A Novel Protein Containing cdc10/SWI6 Motifs Regulates Expression of mRNA Encoding Catecholamine Biosynthesizing Enzymes

Myotrophin/V-1 is a cytosolic protein found at elevated levels in failing human hearts and in postnatal cerebellum. We have previously shown that it disrupts nuclear factor of B (NF B)-DNA complexes in vitro. In this study, we demonstrated that in HeLa cells native myotrophin/V-1 is predominantly present in the cytoplasm and translocates to the nucleus during sustained NF B activation. Three-dimensional alignment studies indicate that myotrophin/V-1 resembles a truncated I B␣ without the signal response domain (SRD) and PEST domains. Co-immunoprecipitation studies reveal that myotrophin/V-1 interacts with NF B proteins in vitro; however, it remains physically associated only with p65 and c-Rel proteins in vivo during NF B activation. In vitro studies indicate that myotrophin/V-1 can promote the formation of p50-p50 homodimers from monomeric p50 proteins and can convert the preformed p50-p65 heterodimers into p50-p50 and p65-p65 homodimers. Furthermore, adenovirus-mediated overexpression of myotrophin/V-1 resulted in elevated levels of both p50-p50 and p65-p65 homodimers exceeding the levels of p50-p65 heterodimers compared with Ad␤gal-infected cells, where the levels of p50-p65 heterodimers exceeded the levels of p50-p50 and p65-p65 homodimers. Thus, overexpression of myotrophin/V-1 during NF B activation resulted in a qualitative shift by quantitatively reducing the level of transactivating heterodimers while elevating the levels of repressive p50-p50 homodimers. Correspondingly, overexpression of myotrophin/V-1 resulted in significantly reduced B-luciferase reporter activity. Because myotrophin/V-1 is found at elevated levels during NF B activation in postnatal cerebellum and in failing human hearts, this study cumulatively suggests that myotrophin/V-1 is a regulatory protein for modulating the levels of activated NF B dimers during this period.

mentioning

confidence: 99%

Myotrophin/V-1, a Protein Up-regulated in the Failing Human Heart and in Postnatal Cerebellum, Converts NFκB p50-p65 Heterodimers to p50-p50 and p65-p65 Homodimers

Knuefermann¹,

Chen²,

Misra

et al. 2002

“…Previous studies suggested the potential role of V-1 in the signal transduction pathways leading to catecholamine synthesis or to cardiac hypertrophy. For example, Yamakuni et al (5)(6)(7) demonstrate that the overexpression of V-1 caused a significant increase in the catecholamine level in PC12 cells, presumably through the transcriptional activation of the genes for catecholamine synthesis. In other reports (8 -11), V-1 was designated as "myotrophin" and was shown to participate in the cell signaling pathways for the NFkB-mediated activation of protein synthesis in the myocytes.…”

mentioning

confidence: 99%

V-1, a Protein Expressed Transiently during Murine Cerebellar Development, Regulates Actin Polymerization via Interaction with Capping Protein

Taoka

Ichimura

Wakamiya-Tsuruta

et al. 2003

V-1 is a 12-kDa protein consisting of three consecutive ANK repeats, which are believed to serve as the surface for protein-protein interactions. It is thought to have a role in neural development for its temporal profile of expression during murine cerebellar development, but its precise role remains unknown. Here we applied the proteomic approach to search for protein targets that interact with V-1. The V-1 cDNA attached with a tandem affinity purification tag was expressed in the cultured 293T cells, and the protein complex formed within the cells were captured and characterized by mass spectrometry. We detected two polypeptides specifically associated with V-1, which were identified as the ␣ and ␤ subunits of the capping protein (CP, alternatively called CapZ or ␤-actinin). CP regulates actin polymerization by capping the barbed end of the actin filament. The V-1⅐CP complex was detected not only in cultured cells transfected with the V-1 cDNA but also endogenously in cells as well as in murine cerebellar extracts. An analysis of the V-1/CP interaction by surface plasmon resonance spectroscopy showed that V-1 formed a stable complex with the CP heterodimer with a dissociation constant of 1.2 ؋ 10 ؊7 M and a molecular stoichiometry of ϳ1:1. In addition, V-1 inhibited the CP-regulated actin polymerization in vitro in a dose-dependent manner. Thus, our results suggest that V-1 is a novel component that regulates the dynamics of actin polymerization by interacting with CP and thereby participates in a variety of cellular processes such as actin-driven cell movements and motility during neuronal development.The V-1 protein was originally identified in the murine cerebellum as one of the proteins expressed significantly at the initial stage of postnatal development (1), particularly in the regions where synaptic formation and neuronal migration occur actively during neurogenesis (2, 3). V-1 consists of 117 amino acids containing three contiguous repeats of the ANK motif, alternatively called the cdc10/SWI6 motif (1, 2), which is crucial for a large number of protein-protein interactions (4). Previous studies suggested the potential role of V-1 in the signal transduction pathways leading to catecholamine synthesis or to cardiac hypertrophy. For example, Yamakuni et al.(5-7) demonstrate that the overexpression of V-1 caused a significant increase in the catecholamine level in PC12 cells, presumably through the transcriptional activation of the genes for catecholamine synthesis. In other reports (8 -11), V-1 was designated as "myotrophin" and was shown to participate in the cell signaling pathways for the NFkB-mediated activation of protein synthesis in the myocytes. Thus, both of these studies (5, 9) suggested the roles of V-1 in the biological events taking place in the nucleus. However, no biological function has been attributed to V-1 in the cytoplasm in which this molecule predominantly resides within the cells and tissues (5, 9).In this study, we screened for V-1-binding proteins by a novel proteomic approach th...

