actin and phosphorylation on the tryptic cleavage pattern of Acanthamoeba myosin IA

Journal of Biological Chemistry

The Mg2+-ATPase activity of Acanthamoeba myosin IA is activated by F-actin only when the myosin h... more The Mg2+-ATPase activity of Acanthamoeba myosin IA is activated by F-actin only when the myosin heavy chain is phosphorylated at a single residue. In order to gain insight into the conformational changes that may be responsible for the effects of F-actin and phosphorylation on myosin I ATPase, we have studied their effects on the proteolysis of the myosin IA heavy chain by trypsin. Trypsin initially cleaves the unphosphorylated, 140-kDa heavy chain of Acanthamoeba myosin IA at sites 38 and 112 kDa from its NH2 terminus and secondarily at sites 64 and 91 kDa from the NH2 terminus. F-actin has no effect on tryptic cleavage at the 91- and 112-kDa sites, but does protect the 38-kDa site and the 64-kDa site. Phosphorylation (which occurs very near the 38-kDa site) has no detectable effect on the tryptic cleavage pattern in the absence of F-actin or on F-actin protection of the 64-kDa site, but significantly enhances F-actin protection of the 38-kDa site. Protection of the 64-kDa site is probably due to direct steric blocking because F-actin binds to this region of the heavy chain. The protection of the 38-kDa site by F-actin may be the result of conformational changes in this region of the heavy chain induced by F-actin binding near the 64-kDa site and by phosphorylation. The conformational changes in the heavy chain of myosin IA that are detected by alterations in its susceptibility to proteolysis are likely to be related to the conformational changes that are involved in the phosphorylation-regulated actin-activated Mg2+-ATPase activities of Acanthamoeba myosins IA and IB.

Research paper thumbnail of Substrate specificity of Acanthamoeba myosin I heavy chain kinase as determined with synthetic peptides

Journal of Biological Chemistry

Research paper thumbnail of 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... more 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.

Research paper thumbnail of Acanthamoeva myosin I heavy chain kinase is activated by phosphatidylserine-enhanced phosphorylation

Journal of Biological Chemistry

ABSTRACT

Research paper thumbnail of Autophosphorylation-independent activation of Acanthamoeba myosin I heavy chain kinase by plasma membranes

Journal of Biological Chemistry

The three isoforms of Acanthamoeba myosin I (nonfilamentous myosin with only a single heavy chain... more 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.

Research paper thumbnail of Immunolocalization of myosin I heavy chain kinase in Acanthamoeba castellanii and binding of purified kinase to isolated plasma membranes

The Journal of Cell Biology

The actin-activated Mg"-ATPase activities of Acanthamoeba myosins I are known to be maximally exp... more 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

Research paper thumbnail of The localization and sequence of the phosphorylation sites of Acanthamoeba myosins I

Journal of Biological Chemistry

The actin-activated Mg2+-ATPase activities of Acanthamoeba myosins IA, IB, and IC are expressed o... more The actin-activated Mg2+-ATPase activities of Acanthamoeba myosins IA, IB, and IC are expressed only when a single site in their heavy chains is phosphorylated by a myosin I heavy chain-specific kinase. We show that phosphorylation occurs at Ser-315 in the myosin IB heavy chain, Ser-311 in myosin IC, and a threonine residue at a corresponding position in myosin IA whose amino acid sequence is as yet unknown. The most obvious feature common to the three substrates is a basic amino acid(s) 2 or 3 residues before the site of phosphorylation. The phosphorylation site is located between the ATP- and actin-binding sites, which corresponds to the middle of the 50-kDa domain of skeletal muscle myosin subfragment 1. The sequence similarity between the region surrounding the phosphorylation site of myosin I and subfragment 1 is much lower than the average sequence similarity between myosin I and subfragment 1. This is consistent with the hypothesis that the conformation of this region of myosin I differs from that of the corresponding region in skeletal muscle myosin and that phosphorylation converts the conformation of the actomyosin I complex into a conformation comparable to that present in actosubfragment 1 without phosphorylation. The protein sequences obtained in the course of this work led to the conclusion that the myosin I genes previously identified as myosin IB and IL (myosin-like) heavy chains actually are the myosin IC and IB heavy chains, respectively. Finally, we report a modification of the method for monitoring the appearance of 32Pi during sequencing of 32P-labeled peptides that results in almost complete recovery of the radioactivity, thus allowing unequivocal assignment of the position of the phosphorylated residue.

