Jennifer Caldwell Busby

Interaction between TAK1–TAB1–TAB2 and RCAN1–calcineurin defines a signalling nodal control point

The calcium-activated protein phosphatase calcineurin is controlled by regulator of calcineurin (RCAN) in organisms ranging from yeast to mammals. Here we performed a yeast two-hybrid screen with RCAN1 as bait, identifying TAK1 binding protein 2 (TAB2) as an interacting partner. TAB2 interacted directly with RCAN1 in vitro and in vivo, recruiting TAK1, TAB1 and calcineurin, forming a macromolecular signalling complex. Overexpression of TAK1 and TAB1, or active TAK1ΔN, promoted direct phosphorylation of RCAN1 in vitro and in vivo. TAK1 phosphorylated RCAN1 at Ser 94 and Ser 136, converting RCAN1 from an inhibitor to a facilitator of calcineurin–NFAT signalling, and enhancing NFATc1 nuclear translocation, NFAT transcriptional activation and the hypertrophic growth of cultured cardiomyocytes. The TAK1–TAB1–TAB2 and the calcineurin–NFAT signalling modules did not interact in Rcan1/2- or Tab2-deficient mouse embryonic fibroblast (MEF) cultures. Calcineurin activation also dephosphorylated and inhibited TAK1 and TAB1, an effect that was absent in Rcan1/2 deficient MEFs. Functionally, TAK1 was indispensable for the cardiomyocyte growth response induced by pro-hypertrophic stimuli through calcineurin. These results describe a signalling relationship between two central regulatory pathways in which TAK1–TAB1–TAB2 selectively induces calcineurin–NFAT signalling through direct phosphorylation of RCAN1, while calcineurin activation diminishes TAK1 signalling by dephosphorylation of TAK1 and TAB1.

Sperm Proteomics Reveals Intensified Selection on Mouse Sperm Membrane and Acrosome Genes

Spermatozoa are a focal point for the impact of sexual selection due to sperm competition and sperm-female interactions in a wide range of sexually reproducing organisms. In-depth molecular investigation of the ramifications of these selective regimes has been limited due to a lack of information concerning the molecular composition of sperm. In this study, we utilize three previously published proteomic data sets in conjunction with our whole mouse sperm proteomic analysis to delineate cellular regions of sperm most impacted by positive selection. Interspecific analysis reveals robust evolutionary acceleration of sperm cell membrane genes (which include genes encoding acrosomal and sperm cell surface proteins) relative to other sperm genes, and evidence for positive selection in approximately 22% of sperm cell membrane components was obtained using maximum likelihood models. The selective forces driving the accelerated evolution of these membrane proteins may occur at a number of locations during sperm development, maturation, and transit through the female reproductive tract where the sperm cell membrane and eventually the acrosome are exposed to the extracellular milieu and available for direct cell-cell interactions.

A coactivator trap identifies NONO (p54nrb) as a component of the cAMP-signaling pathway

Signal transduction pathways often use a transcriptional component to mediate adaptive cellular responses. Coactivator proteins function prominently in these pathways as the conduit to the basic transcriptional machinery. Here we present a high-throughput cell-based screening strategy, termed the “coactivator trap,” to study the functional interactions of coactivators with transcription factors. We applied this strategy to the cAMP signaling pathway, which utilizes two families of coactivators, the cAMP response element binding protein (CREB) binding protein (CBP)/p300 family and the recently identified transducers of regulated CREB activity family (TORCs1–3). In addition to identifying numerous known interactions of these coactivators, this analysis identified NONO (p54nrb) as a TORC-interacting protein. RNA interference experiments demonstrate that NONO is necessary for cAMP-dependent activation of CREB target genes in vivo. Furthermore, TORC2 and NONO complex on cAMP-responsive promoters, and NONO acts as a bridge between the CREB/TORC complex and RNA polymerase II. These data demonstrate the utility of the coactivator trap by identification of a component of cAMP-mediated transcription.

Activation Domain-dependent Degradation of Somatic Wee1 Kinase

Cell cycle progression is dependent upon coordinate regulation of kinase and proteolytic pathways. Inhibitors of cell cycle transitions are degraded to allow progression into the subsequent cell cycle phase. For example, the tyrosine kinase and Cdk1 inhibitor Wee1 is degraded during G2 and mitosis to allow mitotic progression. Previous studies suggested that the N terminus of Wee1 directs Wee1 destruction. Using a chemical mutagenesis strategy, we report that multiple regions of Wee1 control its destruction. Most notably, we find that the activation domain of the Wee1 kinase is also required for its degradation. Mutations in this domain inhibit Wee1 degradation in somatic cell extracts and in cells without affecting the overall Wee1 structure or kinase activity. More broadly, these findings suggest that kinase activation domains may be previously unappreciated sites of recognition by the ubiquitin proteasome pathway.

Proteomics of the nucleus ovoidalis and field L brain regions of zebra finch

The purpose of present study is to analyze the brain proteome of the nucleus ovoidalis (OV) and Field L regions of the zebra finch (Taeniopygia guttata). The OV and Field L are important brain nuclei in song learning in zebra finches; their analyses identified a total of 79 proteins. The zebra finch brain proteome analyses are poised to provide clues about cell and circuit layout as well as possible circuit function.

