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

Kulesza-Lipka, D; Baines, Ivan C.; Brzeska, Hanna; Korn, Edward D.

doi:10.1083/jcb.115.1.109

Cited by 18 publications

(18 citation statements)

References 29 publications

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“…Therefore, the present results leave uncertain the mechanism by which ϳ20% of plasma membrane-associated myosin I remains phosphorylated at steady state in situ (14). One possibility is that membrane-associated myosin I is phosphorylated in situ by cytoplasmic myosin I heavy chain kinase, which is enriched in the subplasma membrane cortex (19), and accounts for about 70% of the total kinase in the cell (19). This is consistent with our earlier results which showed that even though soluble autophosphorylated kinase does not bind to membranes in vitro (19), it can phosphorylate membrane-bound myosin I (21).…”

Section: Figmentioning

confidence: 57%

“…Membrane proteins are probably involved in the specificity of binding of both kinase and the myosin I isoforms to different membranes in situ and in vitro (19,28,29). Moreover, phospholipid vesicles have a higher capacity than isolated plasma membranes for both kinase and myosin I, 2 and autophosphorylation of phospholipid-bound kinase is more rapid and more extensive than autophosphorylation of membranebound kinase (18,19,21). Addition of purified membrane proteins to phospholipid vesicles reduces their binding capacity for myosin I to that of purified plasma membranes.…”

Section: Discussionmentioning

confidence: 99%

“…It is of some interest to compare the data in this paper to the situation in situ. About 70% of the myosin I kinase of Acanthamoeba appears to be in the cytoplasm unassociated with membranes (19). From the kinetic data for soluble kinase presented in this paper, the activity of the cytoplasmic kinase would be expected to vary with the extent to which it is phosphorylated which, presumably, would be a function of the relative rates of its autophosphorylation and dephosphorylation.…”

Section: Figmentioning

confidence: 99%

“…It seemed likely that the increase in myosin I phosphorylation in the presence of phospholipids was due entirely to the effect of phospholipid on the kinase because soluble and phospholipid-bound myosin I are equally good substrates for soluble, phosphorylated kinase (21) which does not bind to phospholipids (19). In order to determine if the enhanced rate of phosphorylation of phospholipid-bound myosin I by phospholipid-bound kinase were due simply to the faster rate of autophosphorylation of phospholipid-bound kinase, the activities of phospholipid-bound and soluble kinase were measured as a function of the extent of kinase phosphorylation.…”

Section: Figmentioning

confidence: 99%

“…The rate of kinase autophosphorylation is ϳ20-fold greater in the presence of acidic phospholipids (18). As determined by immunoelectron microscopy, ϳ30% of myosin I heavy chain kinase is associated with the plasma membrane in situ (19) and both the kinase and its substrate, myosin I, bind to plasma membranes in vitro (19,20). The rate of autophosphorylation of plasma membranebound kinase is only slightly greater (ϳ2-to 5-fold) than the rate of autophosphorylation of soluble kinase, but the membrane-bound kinase is much more active than expected from its level of phosphorylation (21).…”

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confidence: 99%

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

Wang

Brzeska

Baines

et al. 1995

Journal of Biological Chemistry

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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 ϳ20-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 V max relative to soluble, unphosphorylated kinase with either an increase in the apparent K m (when myosin I is the substrate) or no significant change in K m (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.Each of the three isoforms of Acanthamoeba myosin I has a single ϳ110 -140-kDa heavy chain with an ϳ80-kDa N-terminal domain, that is highly similar in sequence to the subfragment 1 domain of conventional myosin II, and a ϳ50-kDa nonfilamentous C-terminal domain that has no sequence similarity to the C-terminal domain of conventional myosin (1, 2; for reviews, see . The N-terminal head domain contains an ATP-binding site (6) and an ATP-sensitive, F-actinbinding site (7,8), as do all myosins. The C-terminal tail domain contains an ATP-insensitive, F-actin-binding site (8 -10) and a membrane (and acidic phospholipid)-binding site (10, 11) which, thus far, appear to be unique to the myosin I family. Actin-activated Mg 2ϩ -ATPase activity is expressed in vitro only when a single serine (myosin IB and IC) or threonine (myosin IA), situated between the ATP-and actin-binding sites in the globular head (6), is phosphorylated (12, 13). The biological importance of phosphorylation of the myosin I heavy chain is evidenced by the observations (14) that ϳ80% of myosin IA and ϳ20% of myosin IB and IC are phosphorylated in situ, that the fraction of phosphorylated myosin IC associated with the contractile vacuole in situ varies with the stage of the contractile vacuole cycle (14), and that antibodies that specifically inhibit phosphorylation ...