Research paper thumbnail of Preparation of a phospholipid-insensitive, autophosphorylation-activated catalytic fragment of Acanthamoeba myosin I heavy chain kinase

Journal of Biological Chemistry

The actin-activated Mg(2+)-ATPase activity of Acanthamoeba myosin I depends on phosphorylation of... more The actin-activated Mg(2+)-ATPase activity of Acanthamoeba myosin I depends on phosphorylation of its single heavy chain. The activity of the myosin I heavy chain kinase is increased about 50-fold by autophosphorylation, and the rate of kinase autophosphorylation is enhanced about 20-fold by acidic phospholipids independent of the presence of Ca2+ (Brzeska, H., Lynch, T. J., and Korn, E. D. (1990) J. Biol. Chem. 265, 3591-3594). In this paper, we show that chymotryptic digestion of the kinase produces a 54-kDa fragment which contains three to four of the approximately 11 original phosphorylation sites and whose activity is greatly stimulated by autophosphorylation. However, both the rate of autophosphorylation and the kinase activity of the 54-kDa fragment are independent of phospholipid and comparable to those of intact kinase in the presence of phospholipid. These data imply that the (probably NH2-terminal) region(s) removed by proteolysis is necessary for phospholipid-sensitive inhibition of autophosphorylation of sites residing within the (probably COOH-terminal) 54-kDa fragment. The 54-kDa fragment contains the catalytic site of the kinase as well as three to four sites whose phosphorylation is necessary for full expression of kinase activity. The middle region of the kinase molecule contains proline-rich regions that are similar to the COOH-terminal tail of the kinase substrate, Acanthamoeba myosin I.

Research paper thumbnail of Differential localization of Acanthamoeba myosin I isoforms

The Journal of Cell Biology

Antibody titers were quantified by a solid phase antibody capture immunoassay after the protocol ... more Antibody titers were quantified by a solid phase antibody capture immunoassay after the protocol of . Purified myosin I was bound to PVC in a 96-well microtiter plate (Falcon Plastics, Cockeysville, MD), antibody was added in a dilution series, and the captured antibody was quantified by binding of a second HRP-coupled anti-irnmunoglobulin antibody with 3',Y,5',5"tetramethylbertzidine as substrate.

Research paper thumbnail of Localization of the actin-binding sites of Acanthamoeba myosin IB and effect of limited proteolysis on its actin-activated Mg2+-ATPase activity

Journal of Biological Chemistry

Research paper thumbnail of Rac-induced increase of phosphorylation of myosin regulatory light chain in HeLa cells

Cell Motility and the Cytoskeleton, 2004

The pathways by which activation of the small GTP-binding protein Rac causes cytoskeletal changes... more The pathways by which activation of the small GTP-binding protein Rac causes cytoskeletal changes are not fully understood but are likely to involve both assembly of new actin filaments and reorganization of actin filaments driven by the actin-dependent ATPase activity of myosin II. Here we show that expression of active RacQ61 in growing HeLa cells, in addition to inducing ruffling, substantially enhances the level of phosphorylation of serine-19 of the myosin II regulatory light chain (MLC), which would increase actomyosin II ATPase and motor activities. Phosphorylated myosin was localized to RacQ61-induced ruffles and stress fibers. RacQ61-induced phosphorylation of MLC was reduced by a maximum of about 38% by an inhibitor (Tat-PAK) of p21-activated kinase (PAK), about 35% by an inhibitor (Y-27632) of Rho kinase, 51% by Tat-PAK plus Y-27632, and 10% by an inhibitor (ML7) of myosin light chain kinase. Staurosporine, a non-specific inhibitor of serine/threonine kinases, reduced RacQ61-induced phosphorylation of MLC by about 58%, at the maximum concentration that did not kill cells. Since Rac activates PAK and PAK can phosphorylate MLC, these data strongly suggest that PAK is responsible for a significant fraction of RacQ61-induced MLC phosphorylation. To our knowledge, this is the first evidence that active Rac causes phosphorylation of MLC in cells, thus implicating activation of the ATPase activity of actomyosin II as one of the ways by which Rac may induce cytoskeletal changes.

