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H Brzeska

Publications and source records attributed to H Brzeska.

At least 19 recordsLinked to original sources
PubMed2001

Calmodulin-binding and autoinhibitory domains of Acanthamoeba myosin I heavy chain kinase, a p21-activated kinase (PAK).

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 MIHCK 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 full-length 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.

Acanthamoeba↗
PubMed2001

Myosin I mutants with only 1% of wild-type actin-activated MgATPase activity retain essential in vivo function(s).

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.

Actins↗
PubMed2000

Functional analysis of tail domains of Acanthamoeba myosin IC by characterization of truncation and deletion mutants.

Acanthamoeba myosin IC has a single 129-kDa heavy chain and a single 17-kDa light chain. The heavy chain comprises a 75-kDa catalytic head domain with an ATP-sensitive F-actin-binding site, a 3-kDa neck domain, which binds a single 17-kDa light chain, and a 50-kDa tail domain, which binds F-actin in the presence or absence of ATP. The actin-activated MgATPase activity of myosin IC exhibits triphasic actin dependence, apparently as a consequence of the two actin-binding sites, and is regulated by phosphorylation of Ser-329 in the head. The 50-kDa tail consists of a basic domain, a glycine/proline/alanine-rich (GPA) domain, and a Src homology 3 (SH3) domain, often referred to as tail homology (TH)-1, -2, and -3 domains, respectively. The SH3 domain divides the TH-3 domain into GPA-1 and GPA-2. To define the functions of the tail domains more precisely, we determined the properties of expressed wild type and six mutant myosins, an SH3 deletion mutant and five mutants truncated at the C terminus of the SH3, GPA-2, TH-1, neck and head domains, respectively. We found that both the TH-1 and GPA-2 domains bind F-actin in the presence of ATP. Only the mutants that retained an actin-binding site in the tail exhibited triphasic actin-dependent MgATPase activity, in agreement with the F-actin-cross-linking model, but truncation reduced the MgATPase activity at both low and high actin concentrations. Deletion of the SH3 domain had no effect. Also, none of the tail domains, including the SH3 domain, affected either the K(m) or V(max) for the phosphorylation of Ser-329 by myosin I heavy chain kinase.

Acanthamoeba↗
PubMed1999

Myosin I heavy chain kinase: cloning of the full-length gene and acidic lipid-dependent activation by Rac and Cdc42.

Acanthamoeba myosin I heavy chain kinase (MIHCK) phosphorylates the heavy chains of amoeba myosins I, increasing their actin-activated ATPase activities. The activity of MIHCK is increased by binding to acidic phospholipids or membranes and by autophosphorylation at multiple sites. Phosphorylation at a single site is necessary and sufficient for full activation of the expressed catalytic domain. The rate of autophosphorylation of native MIHCK is controlled by a region N-terminal to the catalytic domain. By its substrate specificity and the sequence of its C-terminal catalytic domain, MIHCK was identified as a p21-activated kinase (PAK). We have now cloned the full-length genomic DNA and cDNA of MIHCK and have shown it to contain the conserved p21-binding site common to many members of the PAK family. Like some mammalian PAKs, MIHCK is activated by Rac and Cdc42, and this activation is GTP-dependent and accompanied by autophosphorylation. In contrast to mammalian PAKs, activation of MIHCK by Rac and Cdc42 requires the presence of acidic lipids. Also unlike mammalian PAK, MIHCK is not activated by sphingosine or other non-negatively charged lipids. The acidic lipid-binding site is near the N terminus followed by the p21-binding region. The N-terminal regulatory domain of MIHCK contains alternating strongly positive and strongly negative regions. and the extremely Pro-rich middle region of MIHCK has a strongly acidic N-terminal segment and a strongly basic C-terminal segment. We propose that autophosphorylation activates MIHCK by neutralizing the basic segment of the Pro-rich region, thus unfolding the regulatory domain and abolishing its inhibition of the catalytic domain.

Acanthamoeba↗
PubMed1998

Identification of phosphorylation sites in proteins separated by polyacrylamide gel electrophoresis.

We report a fast, sensitive, and robust procedure for the identification of precise phosphorylation sites in proteins separated by polyacrylamide gel electrophoresis by a combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF) and online capillary liquid chromatography electrospray tandem ion trap mass spectrometry (LC/ESI/MS/MS). With this procedure, a single phosphorylation site was identified on as little as 20 ng (500 fmol) of the baculovirus-expressed catalytic domain of myosin I heavy-chain kinase separated by gel electrophoresis. The phosphoprotein is digested in the gel with trypsin, and the resulting peptides are extracted with > 60% yield and analyzed by MALDI/TOF before and after digestion with a phosphatase to identify the phosphopeptides. The phosphopeptides are then separated and fragmented in an on-line LC/ESI ion trap mass spectrometer to identify the precise phosphorylation sites. This procedure eliminates any off-line HPLC separation and minimizes sample handling. The use of MALDI/TOF and LCQ, two types of mass spectrometers that are widely available to the biological community, will make this procedure readily accessible to biologists. We applied this technique to identify two autophosphorylation sites and to assign at least another 12 phosphorylation sites to two tryptic peptides in a series of experiments using a gel slice containing only 200 ng (3 pmol) of human double-stranded RNA-activated protein kinase expressed in a mutant strain of the yeast Saccharomyces cerevisiae.

