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B Barylko

Publications and source records attributed to B Barylko.

At least 19 recordsLinked to original sources
PubMed2001

A novel family of phosphatidylinositol 4-kinases conserved from yeast to humans.

Phosphatidylinositolpolyphosphates (PIPs) are centrally involved in many biological processes, ranging from cell growth and organization of the actin cytoskeleton to endo- and exocytosis. Phosphorylation of phosphatidylinositol at the D-4 position, an essential step in the biosynthesis of PIPs, appears to be catalyzed by two biochemically distinct enzymes. However, only one of these two enzymes has been molecularly characterized. We now describe a novel class of phosphatidylinositol 4-kinases that probably corresponds to the missing element in phosphatidylinositol metabolism. These kinases are highly conserved evolutionarily, but unrelated to previously characterized phosphatidylinositol kinases, and thus represent the founding members of a new family. The novel phosphatidylinositol 4-kinases, which are widely expressed in cells, only phosphorylate phosphatidylinositol, are potently inhibited by adenosine, but are insensitive to wortmannin or phenylarsine oxide. Although they lack an obvious transmembrane domain, they are strongly attached to membranes by palmitoylation. Our data suggest that independent pathways for phosphatidylinositol 4-phosphate synthesis emerged during evolution, possibly to allow tight temporal and spatial control over the production of this key signaling molecule.

1-Phosphatidylinositol 4-Kinase↗
PubMed2000

The mechanism of GTP hydrolysis by dynamin II: a transient kinetic study.

Dynamin II is a 98 kDa protein (870 amino acids) required for the late stages of clathrin-mediated endocytosis. The GTPase activity of dynamin is required for its function in the budding stages of receptor-mediated endocytosis and synaptic vesicle recycling. This activity is stimulated when dynamin self-associates on multivalent binding surfaces, such as microtubules and anionic liposomes. We first investigated the oligomeric state of dynamin II by analytical ultracentrifuge sedimentation equilibrium measurements at high ionic strength and found that it was best described by a monomer-tetramer equilibrium. We then studied the intrinsic dynamin GTPase mechanism by using a combination of fluorescence stopped-flow and HPLC methods using the fluorescent analogue of GTP, mantdGTP (2'-deoxy-3'-O-(N-methylanthraniloyl) guanosine-5'-triphosphate), under the same ionic strength conditions. The results are interpreted as showing that mantdGTP binds to dynamin in a two-step mechanism. The dissociation constant of mantdGTP binding to dynamin, calculated from the ratio of the off-rate to the on-rate (k(off)/k(on)), was 0.5 microM. Cleavage of mantdGTP then occurs to mantdGDP and P(i) followed by the rapid release of mantdGDP and P(i). No evidence of reversibility of hydrolysis was observed. The cleavage step itself is the rate-limiting step in the mechanism. This mechanism more closely resembles that of the Ras family of proteins involved in cell signaling than the myosin ATPase involved in cellular motility.

Animals↗
PubMed2000

Regulation of the enzymatic and motor activities of myosin I.

Myosins I were the first unconventional myosins to be purified and they remain the best characterized. They have been implicated in various motile processes, including organelle translocation, ion channel gating and cytoskeletal reorganization but their exact cellular functions are still unclear. All members of the myosin I family, from yeast to man, have three structural domains: a catalytic head domain that binds ATP and actin; a tail domain believed to be involved in targeting the myosins to specific subcellular locations and a junction or neck domain that connects them and interacts with light chains. In this review we discuss how each of these three domains contributes to the regulation of myosin I enzymatic activity, motor activity and subcellular localization.

Actins↗
PubMed1999

Correlation between self-association modes and GTPase activation of dynamin.

The GTPase activity of dynamin is obligatorily coupled, by a mechanism yet unknown, to the internalization of clathrin-coated endocytic vesicles. Dynamin oligomerizes in vitro and in vivo and both its mechanical and enzymatic activities appear to be mediated by this self-assembly. In this study we demonstrate that dynamin is characterized by a tetramer/monomer equilibrium with an equilibrium constant of 1.67 x 10(17) M(-3). Stopped-flow fluorescence experiments show that the association rate constant for 2'(3')-O-N-methylanthraniloyl (mant)GTP is 7.0 x 10(-5) M(-1) s(-1) and the dissociation rate constant is 2.1 s(-1), whereas the dissociation rate constant for mantdeoxyGDP is 93 s(-1). We also demonstrate the cooperativity of dynamin binding and GTPase activation on a microtubule lattice. Our results indicate that dynamin self-association is not a sufficient condition for the expression of maximal GTPase activity, which suggests that dynamin molecules must be in the proper conformation or orientation if they are to form an active oligomer.