Research paper thumbnail of Activation of myosin in HeLa cells causes redistribution of focal adhesions and F-actin from cell center to cell periphery

Cell Motility and the Cytoskeleton, 2006

Activation of actomyosin II by phosphorylation of its regulatory light chain is one of the main f... more Activation of actomyosin II by phosphorylation of its regulatory light chain is one of the main factors involved in the regulation of cytoskeletal dynamics. Phosphorylation of myosin regulatory light chain may be mediated directly and indirectly by several kinases including myosin light chain kinase (MLCK) and kinases activated by small GTP-binding proteins. Most of the myosin kinases, including PAK, can also interact with other proteins through binding sites located outside of their catalytic domains. In an attempt to study the effects due only to phosphorylation of myosin light chain, we expressed the constitutively active catalytic domain of ameba PAK in HeLa cells. The catalytic domain phosphorylates myosin light chain in vitro with high specific activity but has none of the sequences that target mammalian PAK to other proteins and membranes. Expression of the catalytic domain caused disassembly of focal adhesions and stress fibers in the cell center and accumulation of focal adhesions and F-actin at the cell periphery. There was a twofold increase in the phosphorylation level of endogenous myosin light chain and changes in cell shape consistent with enhanced cell contractility. The phenotype was independent of MLCK, ROCK, MEK, Rac, and Rho activities but was abolished by blebbistatin, a specific inhibitor of myosin II activity. Our data are consistent with myosin being directly phosphorylated by the expressed catalytic domain of ameba PAK with the induced phenotype resulting from cell retraction driven by contraction of peripheral actomyosin. The phenotype induced by expression of the catalytic domain is reminiscent of that caused by expression of active mammalian PAK, suggesting that myosin phosphorylation may play an important role in PAK-induced cytoskeletal changes. The catalytic domain of ameba PAK may be a useful tool for studying the effects of myosin light chain phosphorylation in other cells. Cell Motil.

Research paper thumbnail of Characterization of a calmodulin antiserum by its reactions with fragments of the calmodulin molecule

Journal of immunoassay, 1986

A high affinity antibody, specific to the calcium-free form of calmodulin, which had previously b... more A high affinity antibody, specific to the calcium-free form of calmodulin, which had previously been developed using N-acetyl-muramyl-L-alanyl-D-isoglutamine-calmodulin conjugate as an immunogen, was tested for cross-reactivity with tryptic fragments of calmodulin (CaM1-77, CaM1-90, CaM78-149, and CaM106-149) as well as with synthetic peptides corresponding to the 1st, 2nd, and 3rd calcium binding loop of calmodulin. The results showed that the antigenic determinant involves a special conformation of amino acid residues 90-106 in the 3rd calcium-binding domain.

Research paper thumbnail of Effect of Mutating the Regulatory Phosphoserine and Conserved Threonine Onthe Activity of the Expressed Catalytic Domain of Acanthamoeba Myosin I Hea Vy Chain Kinase

Proceedings of the National Academy of Sciences of the United States of America, 1998

Phosphorylation of Ser-627 is both necessary and sufficient for full activity of the expressed 35... more 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.

Research paper thumbnail of Properties of Acanthamoeba Myosin I Heavy Chain Kinase Bound to Phospholipid Vesicles

