RESUMO
Rho family GTPases are important regulators of neuronal morphology, but the proteins directly controlling their activity in neurons are still poorly defined. We report the identification of myr 7, a novel unconventional myosin IX-RhoGAP expressed in rat brain. Myr 7 is a multidomain protein related to myr 5, the first class IX myosin to be characterized. It exhibits a myosin head domain with an N-terminal extension and a large insertion at loop 2, an actin contact site and regulator of myosin ATPase rate. The myosin head domain is followed by a neck domain consisting of six unevenly spaced consecutive IQ motifs representing light chain binding sites. The tail domain contains a C6H2-zinc binding motif and a region that specifically stimulates the GTPase-activity of Rho followed by a short stretch predicted to adopt a coiled-coil structure. Five alternatively spliced regions, one in the 5'-noncoding region, two in the myosin head and two in the tail domain, were noted. Analysis of myr 7 and myr 5 expression in different tissues revealed that myr 7 is expressed at high levels in developing and adult brain tissue whereas myr 5 is expressed only at moderate levels in embryonic brain tissue and at even further reduced levels in adult brain tissue. Myr 5 is, however, highly expressed in lung, liver, spleen and testis. Myr 7 is expressed in all brain regions and is localized in the cytoplasm of cell bodies, dendrites and axons. Myr 5 exhibits an overlapping, but not identical cellular distribution. Finally, a myr 7 fusion protein encompassing the GAP domain specifically activates the GTPase-activity of Rho in vitro, and overexpression of myr 7 in HtTA1-HeLa cells leads to inactivation of Rho in vivo. These results are compatible with a role for myr 7 (and myr 5) in regulating Rho activity in neurons and hence in regulating neuronal morphology and function.
Assuntos
Encéfalo/metabolismo , Proteínas de Ligação ao GTP/genética , Proteínas Ativadoras de GTPase , Miosinas/biossíntese , Miosinas/genética , Glândulas Suprarrenais/metabolismo , Sequência de Aminoácidos , Animais , Encéfalo/enzimologia , Células Cultivadas , Clonagem Molecular , Embrião de Mamíferos , Ativação Enzimática , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Ligação ao GTP/biossíntese , Proteínas de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células HeLa , Humanos , Imuno-Histoquímica , Masculino , Dados de Sequência Molecular , Miosinas/química , Miosinas/imunologia , Especificidade de Órgãos , Ratos , Ratos Wistar , Testículo/metabolismo , Proteínas rho de Ligação ao GTPRESUMO
Myr 3, a member of the myosin-I family from rat, is shown in this study to be localized at adherens-type intercellular junctions in epithelial and nonepithelial tissues. Formation of intercellular junctions and the accompanying recruitment of myr 3 to these junctions involves signaling by the Rho subfamily of small GTP-binding proteins. This conclusion is based on studies with HtTA-1 HeLa cells that were induced by overexpression of constitutively active Cdc42Hs to form typical adherens-type intercellular junctions enriched in cadherins (N-cadherin), beta-catenin, filamentous actin and myr 3. Recruitement of myr 3 to Cdc42-induced adherens junctions in HeLa cells was dependent on a short region of the tail domain and a functional myosin motor domain, but was independent of its myosin-I tail homology and SH3 regions. Overexpression of constitutively active Rac1 induced a distinct type of adherens junction in HeLa cells that was characterized by elaborate intercellular interdigitations enriched in N-cadherin, beta-catenin and F-actin. Myr 3 was often present, but not specifically enriched in the intercellular junctions induced by constitutively active Rac1.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Junções Intercelulares/metabolismo , Miosina Tipo I , Miosinas/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Proteínas de Ciclo Celular/genética , Proteínas de Ligação ao GTP/genética , Expressão Gênica , Células HeLa , Humanos , Junções Intercelulares/ultraestrutura , Microscopia Eletrônica , Proteínas Motores Moleculares/química , Proteínas Motores Moleculares/genética , Proteínas Motores Moleculares/metabolismo , Miosinas/química , Miosinas/genética , Plasmídeos/genética , Ratos , Transfecção , Proteína cdc42 de Ligação ao GTP , Proteínas rac de Ligação ao GTPRESUMO
We purified Myr3 (third unconventional myosin from rat), a mammalian "amoeboid" subclass myosin I, from rat liver. The heavy chain of purified Myr3 is associated with a single calmodulin light chain. Myr3 exhibits K/EDTA-ATPase and Mg-ATPase activity. The Mg-ATPase activity is stimulated by increasing F-actin concentrations in a complex triphasic manner similar to the Mg-ATPase activity of myosin I molecules from protozoa. Although purified Myr3 was observed to cross-link actin filaments, it bound in an ATP regulated manner to F-actin, and no evidence for a nucleotide-independent high affinity actin binding site that could explain the triphasic activation pattern was obtained. Micromolar concentrations of free Ca2+ reversibly inhibit the Mg-ATPase activity of Myr3 by binding to its light chain calmodulin, which remains bound to the Myr3 heavy chain irrespective of the free Ca2+ concentration. Polyclonal antibodies and Fab fragments directed against the tail domain were found to stimulate the Mg-ATPase activity. A similar stimulation of the Myr3 Mg-ATPase activity is observed upon proteolytic removal of the very C-terminal SH3 domain. These results demonstrate that Myr3 is subject to negative regulation by free calcium and its own tail domain and possibly positive regulation by a tail-domain binding partner.