Animals↗
PubMed1999

Essential role of the dynamin pleckstrin homology domain in receptor-mediated endocytosis.

Pleckstrin homology (PH) domains are found in numerous membrane-associated proteins and have been implicated in the mediation of protein-protein and protein-phospholipid interactions. Dynamin, a GTPase required for clathrin-dependent endocytosis, contains a PH domain which binds to phosphoinositides and participates in the interaction between dynamin and the betagamma subunits of heterotrimeric G proteins. The PH domain is essential for expression of phosphoinositide-stimulated GTPase activity of dynamin in vitro, but its involvement in the endocytic process is unknown. We expressed a series of dynamin PH domain mutants in cultured cells and determined their effect on transferrin uptake by those cells. Endocytosis is blocked in cells expressing a PH domain deletion mutant and a point mutant that fails to interact with phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. In contrast, expression of a point mutant with unimpaired PI(4,5)P2 interaction has no effect on transferrin uptake. These results demonstrate the significance of the PH domain for dynamin function and suggest that its role may be to mediate interactions between dynamin and phosphoinositides.

Animals↗
PubMed1998

Synergistic activation of dynamin GTPase by Grb2 and phosphoinositides.

Hydrolysis of GTP by dynamin is essential for budding clathrin-coated vesicles from the plasma membrane. Two distinct domains of dynamin are implicated in the interactions with dynamin GTPase activators. Microtubules and Grb2 bind to the carboxyl-terminal proline/arginine-rich domain (PRD), whereas phosphoinositides bind to the pleckstrin homology (PH) domain. In this study we tested the effect of different phosphoinositides on dynamin GTPase activity and found that the best activator is phosphatidylinositol 4,5-bisphosphate followed by 1-O-(1, 2-di-O-palmitoyl-sn-glycerol-3-benzyloxyphosphoryl)-D-myo-inositol 3,4,5-triphosphate. Phosphatidylinositol 4-phosphate was a weak activator and phosphatidylinositol 3,4-bisphosphate did not activate GTPase at all. We then addressed the question of whether both domains of dynamin, PRD and PH, can be engaged simultaneously, and determined the effects of dual occupancy on dynamin GTPase activity. We found that Grb2 and phosphatidylinositol 4,5-bisphosphate together increased the dynamin GTPase activity up to 4-fold higher than that obtained by these activators tested separately, and also reduced the dynamin concentration required for half-maximal activities by 3-fold. These results indicate that both stimulators can bind to dynamin simultaneously resulting in superactivation of dynamin GTPase activity. We propose that SH3-containing proteins such as Grb2 bind to the dynamin PRD to target it to clathrin-coated pits and prime it for superactivation by phosphoinositides.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Phosphatidylinositol (4,5)-bisphosphate-dependent activation of dynamins I and II lacking the proline/arginine-rich domains.

Dynamins comprise a family of GTPases that participate in the early stages of endocytosis. The GTPase activity of neuronal specific dynamin I is stimulated by microtubules, negatively charged phospholipid vesicles, and Src homology 3-containing proteins, including Grb2. These activators were previously shown to bind to a proline/arginine-rich domain (PRD) in the carboxyl-terminal region of the enzyme. Dynamin II, which is ubiquitously expressed, had not been purified or characterized previously. In this study, the enzymatic properties of rat dynamin II and of D746, a dynamin II truncation mutant lacking the PRD, have been characterized. Dynamin II has a higher basal activity than dynamin I, but the two types of dynamin are stimulated similarly by microtubules, Grb2, and phospholipids. D746 is not activated by microtubules or Grb2, highlighting the significance of the PRD for these interactions, but it is activated by phospholipid vesicles containing phosphatidylserine or phosphatidylinositol-4,5- bisphosphate. Moreover, in contrast to previous reports, the PRD appears not to be required for phospholipid-stimulated self-assembly of dynamin, which is a key element in the regulation of its activity. Similar results were obtained with bovine brain dynamin I that had been subjected to limited proteolytic digestion to remove the PRD. Our data highlight the potential involvement of dynamin pleckstrin homology domains in the regulation of GTPase activity by phospholipids.

Animals↗
PubMed1996

Phosphorylation of dynamin by ERK2 inhibits the dynamin-microtubule interaction.