J Biol Chem, 1995

The actin-activated Mg(2+)-ATPase and in vitro motility activities of the three Acanthamoeba myos... more The actin-activated Mg(2+)-ATPase and in vitro motility activities of the three Acanthamoeba myosin I isozymes depend upon phosphorylation of their single heavy chains by myosin I heavy chain kinase. Previously, the kinase had been shown to be activated by autophosphorylation, which is enhanced by acidic phospholipids, or simply by binding to purified plasma membranes in the absence of significant autophosphorylation. In this paper, we show that the rate of phosphorylation of myosin I by unphosphorylated kinase is approximately 20-fold faster when both the myosin I and the kinase are bound to acidic phospholipid vesicles than when both are soluble. This activation is not due to an increase in the local concentrations of vesicle-bound kinase and myosin I. Thus, acidic phospholipids, like membranes, can activate myosin I heavy chain kinase in the absence of significant autophosphorylation, i.e. membrane proteins are not required. Kinetic studies show that both binding of kinase to phospholipid vesicles and autophosphorylation of kinase in the absence of phospholipid increase the Vmax relative to soluble, unphosphorylated kinase with either an increase in the apparent Km (when myosin I is the substrate) or no significant change in Km (when a synthetic peptide is the substrate). Kinetic data showed that autophosphorylation of phospholipid-bound kinase is both intermolecular and intervesicular, and that phosphorylation of phospholipid-bound myosin I by phospholipid-bound kinase is also intervesicular even when the kinase and myosin are bound to the same vesicles. The relevance of these results to the activation of myosin I heavy chain kinase and phosphorylation of myosin I isozymes in situ are discussed.

Research paper thumbnail of Selective Localization of Myosin-I Proteins in Macropinosomes and Actin Waves

Cytoskeleton, 2016

Class I myosins are widely expressed with roles in endocytosis and cell migration in a variety of... more Class I myosins are widely expressed with roles in endocytosis and cell migration in a variety of cell types. Dictyostelium express multiple myosin Is, including three short-tailed (Myo1A, Myo1E, Myo1F) and three long-tailed (Myo1B, Myo1C, Myo1D). Here we report the molecular basis of the specific localizations of short-tailed Myo1A, Myo1E and Myo1F compared to our previously determined localization of long-tailed Myo1B. Myo1A and Myo1B have common and unique localizations consistent with the various features of their tail region; specifically the BH sites in their tails are required for their association with the plasma membrane and heads are sufficient for relocalization to the front of polarized cells. Myo1A does not localize to actin waves and macropinocytic protrusions, in agreement with the absence of a tail region which is required for these localizations of Myo1B. However, in spite of the overall similarity of their domain structures, the cellular distributions of Myo1E and Myo1F are quite different from Myo1A. Myo1E and Myo1F, but not Myo1A, are associated with macropinocytic cups and actin waves. The localizations of Myo1E and Myo1F in macropinocytic structures and actin waves differ from the localization of Myo1B. Myo1B colocalizes with F-actin in the actin waves and at the tips of mature macropinocytic cups whereas Myo1E and Myo1F are in the interior of actin waves and along the entire surface of macropinocytic cups. Our results point to different mechanisms of targeting of short- and long-tailed myosin Is, and are consistent with these myosins having both shared and divergent cellular functions. This article is protected by copyright. All rights reserved.

Research paper thumbnail of Cardiac troponin-C: a rapid and effective method of purification

Febs Letters, Feb 1, 1985

A new purification procedure for cardiac troponin-C is described which has several advantages ove... more A new purification procedure for cardiac troponin-C is described which has several advantages over previous methods. High purity of the final product was assessed by electrophoretic, enzymatic and spectroscopic methods.

Research paper thumbnail of Calmodulin-binding and Autoinhibitory Domains of Acanthamoeba Myosin I Heavy Chain Kinase, a p21-activated Kinase (PAK)

J Biol Chem, 2001

The sequence homology between Acanthamoeba myosin I heavy chain kinase (MIHCK) and other p21-acti... more The sequence homology between Acanthamoeba myosin I heavy chain kinase (MIHCK) and other p21-activated kinases (PAKs) is relatively low, including only the catalytic domain and a short PAK N-terminal motif (PAN), and even these regions are not highly homologous. In this paper, we report the expression in insect cells of full-length, fully regulated Acanthamoeba MI-HCK and further characterize the regulation of this PAK by Rac, calmodulin, and autoinhibition. We map the autoinhibitory region of MIHCK to its PAN region and show that the PAN region inhibits autophosphorylation and kinase activity of unphosphorylated fulllength MIHCK and its expressed catalytic domain but has very little effect on either when they are phosphorylated. These properties are similar to those reported for mammalian PAK1. Unlike PAK1, MIHCK is activated by Rac only in the presence of phospholipid. However, peptides containing the PAN region of MIHCK bind Rac in the absence of lipid, and Rac binding reverses the inhibition of the MIHCK catalytic domain by PAN peptides. Our data suggest that a region N-terminal to PAN is required for optimal binding of Rac. Also unlike mammalian PAK, phospholipid stimulation of Acanthamoeba MIHCK and Dictyostelium MIHCK) (which is also a PAK) is inhibited by Ca 2؉ -calmodulin. In contrast to Dictyostelium MIHCK, however, Ca 2؉ -calmodulin also inhibits Rac-induced activity of Acanthamoeba MIHCK. The basic region N-terminal to PAN is essential for calmodulin binding.