Acanthamoeba↗
PubMed1998

Effect of mutating the regulatory phosphoserine and conserved threonine on the activity of the expressed catalytic domain of Acanthamoeba myosin I heavy chain kinase.

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 Ser/Thr also occurs in many other Ser/Thr protein kinases. The variable loop of MIHCK contains two other hydroxyamino acids: Thr-631, which is conserved in almost all Ser/Thr 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 kcat and higher Km 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 Ser/Thr protein kinases as well as for the activity of MIHCK.

Acanthamoeba↗
PubMed1997

Identification by mass spectrometry of the phosphorylated residue responsible for activation of the catalytic domain of myosin I heavy chain kinase, a member of the PAK/STE20 family.

Myosin I heavy chain kinase from Acanthamoeba castellanii is activated in vitro by autophosphorylation (8-10 mol of P per mol). The catalytically active C-terminal domain produced by trypsin cleavage of the phosphorylated kinase contains 2-3 mol of P per mol. However, the catalytic domain expressed in a baculovirus-insect cell system is fully active as isolated without autophosphorylation in vitro. We now show that the expressed catalytic domain is inactivated by incubation with acid phosphatase and regains activity upon autophosphorylation. The state of phosphorylation of all of the hydroxyamino acids in the catalytic domain were determined by mass spectrometry of unfractionated protease digests. Ser-627 was phosphorylated in the active, expressed catalytic domain, lost its phosphate when the protein was incubated with phosphatase, and was rephosphorylated when the dephosphorylated protein was incubated with ATP. No other residue was significantly phosphorylated in any of the three samples. Thus, phosphorylation of Ser-627, which is in the same position as the Ser and Thr residues that are phosphorylated in many other kinases, is necessary and sufficient for full activity of the catalytic domain. Ser-627 is also phosphorylated when full-length, native kinase is activated by autophosphorylation.

Acanthamoeba↗
PubMed1997

p21-activated kinase has substrate specificity similar to Acanthamoeba myosin I heavy chain kinase and activates Acanthamoeba myosin I.

Acanthamoeba class I myosins are unconventional, single-headed myosins that express actin-activated Mg2+-ATPase and in vitro motility activities only when a single serine or threonine in the heavy chain is phosphorylated by myosin I heavy chain kinase (MIHCK). Some other, but not most, class I myosins have the same consensus phosphorylation site sequence, and the two known class VI myosins have a phosphorylatable residue in the homologous position, where most myosins have an aspartate or glutamate residue. Recently, we found that the catalytic domain of Acanthamoeba MIHCK has extensive sequence similarity to the p21-activated kinase (PAK)/STE20 family of kinases from mammals and yeast, which are activated by small GTP-binding proteins. The physiological substrates of the PAK/STE20 kinases are not well characterized. In this paper we show that PAK1 has similar substrate specificity as MIHCK when assayed against synthetic substrates and that PAK1 phosphorylates the heavy chain (1 mol of P(i) per mol) and activates Acanthamoeba myosin I as MIHCK does. These results, together with the known involvement of Acanthamoeba myosin I, yeast myosin I, STE20, PAK, and small GTP-binding proteins in membrane- and cytoskeleton-associated morphogenetic transformations and activities, suggest that myosins may be physiological substrates for the PAK/STE20 family and thus mediators of these events.

Acanthamoeba↗
PubMed1996

The catalytic domain of Acanthamoeba myosin I heavy chain kinase. I. Identification and characterization following tryptic cleavage of the native enzyme.

The actin-activated Mg2+-ATPase activities of the myosin I isoenzymes from Acanthamoeba castellanii are greatly increased by phosphorylation catalyzed by myosin I heavy chain kinase (MIHC kinase), a monomeric 97-kDa protein whose activity is greatly enhanced by acidic phospholipids and by autophosphorylation of multiple sites. In this paper, we show that the 35-kDa COOH-terminal fragment obtained by trypsin cleavage of maximally activated, autophosphorylated kinase retains the full activity and two to three of the autophosphorylation sites of the native enzyme. Other autophosphorylation sites occur in the middle third of the native enzyme. A trypsin cleavage site within the 35-kDa region is protected in phosphorylated kinase but is readily cleaved in unphosphorylated kinase producing catalytically inactive 25- and 11-kDa fragments from the NH2- and COOH-terminal ends, respectively, of the 35-kDa peptide. This implies that the conformation around the "25/11" cleavage site changes upon phosphorylation of the native enzyme. The position of this site corresponds to the activation loop of protein kinase A (see the accompanying paper: Brzeska, H., Szczepanowska, J., Hoey, J., and Korn, E. D. (1996) J. Biol. Chem. 271, 27056-27062). Exogenously added MIHC kinase phosphorylates the 11-kDa fragment, but not the 25-kDa fragment, indicating that the phosphorylation sites of the 35-kDa catalytic fragment are located within the COOH-terminal 11 kDa. The accompanying paper describes the cloning, sequencing, and expression of a fully active 35-kDa catalytic domain.