In the present study we show that purified bovine brain dynamin can be phosphorylated by MAP kinase, ERK2, with a stoichiometry of 1 mol phosphate/mol dynamin. The phosphorylated serine residue is located within the C-terminal 10 kDa of dynamin. Dynamin I phosphorylated by ERK2 can be specifically dephosphorylated by calcineurin but not by protein phosphatase 2A (PP2A). Phosphorylation of dynamin by ERK2 weakens the binding of dynamin to microtubules and inhibits dynamin's microtubule-activated GTPase activity. Stimulation of GTPase activity by either Grb2 or phospholipids was not affected by ERK2 phosphorylation, suggesting that the binding sites for Grb2 and phospholipids do not overlap with that for microtubules.

Binding Sites↗
PubMed1994

Domain structure of a mammalian myosin I beta.

We have determined the primary structure of a myosin I (called mammalian myosin I beta, MMI beta) from bovine brain and identified its functional domains. The protein was previously purified from brain and adrenal gland. Several constructs were generated and expressed in Escherichia coli as glutathione S-transferase fusion proteins and the recombinant proteins were recognized by monoclonal antibodies that recognize either "head" or "tail" domains of native myosin I. A gel overlay method was used to confirm that calmodulin binds to the consensus calmodulin-binding sequence in MMI beta. Binding assays were used to detect interaction with anionic phospholipid vesicles. We conclude that MMI beta consists of an amino-terminal 80.5-kDa domain that contains the ATP- and actin-binding sites, followed by an 8.5-kDa domain with three calmodulin-binding sequences and a basic 30-kDa carboxyl-terminal tail segment that binds to anionic phospholipids and membranes.

Actins↗
PubMed1992

Purification and characterization of a mammalian myosin I.

Myosin I, an actin-dependent force-generating enzyme, has been purified from three mammalian sources: bovine adrenal medulla, adrenal cortex, and brain. The purification procedure includes extraction of tissue with ATP at low ionic strength and coprecipitation with actin, followed by gel filtration on Sepharose 4B, anion-exchange chromatography on Q Sepharose, and affinity chromatography on ATP-agarose. Mammalian myosin I molecules are composed of a heavy chain of 116 kDa and multiple low molecular weight polypeptides identified as calmodulin. The structural and enzymatic properties of adrenal medulla myosin I were further characterized. This enzyme exhibits high K+,EDTA- and Ca(2+)-ATPase specific activities (about 0.2 mumol.min-1 per mg of protein), whereas the Mg(2+)-ATPase activity is very low (1-3 nmol.min-1.mg-1). The Mg(2+)-ATPase of medulla myosin I is activated by F-actin in a Ca(2+)-dependent manner: activity is stimulated 40-fold in the presence of EGTA and 90-fold in the presence of 10 microM Ca2+. Two structural domains of the myosin I heavy chain were identified. A 74-kDa chymotryptic fragment contains the catalytic site, while a 36-kDa polypeptide contains the calmodulin-binding sites. These results indicate that mammalian myosin I is more closely related to myosin I from the avian intestinal brush border than to the enzymes isolated from the protozoans Acanthamoeba and Dictyostelium.

Actins↗
PubMed1992

Tissue distribution and subcellular localization of mammalian myosin I.

Myosin I, a nonfilamentous single-headed actin-activated ATPase, has recently been purified from mammalian tissue (Barylko, B., M. C. Wagner, O. Reizes, and J. P. Albanesi. 1992. Proc. Natl. Acad. Sci. USA. 89:490-494). To investigate the distribution of this enzyme in cells and tissues mAbs were generated against myosin I purified from bovine adrenal gland. Eight antibodies were characterized, five of them (M4-M8) recognize epitope(s) on the catalytic "head" portion of myosin I while the other three (M1-M3) react with the "tail" domain. Immunoblot analysis using antiadrenal myosin I antibody M2 demonstrates the widespread distribution of the enzyme in mammalian tissues. Myosin I was immunolocalized in several cell types including bovine kidney (MDBK), rat kidney (NRK), rat brain, rat phaeochromocytoma (PC12), fibroblast (Swiss 3T3), and CHO cells. In all cases, myosin I was concentrated at the cell periphery. The most intense labeling was observed in regions of the cell usually associated with motile activity (i.e., filopodia, lamellipodia and growth cones). These results are consistent with earlier observations on protozoan myosin I that suggest a motile role for the enzyme at the plasma membrane.

Animals↗
PubMed1986

Proteolytic fragmentation of brain myosin and localisation of the heavy-chain phosphorylation site.