Research paper thumbnail of Studies on calmodulin structure using its tryptic fragments. In: Calcium-Binding Proteins: Structure and Function

Research paper thumbnail of Inhibition of Acanthamoeba myosin I heavy chain kinase by Ca(2+)-calmodulin

Journal of Biological Chemistry

vesicles and purified plasma membranes. Proteolytic removal of a 7-kDa NHz-terminal segment from ... more vesicles and purified plasma membranes. Proteolytic removal of a 7-kDa NHz-terminal segment from the 97-kDa kinase prevents binding of both calmodulin and phospholipid; therefore, we propose that they bind to the same or overlapping sites. These data provide a mechanism by which Ca2+ could inhibit the actin-activated Mg2+-ATPase activity of the myosin I isozymes in vivo and thus regulate myosin I-dependent motile activities.

Research paper thumbnail of The effect of actin and phosphorylation on the tryptic cleavage pattern of Acanthamoeba myosin IA

Journal of Biological Chemistry

The Mg2+-ATPase activity of Acanthamoeba myosin IA is activated by F-actin only when the myosin h... more The Mg2+-ATPase activity of Acanthamoeba myosin IA is activated by F-actin only when the myosin heavy chain is phosphorylated at a single residue. In order to gain insight into the conformational changes that may be responsible for the effects of F-actin and phosphorylation on myosin I ATPase, we have studied their effects on the proteolysis of the myosin IA heavy chain by trypsin. Trypsin initially cleaves the unphosphorylated, 140-kDa heavy chain of Acanthamoeba myosin IA at sites 38 and 112 kDa from its NH2 terminus and secondarily at sites 64 and 91 kDa from the NH2 terminus. F-actin has no effect on tryptic cleavage at the 91- and 112-kDa sites, but does protect the 38-kDa site and the 64-kDa site. Phosphorylation (which occurs very near the 38-kDa site) has no detectable effect on the tryptic cleavage pattern in the absence of F-actin or on F-actin protection of the 64-kDa site, but significantly enhances F-actin protection of the 38-kDa site. Protection of the 64-kDa site is probably due to direct steric blocking because F-actin binds to this region of the heavy chain. The protection of the 38-kDa site by F-actin may be the result of conformational changes in this region of the heavy chain induced by F-actin binding near the 64-kDa site and by phosphorylation. The conformational changes in the heavy chain of myosin IA that are detected by alterations in its susceptibility to proteolysis are likely to be related to the conformational changes that are involved in the phosphorylation-regulated actin-activated Mg2+-ATPase activities of Acanthamoeba myosins IA and IB.

Research paper thumbnail of Substrate specificity of Acanthamoeba myosin I heavy chain kinase as determined with synthetic peptides

Journal of Biological Chemistry

Research paper thumbnail of 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... more 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.

Research paper thumbnail of Acanthamoeva myosin I heavy chain kinase is activated by phosphatidylserine-enhanced phosphorylation

Journal of Biological Chemistry

ABSTRACT

Research paper thumbnail of Autophosphorylation-independent activation of Acanthamoeba myosin I heavy chain kinase by plasma membranes

Journal of Biological Chemistry

The three isoforms of Acanthamoeba myosin I (nonfilamentous myosin with only a single heavy chain... more 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.

