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Domain organization of D-AKAP2 revealed by enhanced deuterium exchange-mass spectrometry (DXMS
- J Mol Biol
"... Dual specific A-kinase anchoring protein 2 (D-AKAP2) is a scaffold protein that coordinates cAMP-mediated signaling complexes by binding to type I and type II protein kinase A (PKA). While information is unfold-ing regarding specific binding motifs, very little is known about the over-all structure ..."
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Cited by 11 (4 self)
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Dual specific A-kinase anchoring protein 2 (D-AKAP2) is a scaffold protein that coordinates cAMP-mediated signaling complexes by binding to type I and type II protein kinase A (PKA). While information is unfold-ing regarding specific binding motifs, very little is known about the over-all structure and dynamics of these scaffold proteins. We have used deuterium exchange-mass spectrometry (DXMS) and limited proteolysis to probe the folded regions of D-AKAP2, providing for the first time insight into the intra-domain dynamics of a scaffold protein. Deuterium on-exchange revealed two regions of low deuterium exchange that were surrounded by regions of high exchange, suggestive of two distinctly folded regions, flanked by disordered or solvent accessible regions. Simi-lar folded regions were detected by limited proteolysis. The first folded region contained a putative regulator of G-protein signaling (RGS) domain. A structural model of the RGS domain revealed that the more deuterated regions mapped onto loops and turns, whereas less deuterated regions mapped onto a-helices, consistent with this region folding into an RGS domain. The second folded region contained a highly protected PKA binding site and a more solvent-accessible PDZ binding motif, which may serve as a potential targeting domain for D-AKAP2. DXMS has verified the multi-domain architecture of D-AKAP2 implied by sequence homology and has provided unique insight into the accessibility of the PKA binding site.
During Labor
"... OBJECTIVE: We have previously shown that the association ofprotein kinase A (PKA) with purfied myometrial plasma membrane declined at the end of pregnancy in the rat. This study was designed to determine if a similar decline in PKA occurred in pregnant human myometrium. METHODS: Myometrial plasma me ..."
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OBJECTIVE: We have previously shown that the association ofprotein kinase A (PKA) with purfied myometrial plasma membrane declined at the end of pregnancy in the rat. This study was designed to determine if a similar decline in PKA occurred in pregnant human myometrium. METHODS: Myometrial plasma membranes were isolated from lower uterine segment tissues from not-in-labor (NIL) and in-labor (IL) patients undergoing cesarean delivery. Membrane proteins were subjected to Western blot analysis to detect PKA-catalytic (PKA-cat) and PKA-regulatory (PKA-reg) subunits, the PKA4 binding protein A-kinase anchoring protein 79 (AKAP79), protein phosphatase 2B (PP2B), and Gotq, a guanosine triphosphate (GTP)-binding protein. Protein levels were expressed relative to caveolin- 1, which was invariant between the two groups. RESULTS: The amount of PKA-cat, PKA-reg, AKAP79, and PP2B in plasma membranes fiom myometrium of women in early labor decreased signficantly compared with that in tissuesfrom women not in labor. In contrast, Gaq did not change. All proteins were localized to myometrial smooth muscle cells by immunohistochemistry. CONCLUSIONS: Expression of PKA, PP2B, and AKAP79 is consistent with the presence of a fiunctional AKAP-mediated signaling complex in pregnant human myometrial membranes. A small but significant decrease in PKA, AKAP79, and PP2B in myometrial tissues from women in labor may contribute to a decrease in negative feedback on and enhancement of contractant signals at term. (U Soc
Two-dimensional differential in-gel electrophoresis-based proteomics of male gametes in relation to oxidative stress
"... Objective: To identify the relative abundance of proteins in pooled reactive oxygen species (ROS)-positive (ROSþ) and ROS-negative (ROSÀ) semen samples with the use of two-dimensional differential in-gel electrophoresis (2D-DIGE). Design: Spermatozoa suspensions from ROSþ and ROSÀ groups by 2D-DIGE ..."
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Objective: To identify the relative abundance of proteins in pooled reactive oxygen species (ROS)-positive (ROSþ) and ROS-negative (ROSÀ) semen samples with the use of two-dimensional differential in-gel electrophoresis (2D-DIGE). Design: Spermatozoa suspensions from ROSþ and ROSÀ groups by 2D-DIGE analysis. Setting: Tertiary hospital. Patient(s): 20 donors and 32 infertile men. Intervention(s): Seminal ejaculates evaluated for semen and proteomic analysis. Main Outcome Measure(s): Semen samples from 20 donors and 32 infertile men were pooled, divided into ROSþ and ROSÀ groups based on the cutoff value of <20 relative light units/s/10 6 sperm and frozen. From each pooled group, spermatozoa were labeled with Cy3/Cy5 fluorescent dye. Duplicate 2D-DIGE gels were run. Image analysis was performed with the use of Decider software. Protein spots exhibiting R1.5-fold difference in intensity were excised from the preparatory gel and identified by liquid chromatographymass spectrometry. Data were analyzed with the use of Sequest and Blast programs. Result(s): A total of 1,343 protein spots in gel 1 (ROSÀ) and 1,265 spots in gel 2 (ROSþ) were detected. The majority of protein spots had similar expression, with 31 spots were differentially expressed. Six spots were significantly decreased and 25 increased in the ROSÀ sample compared with the ROSþ sample. Conclusion(s): Significantly different expression of protective proteins against oxidative stress was found in ROSÀcompared with ROSþ samples. These differences may explain the role of oxidation species in the pathology of male infertility. (Fertil Steril Ò 2013;99:1216-26. Ó2013 by American Society for Reproductive Medicine.)
www.elsevier.com/locate/neuropharm Review
, 2003
"... Orchestration of synaptic plasticity through AKAP signaling complexes ..."
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Madame Sylvie MAZAN Directeur de Recherche, CNRS.
"... Discipline / Spécialité: Biologie Cellulaire et Moléculaire Caractérisation moléculaire de la rétine embryonnaire et larvaire de la lamproie Petromyzon marinus. THÈSE dirigée par: ..."
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Discipline / Spécialité: Biologie Cellulaire et Moléculaire Caractérisation moléculaire de la rétine embryonnaire et larvaire de la lamproie Petromyzon marinus. THÈSE dirigée par:
RESEARCH ARTICLE Open Access
"... Myomegalin is a novel A-kinase anchoring protein involved in the phosphorylation of cardiac myosin binding protein C ..."
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Myomegalin is a novel A-kinase anchoring protein involved in the phosphorylation of cardiac myosin binding protein C
RESEARCH ARTICLE k c
"... specificity serine/threonine protein kinase that is ubiqui- showing that PKA is concentrated in discrete intracellu- ..."
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specificity serine/threonine protein kinase that is ubiqui- showing that PKA is concentrated in discrete intracellu-
PROPERTIES OF THE INSECT NEUTRAL AMINO ACID COTRANSPORTER KAAT1 STUDIED BY ALANINE SCANNING MUTAGENESIS AND TEMPERATURE
, 2009
"... Dottorato di ricerca XXV ciclo in ..."
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"... Ion channels are the targets of many intra-cellular signaling pathways, including pro-tein phosphorylation and dephosphorylation. Indeed, nearly every type of voltage-gated K+, ..."
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Ion channels are the targets of many intra-cellular signaling pathways, including pro-tein phosphorylation and dephosphorylation. Indeed, nearly every type of voltage-gated K+,