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Section: Figmentioning

confidence: 57%

Section: Discussionmentioning

confidence: 99%

Section: Figmentioning

confidence: 99%

Section: Figmentioning

confidence: 99%

mentioning

confidence: 99%

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

Wang

Brzeska

Baines

et al. 1995

Journal of Biological Chemistry

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Characterization of Amoeba proteus myosin VI immunoanalog

Dominik

Kłopocka

Pomorski

et al. 2005

Cell Motil. Cytoskeleton

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Amoeba proteus, the highly motile free-living unicellular organism, has been widely used as a model to study cell motility. However, molecular mechanisms underlying its unique locomotion and intracellular actin-based-only trafficking remain poorly understood. A search for myosin motors responsible for vesicular transport in these giant cells resulted in detection of 130-kDa protein interacting with several polyclonal antibodies against different tail regions of human and chicken myosin VI. This protein was binding to actin in the ATP-dependent manner, and immunoprecipitated with anti-myosin VI antibodies. In order to characterize its possible functions in vivo, its cellular distribution and colocalization with actin filaments and dynamin II during migration and pinocytosis were examined. In migrating amoebae, myosin VI immunoanalog localized to vesicular structures, particularly within the perinuclear and sub-plasma membrane areas, and colocalized with dynamin II immunoanalog and actin filaments. The colocalization was even more evident in pinocytotic cells as proteins concentrated within pinocytotic pseudopodia. Moreover, dynamin II and myosin VI immunoanalogs cosedimented with actin filaments, and were found on the same isolated vesicles. Blocking endogenous myosin VI immunoanalog with anti-myosin VI antibodies inhibited the rate of pseudopodia protrusion (about 19% decrease) and uroidal retraction (about 28% decrease) but did not affect cell morphology and the manner of cell migration. Treatment with anti-human dynamin II antibodies led to changes in directionality of amebae migration and affected the rate of only uroidal translocation (about 30% inhibition). These results indicate that myosin VI immunoanalog is expressed in protist Amoeba proteus and may be involved in vesicle translocation and cell locomotion.

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Class I myosins: Highly versatile proteins with specific functions in the immune system

Girón-Pérez

Piedra-Quintero

Santos-Argumedo

2019

Journal of Leukocyte Biology

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Connections established between cytoskeleton and plasma membrane are essential in cellular processes such as cell migration, vesicular trafficking, and cytokinesis. Class I myosins are motor proteins linking the actin‐cytoskeleton with membrane phospholipids. Previous studies have implicated these molecules in cell functions including endocytosis, exocytosis, release of extracellular vesicles and the regulation of cell shape and membrane elasticity. In immune cells, those proteins also are involved in the formation and maintenance of immunological synapse‐related signaling. Thus, these proteins are master regulators of actin cytoskeleton dynamics in different scenarios. Although the localization of class I myosins has been described in vertebrates, their functions, regulation, and mechanical properties are not very well understood. In this review, we focused on and summarized the current understanding of class I myosins in vertebrates with particular emphasis in leukocytes.

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

Cited by 18 publications

References 29 publications

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

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

Characterization of Amoeba proteus myosin VI immunoanalog

Class I myosins: Highly versatile proteins with specific functions in the immune system

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