Research paper thumbnail of Immunolocalization of myosin I heavy chain kinase in Acanthamoeba castellanii and binding of purified kinase to isolated plasma membranes

The Journal of Cell Biology

The actin-activated Mg"-ATPase activities of Acanthamoeba myosins I are known to be maximally exp... more 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

Research paper thumbnail of The localization and sequence of the phosphorylation sites of Acanthamoeba myosins I

Journal of Biological Chemistry

The actin-activated Mg2+-ATPase activities of Acanthamoeba myosins IA, IB, and IC are expressed o... more The actin-activated Mg2+-ATPase activities of Acanthamoeba myosins IA, IB, and IC are expressed only when a single site in their heavy chains is phosphorylated by a myosin I heavy chain-specific kinase. We show that phosphorylation occurs at Ser-315 in the myosin IB heavy chain, Ser-311 in myosin IC, and a threonine residue at a corresponding position in myosin IA whose amino acid sequence is as yet unknown. The most obvious feature common to the three substrates is a basic amino acid(s) 2 or 3 residues before the site of phosphorylation. The phosphorylation site is located between the ATP- and actin-binding sites, which corresponds to the middle of the 50-kDa domain of skeletal muscle myosin subfragment 1. The sequence similarity between the region surrounding the phosphorylation site of myosin I and subfragment 1 is much lower than the average sequence similarity between myosin I and subfragment 1. This is consistent with the hypothesis that the conformation of this region of myosin I differs from that of the corresponding region in skeletal muscle myosin and that phosphorylation converts the conformation of the actomyosin I complex into a conformation comparable to that present in actosubfragment 1 without phosphorylation. The protein sequences obtained in the course of this work led to the conclusion that the myosin I genes previously identified as myosin IB and IL (myosin-like) heavy chains actually are the myosin IC and IB heavy chains, respectively. Finally, we report a modification of the method for monitoring the appearance of 32Pi during sequencing of 32P-labeled peptides that results in almost complete recovery of the radioactivity, thus allowing unequivocal assignment of the position of the phosphorylated residue.

Research paper thumbnail of Preparation of a phospholipid-insensitive, autophosphorylation-activated catalytic fragment of Acanthamoeba myosin I heavy chain kinase

Journal of Biological Chemistry

The actin-activated Mg(2+)-ATPase activity of Acanthamoeba myosin I depends on phosphorylation of... more The actin-activated Mg(2+)-ATPase activity of Acanthamoeba myosin I depends on phosphorylation of its single heavy chain. The activity of the myosin I heavy chain kinase is increased about 50-fold by autophosphorylation, and the rate of kinase autophosphorylation is enhanced about 20-fold by acidic phospholipids independent of the presence of Ca2+ (Brzeska, H., Lynch, T. J., and Korn, E. D. (1990) J. Biol. Chem. 265, 3591-3594). In this paper, we show that chymotryptic digestion of the kinase produces a 54-kDa fragment which contains three to four of the approximately 11 original phosphorylation sites and whose activity is greatly stimulated by autophosphorylation. However, both the rate of autophosphorylation and the kinase activity of the 54-kDa fragment are independent of phospholipid and comparable to those of intact kinase in the presence of phospholipid. These data imply that the (probably NH2-terminal) region(s) removed by proteolysis is necessary for phospholipid-sensitive inhibition of autophosphorylation of sites residing within the (probably COOH-terminal) 54-kDa fragment. The 54-kDa fragment contains the catalytic site of the kinase as well as three to four sites whose phosphorylation is necessary for full expression of kinase activity. The middle region of the kinase molecule contains proline-rich regions that are similar to the COOH-terminal tail of the kinase substrate, Acanthamoeba myosin I.

Research paper thumbnail of Differential localization of Acanthamoeba myosin I isoforms

The Journal of Cell Biology

Antibody titers were quantified by a solid phase antibody capture immunoassay after the protocol ... more Antibody titers were quantified by a solid phase antibody capture immunoassay after the protocol of . Purified myosin I was bound to PVC in a 96-well microtiter plate (Falcon Plastics, Cockeysville, MD), antibody was added in a dilution series, and the captured antibody was quantified by binding of a second HRP-coupled anti-irnmunoglobulin antibody with 3',Y,5',5"tetramethylbertzidine as substrate.