“…V-1 is important for expression of catecholamine-synthesizing enzymes (7), differentiation and regeneration of skeletal muscle (8), folliculogenesis and corpus luteum formation in the ovary (9), and regulation of insulin secretion (10). We will refer to the myotrophin/V-1 protein as V-1 for simplicity.…”

mentioning

confidence: 99%

Binding of Myotrophin/V-1 to Actin-capping Protein

Bhattacharya

Ghosh

Sept

et al. 2006

The heterodimeric actin-capping protein (CP) regulates actin assembly and cell motility by binding tightly to the barbed end of the actin filament. Here we demonstrate that myotrophin/V-1 binds directly to CP in a 1:1 molar ratio with a K d of 10 -50 nM. V-1 binding inhibited the ability of CP to cap the barbed ends of actin filaments. The actin-binding COOH-terminal region, the "tentacle," of the CP ␤ subunit was important for binding V-1, with lesser contributions from the ␣ subunit COOH-terminal region and the body of the protein. V-1 appears to be unable to bind to CP that is on the barbed end, based on the observations that V-1 had no activity in an uncapping assay and that the V-1⅐CP complex had no capping activity. Two loops of V-1, which extend out from the ␣-helical backbone of this ankyrin repeat protein, were necessary for V-1 to bind CP. Parallel computational studies determined a bound conformation of the ␤ tentacle with V-1 that is consistent with these findings, and they offered insight into experimentally observed differences between the ␣1 and ␣2 isoforms as well as the mutant lacking the ␣ tentacle. These results support and extend our "wobble" model for CP binding to the actin filament, in which the two COOH-terminal regions of CP bind independently to the actin filament, and bound CP is able to wobble when attached only via its mobile ␤-subunit tentacle. This model is also supported by molecular dynamics simulations of CP reported here. The existence of the wobble state may be important for actin dynamics in cells.Myotrophin is a 12-kDa protein identified in hypertrophied rat hearts (1) and dilated cardiomyopathic human hearts (2). Myotrophin was named for its ability to stimulate hypertrophy in cardiac myocytes when added to cells in culture (1). Transgenic mice overexpressing myotrophin in cardiac myocytes develop cardiac hypertrophy and heart failure (3). Myotrophin was found to be identical to the protein V-1 (4), which had been identified based on increased expression in granule cell neurons during development of the rat cerebellum (5). V-1 expression correlates with morphogenetic changes of cerebellar differentiation. V-1 is expressed in many vertebrate cells and tissues (6).V-1 homologues are present and highly conserved across vertebrates, and similar protein sequences are predicted by the genomes of other animal species, but not by those of fungi or plants. V-1 is important for expression of catecholamine-synthesizing enzymes (7), differentiation and regeneration of skeletal muscle (8), folliculogenesis and corpus luteum formation in the ovary (9), and regulation of insulin secretion (10). We will refer to the myotrophin/V-1 protein as V-1 for simplicity.Structurally, V-1 is a small ankyrin repeat protein. A solution structure of V-1 shows two full ankyrin repeat motifs in tandem, with additional incomplete repeats at the amino and carboxyl termini (11,12). An ankyrin repeat consists of a ␤ hairpin loop followed by a pair of anti-parallel ␣ helices connected by a short turn sequence ...