A cell based screening approach for identifying protein degradation regulators

ABSTRACT Cellular transitions are achieved by the concerted actions of regulated degradation pathways. In the case of the cell cycle, ubiquitin mediated degradation ensures unidirectional transition from one phase to another. For instance, turnover of the cell cycle regulator cyclin B1 occurs after metaphase to induce mitotic exit. To better understand pathways controlling cyclin B1 turnover, the N-terminal domain of cyclin B1 was fused to luciferase to generate an N-cyclin B1-luciferase protein that can be used as a reporter for protein turnover. Prior studies demonstrated that cell-based screens using this reporter identified small molecules inhibiting the ubiquitin ligase controlling cyclin B1-turnover. Our group adapted this approach for the G2-M regulator Wee1 where a Wee1-luciferase construct was used to identify selective small molecules inhibiting an upstream kinase that controls Wee1 turnover. In the present study we present a screening approach where cell cycle regulators are fused to luciferase and overexpressed with cDNAs to identify specific regulators of protein turnover. We overexpressed approximately 14,000 cDNAs with the N-cyclin B1-luciferase fusion protein and determined its steady-state level relative to other luciferase fusion proteins. We identified the known APC/C regulator Cdh1 and the F-box protein Fbxl15 as specific modulators of N-cyclin B1-luciferase steady-state levels and turnover. Collectively, our studies suggest that analyzing the steady-state levels of luciferase fusion proteins in parallel facilitates identification of specific regulators of protein turnover.

Interaction between TAK1|[ndash]|TAB1|[ndash]|TAB2 and RCAN1|[ndash]|calcineurin defines a signalling nodal control point

The calcium-activated protein phosphatase calcineurin is controlled by regulator of calcineurin (RCAN) in organisms ranging from yeast to mammals. Here we performed a yeast two-hybrid screen with RCAN1 as bait, identifying TAK1 binding protein 2 (TAB2) as an interacting partner. TAB2 interacted directly with RCAN1 in vitro and in vivo, recruiting TAK1, TAB1 and calcineurin, forming a macromolecular signalling complex. Overexpression of TAK1 and TAB1, or active TAK1ΔN, promoted direct phosphorylation of RCAN1 in vitro and in vivo. TAK1 phosphorylated RCAN1 at Ser 94 and Ser 136, converting RCAN1 from an inhibitor to a facilitator of calcineurin–NFAT signalling, and enhancing NFATc1 nuclear translocation, NFAT transcriptional activation and the hypertrophic growth of cultured cardiomyocytes. The TAK1–TAB1–TAB2 and the calcineurin–NFAT signalling modules did not interact in Rcan1/2- or Tab2-deficient mouse embryonic fibroblast (MEF) cultures. Calcineurin activation also dephosphorylated and inhibited TAK1 and TAB1, an effect that was absent in Rcan1/2 deficient MEFs. Functionally, TAK1 was indispensable for the cardiomyocyte growth response induced by pro-hypertrophic stimuli through calcineurin. These results describe a signalling relationship between two central regulatory pathways in which TAK1–TAB1–TAB2 selectively induces calcineurin–NFAT signalling through direct phosphorylation of RCAN1, while calcineurin activation diminishes TAK1 signalling by dephosphorylation of TAK1 and TAB1.

Novel interaction between the TAK1-TAB1-TAB2 and the RCAN1-calcineurin regulatory pathways defines a signaling nodal control point

The calcium-activated protein phosphatase calcineurin is controlled by regulator of calcineurin (RCAN) in organisms ranging from yeast to mammals. Here we performed a yeast two-hybrid screen with RCAN1 as bait, identifying TAK1 binding protein 2 (TAB2) as an interacting partner. TAB2 interacted directly with RCAN1 in vitro and in vivo, recruiting TAK1, TAB1 and calcineurin, forming a macromolecular signalling complex. Overexpression of TAK1 and TAB1, or active TAK1ΔN, promoted direct phosphorylation of RCAN1 in vitro and in vivo. TAK1 phosphorylated RCAN1 at Ser 94 and Ser 136, converting RCAN1 from an inhibitor to a facilitator of calcineurin–NFAT signalling, and enhancing NFATc1 nuclear translocation, NFAT transcriptional activation and the hypertrophic growth of cultured cardiomyocytes. The TAK1–TAB1–TAB2 and the calcineurin–NFAT signalling modules did not interact in Rcan1/2- or Tab2-deficient mouse embryonic fibroblast (MEF) cultures. Calcineurin activation also dephosphorylated and inhibited TAK1 and TAB1, an effect that was absent in Rcan1/2 deficient MEFs. Functionally, TAK1 was indispensable for the cardiomyocyte growth response induced by pro-hypertrophic stimuli through calcineurin. These results describe a signalling relationship between two central regulatory pathways in which TAK1–TAB1–TAB2 selectively induces calcineurin–NFAT signalling through direct phosphorylation of RCAN1, while calcineurin activation diminishes TAK1 signalling by dephosphorylation of TAK1 and TAB1.