Research paper thumbnail of Localization of the actin-binding sites of Acanthamoeba myosin IB and effect of limited proteolysis on its actin-activated Mg2+-ATPase activity

Journal of Biological Chemistry

Research paper thumbnail of Rac-induced increase of phosphorylation of myosin regulatory light chain in HeLa cells

Cell Motility and the Cytoskeleton, 2004

The pathways by which activation of the small GTP-binding protein Rac causes cytoskeletal changes... more The pathways by which activation of the small GTP-binding protein Rac causes cytoskeletal changes are not fully understood but are likely to involve both assembly of new actin filaments and reorganization of actin filaments driven by the actin-dependent ATPase activity of myosin II. Here we show that expression of active RacQ61 in growing HeLa cells, in addition to inducing ruffling, substantially enhances the level of phosphorylation of serine-19 of the myosin II regulatory light chain (MLC), which would increase actomyosin II ATPase and motor activities. Phosphorylated myosin was localized to RacQ61-induced ruffles and stress fibers. RacQ61-induced phosphorylation of MLC was reduced by a maximum of about 38% by an inhibitor (Tat-PAK) of p21-activated kinase (PAK), about 35% by an inhibitor (Y-27632) of Rho kinase, 51% by Tat-PAK plus Y-27632, and 10% by an inhibitor (ML7) of myosin light chain kinase. Staurosporine, a non-specific inhibitor of serine/threonine kinases, reduced RacQ61-induced phosphorylation of MLC by about 58%, at the maximum concentration that did not kill cells. Since Rac activates PAK and PAK can phosphorylate MLC, these data strongly suggest that PAK is responsible for a significant fraction of RacQ61-induced MLC phosphorylation. To our knowledge, this is the first evidence that active Rac causes phosphorylation of MLC in cells, thus implicating activation of the ATPase activity of actomyosin II as one of the ways by which Rac may induce cytoskeletal changes.

Research paper thumbnail of Activation of myosin in HeLa cells causes redistribution of focal adhesions and F-actin from cell center to cell periphery

Cell Motility and the Cytoskeleton, 2006

Activation of actomyosin II by phosphorylation of its regulatory light chain is one of the main f... more Activation of actomyosin II by phosphorylation of its regulatory light chain is one of the main factors involved in the regulation of cytoskeletal dynamics. Phosphorylation of myosin regulatory light chain may be mediated directly and indirectly by several kinases including myosin light chain kinase (MLCK) and kinases activated by small GTP-binding proteins. Most of the myosin kinases, including PAK, can also interact with other proteins through binding sites located outside of their catalytic domains. In an attempt to study the effects due only to phosphorylation of myosin light chain, we expressed the constitutively active catalytic domain of ameba PAK in HeLa cells. The catalytic domain phosphorylates myosin light chain in vitro with high specific activity but has none of the sequences that target mammalian PAK to other proteins and membranes. Expression of the catalytic domain caused disassembly of focal adhesions and stress fibers in the cell center and accumulation of focal adhesions and F-actin at the cell periphery. There was a twofold increase in the phosphorylation level of endogenous myosin light chain and changes in cell shape consistent with enhanced cell contractility. The phenotype was independent of MLCK, ROCK, MEK, Rac, and Rho activities but was abolished by blebbistatin, a specific inhibitor of myosin II activity. Our data are consistent with myosin being directly phosphorylated by the expressed catalytic domain of ameba PAK with the induced phenotype resulting from cell retraction driven by contraction of peripheral actomyosin. The phenotype induced by expression of the catalytic domain is reminiscent of that caused by expression of active mammalian PAK, suggesting that myosin phosphorylation may play an important role in PAK-induced cytoskeletal changes. The catalytic domain of ameba PAK may be a useful tool for studying the effects of myosin light chain phosphorylation in other cells. Cell Motil.

Research paper thumbnail of Characterization of a calmodulin antiserum by its reactions with fragments of the calmodulin molecule

Journal of immunoassay, 1986

A high affinity antibody, specific to the calcium-free form of calmodulin, which had previously b... more A high affinity antibody, specific to the calcium-free form of calmodulin, which had previously been developed using N-acetyl-muramyl-L-alanyl-D-isoglutamine-calmodulin conjugate as an immunogen, was tested for cross-reactivity with tryptic fragments of calmodulin (CaM1-77, CaM1-90, CaM78-149, and CaM106-149) as well as with synthetic peptides corresponding to the 1st, 2nd, and 3rd calcium binding loop of calmodulin. The results showed that the antigenic determinant involves a special conformation of amino acid residues 90-106 in the 3rd calcium-binding domain.