The heavy chains and the 19-kDa and 20-kDa light chains of bovine brain myosin can by phosphorylated. To localise the site of heavy-chain phosphorylation, the myosin was initially subjected to digestion with chymotrypsin and papain under a variety of conditions and the fragments thus produced were identified. Irrespective of the ionic strength, i.e. whether the myosin was monomeric or filamentous, chymotryptic digestion produced two major fragments of 68 kDa and 140 kDa; the 140-kDa fragment was further digested by papain to yield a 120-kDa and a 23-kDa fragment. These fragments were characterised by (a) a gel overlay technique using 125I-labelled light chains, which showed that the 140-kDa and 23-kDa polypeptides contain the light-chain-binding sites; (b) using myosin photoaffinity labelled at the active site with [3H]UTP, which showed that the 68-kDa fragment contained the catalytic site, and (c) electron microscopy, using rotary shadowing and negative-staining techniques, which demonstrated that after chymotryptic digestion the myosin head remains attached to the tail whereas on papain digestion isolated heads and tails were observed. Thus the 120-kDa polypeptide derived from the 140-kDa fragment is the tail of the myosin, and the 68-kDa fragment containing the catalytic site and the 23-kDa fragment, with the light-chain-binding sites, form the head (S1) portion of the myosin. When [32P]-phosphorylated brain myosin was digested with chymotrypsin and papain it was shown that the heavy-chain phosphorylation site is located in a 5-kDa peptide at the C-terminal end of the heavy chain, i.e. the end of the myosin tail. Using hydrodynamic and electron microscopic techniques, no significant effect of either light-chain or heavy-chain phosphorylation on the stability of brain myosin filaments was observed, even in the presence of MgATP. Brain myosin filaments appear to be more stable than those of other non-muscle myosins. Light-chain phosphorylation did, however, have an effect on the conformation of brain myosin, for example in the presence of MgATP non-phosphorylated myosin molecules were induced to fold into a very compact folded state.

Adenosine Triphosphate↗
PubMed1983

Phosphorylation and actin activation of brain myosin.

A method is described for obtaining brain myosin that shows significant actin activation, after phosphorylation with chicken gizzard myosin light chain kinase. Myosin with this activity could be obtained only via the initial purification of brain actomyosin. The latter complex, isolated by a method similar to that used for smooth muscle, contained actin, myosin, tropomyosin of the non-muscle type and another actin-binding protein of approximately 100,000 daltons. From the presence of a specific myosin light chain kinase and phosphatase in brain tissue it is suggested that the regulation of actin-myosin interaction operates via phosphorylation and dephosphorylation of myosin.

Actins↗
PubMed1977

Degradation of TN-C component of troponin by trypsin.

The peptide pattern obtained during degradation of TN-C component of troponin by trypsin markedly depends on the concentration of calcium ions. In the presence of calcium higher than 0.1 mM a peptide with a mass of about 16 000 daltons is formed, followed by its splitting into two peptides (8500 and 7500 daltons), which accumulate during further digestion. When calcium is sequestred by EGTA the degradation of TN-C is much faster. The first products of digestion are the 11 000 and 7500 dalton peptides. The former one is degraded to 9700 dalton peptide and, subsequently, both peptides are split to small fragments. In the presence of calcium only three -NH2 groups per molecule of TN-C appear during digestion, whereas in its absence almost all peptide bonds available for trypsin are cleaved. The fragments of TN-C obtained in the presence of calcium are still able to interact with TN-I component as judged by disc-electrophoresis performed in the presence of calcium. During digestion in the presence of calcium only a slight decrease of tyrosyl intrinsic fluorescence occurs and the further decrease after removal of calcium bound to TN-C is fully reversible. Similarly, the content of alpha-helix decreases only slowly during digestion in the presence of calcium. All the results suggest that calcium stabilizes the structure of TN-C molecule, so that trypsin in the presence of calcium releases one small peptide from N-terminal not involved in the first "EF-hand" and the other from the region between pairs 1-2 and 3-4 of "EF-hands" in the nomenclature of Kretsinger (see Kretsinger and Barry [16]). The obtained fragments, containing almost exclusively pairs of "EF-hands", restore most of the properties of original TN-C molecule.

Animals↗
PubMed1975

Composition of cardiac muscle troponin.

Preparations of troponin from cardiac muscle consist of functionally the same subunits as skeletal troponin, that is TN-T, TN-I, and TN-C. Cardiac TN-T and TN-I have, however, higher molecular weights than those of skeletal muscle. Only TN-C from both sources does not differ in respect to the molecular weight, isoelectric point, and ability to bind calcium or strontium.

Animals↗