Acanthamoeba↗
PubMed1996

The catalytic domain of acanthamoeba myosin I heavy chain kinase. II. Expression of active catalytic domain and sequence homology to p21-activated kinase (PAK).

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 Ca2+-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.

Acanthamoeba↗
PubMed1995

Properties of Acanthamoeba myosin I heavy chain kinase bound to phospholipid vesicles.

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.

Acanthamoeba↗
PubMed1993

Autophosphorylation-independent activation of Acanthamoeba myosin I heavy chain kinase by plasma membranes.

The three isoforms of Acanthamoeba myosin I (non-filamentous myosin with only a single heavy chain) express actin-activated Mg(2+)-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 situ with membranes, and all four enzymes bind to purified membranes in vitro. We 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 Mg(2+)-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.

Acanthamoeba↗
PubMed1992

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

The actin-activated Mg(2+)-ATPase activity of Acanthamoeba myosins I depends on phosphorylation of their single heavy chains by myosin I heavy chain kinase. Kinase activity is enhanced > 50-fold by autophosphorylation at multiple sites. The rate of kinase autophosphorylation is increased approximately 20-fold by acidic phospholipids independent of the presence of Ca2+ and diglycerides. We show in this paper that Ca(2+)-calmodulin inhibits phospholipid-stimulated autophosphorylation of myosin I heavy chain kinase and hence also inhibits the catalytic activity of unphosphorylated kinase in the presence of phospholipid. Ca(2+)-calmodulin does not inhibit kinase activity in the absence of phospholipid. Micromolar Ca(2+)-calmodulin also inhibits binding of myosin I heavy chain kinase to phospholipid vesicles and purified plasma membranes. Proteolytic removal of a 7-kDa NH2-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 Mg(2+)-ATPase activity of the myosin I isozymes in vivo and thus regulate myosin I-dependent motile activities.

Acanthamoeba↗
PubMed1992

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

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.

Acanthamoeba↗
PubMed1992

Differential localization of Acanthamoeba myosin I isoforms.

Acanthamoeba myosins IA and IB were localized by immunofluorescence and immunoelectron microscopy in vegetative and phagocytosing cells and the total cell contents of myosins IA, IB, and IC were quantified by immunoprecipitation. The quantitative distributions of the three myosin I isoforms were then calculated from these data and the previously determined localization of myosin IC. Myosin IA occurs almost exclusively in the cytoplasm, where it accounts for approximately 50% of the total myosin I, in the cortex beneath phagocytic cups and in association with small cytoplasmic vesicles. Myosin IB is the predominant isoform associated with the plasma membrane, large vacuole membranes and phagocytic membranes and accounts for almost half of the total myosin I in the cytoplasm. Myosin IC accounts for a significant fraction of the total myosin I associated with the plasma membrane and large vacuole membranes and is the only myosin I isoform associated with the contractile vacuole membrane. These data suggest that myosin IA may function in cytoplasmic vesicle transport and myosin I-mediated cortical contraction, myosin IB in pseudopod extension and phagocytosis, and myosin IC in contractile vacuole function. In addition, endogenous and exogenously added myosins IA and IB appeared to be associated with the cytoplasmic surface of different subpopulations of purified plasma membranes implying that the different myosin I isoforms are targeted to specific membrane domains through a mechanism that involves more than the affinity of the myosins for anionic phospholipids.

Acanthamoeba↗
PubMed1991

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

The actin-activated Mg(2+)-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 highly purified plasma membranes with an apparent KD of approximately 17 nM and a capacity of approximately 0.8 nmol/mg of plasma membrane protein, values that are similar to the affinity and capacity of plasma membranes for myosins I. Binding of kinase to membranes is inhibited by elevated ionic strength and by extensive autophosphorylation but not by substrate-level concentrations of ATP. Membrane-bound kinase autophosphorylates to a lesser extent than free kinase and does not dissociate from the membranes after autophosphorylation. The co-localization of myosin I heavy chain kinase and myosin I at the plasma membrane is of interest in relation to the possible functions of myosin I especially as phospholipids increase kinase activity.

Acanthamoeba↗