Assuntos
ATPase de Ca(2+) e Mg(2+)/antagonistas & inibidores , Cálcio/farmacologia , Calmodulina/metabolismo , Miosina Tipo I , Miosinas/química , Actinas/metabolismo , Actinas/ultraestrutura , Regulação Alostérica/fisiologia , Sequência de Aminoácidos , Animais , Anticorpos/farmacologia , Ativação Enzimática/fisiologia , Fragmentos Fab das Imunoglobulinas/farmacologia , Fígado/química , Masculino , Microscopia Eletrônica , Dados de Sequência Molecular , Cadeias Leves de Miosina/metabolismo , Miosinas/ultraestrutura , Fragmentos de Peptídeos/química , Ratos , Ratos Sprague-Dawley , Domínios de Homologia de src/fisiologiaRESUMO
In an effort to determine diversity and function of mammalian myosin I molecules, we report here the cloning and characterization of myr 3 (third unconventional myosin from rat), a novel mammalian myosin I from rat tissues that is related to myosin I molecules from protozoa. Like the protozoan myosin I molecules, myr 3 consists of a myosin head domain, a single light chain binding motif, and a tail region that includes a COOH-terminal SH3 domain. However, myr 3 lacks the regulatory phosphorylation site present in the head domain of protozoan myosin I molecules. Evidence was obtained that the COOH terminus of the tail domain is involved in regulating F-actin binding activity of the NH2-terminal head domain. The light chain of myr 3 was identified as the Ca(2+)-binding protein calmodulin. Northern blot and immunoblot analyses revealed that myr 3 is expressed in many tissues and cell lines. Immunofluorescence studies with anti-myr 3 antibodies in NRK cells demonstrated that myr 3 is localized in the cytoplasm and in elongated structures at regions of cell-cell contact. These elongated structures contained F-actin and alpha-actinin but were devoid of vinculin. Incubation of NRK cells with Con A stimulated the formation of myr 3-containing structures along cell-cell contacts. These results suggest for myr 3 a function mediated by cell-cell contact.
Assuntos
Actinas/isolamento & purificação , Compartimento Celular , Junções Intercelulares/química , Proteínas de Membrana/isolamento & purificação , Miosina Tipo I , Miosinas/isolamento & purificação , Sequência de Aminoácidos , Animais , Sequência de Bases , Calmodulina/análise , Linhagem Celular , Concanavalina A/farmacologia , Análise Mutacional de DNA , DNA Complementar/genética , Imunofluorescência , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Miosinas/química , Miosinas/genética , Miosinas/metabolismo , Proteínas de Protozoários/genética , Ratos , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Solubilidade , Relação Estrutura-Atividade , Distribuição TecidualRESUMO
We report the identification and characterization of myr 4 (myosin from rat), the first mammalian myosin I that is not closely related to brush border myosin I. Myr 4 contains a myosin head (motor) domain, a regulatory domain with light chain binding sites and a tail domain. Sequence analysis of myosin I head (motor) domains suggested that myr 4 defines a novel subclass of myosin I's. This subclass is clearly different from the vertebrate brush border myosin I subclass (which includes myr 1) and the myosin I subclass(es) identified from Acanthamoeba castellanii and Dictyostelium discoideum. In accordance with this notion, a detailed sequence analysis of all myosin I tail domains revealed that the myr 4 tail is unique, except for a newly identified myosin I tail homology motif detected in all myosin I tail sequences. The Ca(2+)-binding protein calmodulin was demonstrated to be associated with myr 4. Calmodulin binding activity of myr 4 was mapped by gel overlay assays to the two consecutive light chain binding motifs (IQ motifs) present in the regulatory domain. These two binding sites differed in their Ca2+ requirements for optimal calmodulin binding. The NH2-terminal IQ motif bound calmodulin in the absence of free Ca2+, whereas the COOH-terminal IQ motif bound calmodulin in the presence of free Ca2+. A further Ca(2+)-dependent calmodulin binding site was mapped to amino acids 776-874 in the myr 4 tail domain. These results demonstrate a differential Ca2+ sensitivity for calmodulin binding by IQ motifs, and they suggest that myr 4 activity might be regulated by Ca2+/calmodulin. Myr 4 was demonstrated to be expressed in many cell lines and rat tissues with the highest level of expression in adult brain tissue. Its expression was developmentally regulated during rat brain ontogeny, rising 2-3 wk postnatally, and being maximal in adult brain. Immunofluorescence localization demonstrated that myr 4 is expressed in subpopulations of neurons. In these neurons, prominent punctate staining was detected in cell bodies and apical dendrites. A punctate staining that did not obviously colocalize with the bulk of F-actin was also observed in C6 rat glioma cells. The observed punctate staining for myr 4 is reminiscent of a membranous localization.