An ending is a new beginning: Transcription termination supports re-initiation

Comment on: Mapendano CK, et al. Mol Cell 2010; 40:410-22.     

Spatiotemporal control of cyclic AMP immunomodulation through the PKA-Csk inhibitory pathway is achieved by anchoring to an Ezrin-EBP50-PAG scaffold in effector T cells

A variety of immunoregulatory signals to effector T cells from monocytes, macrophages and regulatory T cells act through cyclic adenosine monophosphate. In the effector T cell, the protein kinase A (PKA) type I isoenzyme localizes to lipid rafts during T cell activation and modulates directly the proximal events that take place after engagement of the T cell receptor. The most proximal target for PKA phosphorylation is C-terminal Src kinase (Csk), which initiates a negative signal pathway that fine-tunes the T cell activation process. The A kinase anchoring protein Ezrin colocalizes PKA and Csk by forming a supramolecular signaling complex consisting of PKA, Ezrin, Ezrin/radixin/moesin (ERM) binding protein of 50 kDa (EBP50), phosphoprotein associated with glycosphingolipid-enriched membrane microdomains (GEMs) (PAG) and Csk.     

Rab32 is an A-kinase anchoring protein and participates in mitochondrial dynamics

A-kinase anchoring proteins (AKAPs) tether the cAMP-dependent protein kinase (PKA) and other signaling enzymes to distinct subcellular organelles. Using the yeast two-hybrid approach, we demonstrate that Rab32, a member of the Ras superfamily of small molecular weight G-proteins, interacts directly with the type II regulatory subunit of PKA. Cellular and biochemical studies confirm that Rab32 functions as an AKAP inside cells. Anchoring determinants for PKA have been mapped to sites within the conserved alpha5 helix that is common to all Rab family members. Subcellular fractionation and immunofluorescent approaches indicate that Rab32 and a proportion of the cellular PKA pool are associated with mitochondria. Transient transfection of a GTP binding-deficient mutant of Rab32 promotes aberrant accumulation of mitochondria at the microtubule organizing center. Further analysis of this mutant indicates that disruption of the microtubule cytoskeleton results in aberrantly elongated mitochondria. This implicates Rab32 as a participant in synchronization of mitochondrial fission. Thus, Rab32 is a dual function protein that participates in both mitochondrial anchoring of PKA and mitochondrial dynamics.     

Hydroacoustic observation of fish spatial patterns and behavior in the ship lock and adjacent areas of Gezhouba Dam, Yangtze River

The split-beam echo sounders were used in the No. 1 ship lock and the adjacent areas of the Gezhouba Dam Reach separately, to observe fish spatial distribution and the behavior of local fish about the Gezhouba Dam, and figure out whether the ship lock could served as a channel for fish migration in a way. The resulted showed that there were significance difference in both fish size (converted from the TS of individual fish) and abundance in the five marked sub-areas. Beyond that, there was an analogical fish distribution format in the analogical environmental condition. It might well be that fish species distributed in this area with the regioselectivity, which might resulted in the spatial differentiation. The horizontal fixed-location detection of the No. 1 ship lock revealed that, there were many ups and downs in the fish density with significant difference during a diurnal period, which might be resulted by the law of diel vertical distribution. There were all together 38 fishes moving up- or downstream the ship lock were detected during the period of ship-lock opening. In spite of the fact that, we cannot regard that the ship lock can served as the channel for fish migration. Because the evidence originated from the fish spatial differentiation and behavior in preference to that, it was the general behavior of that the fish swam backwards and forwards across the ship lock.     

FRET-based direct detection of dynamic protein kinase A activity on the sarcoplasmic reticulum in cardiomyocytes

The second messenger cAMP-dependent protein kinase A (PKA) plays an important role in the various cellular and physiological responses. On the sarcoplasmic reticulum (SR) in cardiomyocytes, PKA regulates the calcium cycling for exciting–contraction coupling, which is often dysfunctional in a variety of heart diseases including heart failure. Here, we have developed a novel FRET-based A-kinase activity biosensor (AKAR), termed SR-AKAR3, to visualize the PKA dynamics on the SR. Activation of adrenergic receptor induces a rapid and significant increase in SR-AKAR3 FRET ratio, which is dependent on agonist occupation of the receptor and inhibited by H-89, a PKA inhibitor. Interestingly, direct activation of adenylyl cyclases or application of a cAMP analog 8-Br-cAMP induced much slower and smaller increases in SR-AKAR3 FRET ratio. These data indicate that the signaling induced by adrenergic stimulation displays a preferential access to the SR in comparison to those by direct activation of adenylyl cyclases. More, SR-AKAR3 mimics endogenous protein phospholamban on the SR for PKA-mediated phosphorylation and myocyte contraction response under adrenergic stimulation. Together, this new PKA activity biosensor provides a useful tool to directly visualize the dynamic regulation of PKA activity on the SR in cardiomyocytes under various physiological and clinical conditions.     

Decreased mAKAP, ryanodine receptor, and SERCA2a gene expression in mdx hearts

Duchenne muscular dystrophy (DMD) is a common genetic disease resulting from mutations in the dystrophin gene. The lack of dystrophin function as a cytoskeletal protein leads to abnormal intracellular Ca(2+) homeostasis, the actual source and functional consequences of which remain obscure. The mdx mouse, a mouse model of DMD, revealed alterations in contractile properties that are likely due to defective Ca(2+) handling. However, the exact mechanisms of the Ca(2+) handling defect are unclear. We performed suppressive subtractive hybridization to isolate differentially expressed genes between 5-month-old mdx and control mice. We observed a decrease in muscle A-kinase anchoring protein (mAKAP) in the mdx hearts. We noticed not only down-regulation of mAKAP mRNA but also decreased mRNA level of the molecules involved in Ca(2+) handling and excitation-contraction (E-C) coupling in the sarcoplasmic reticulum (SR), the cardiac ryanodine receptor, and the sarcoplasmic reticulum Ca(2+) ATPase. Therefore, dystrophin deficiency may cause an impairment of SR Ca(2+) homeostasis and E-C coupling in mdx hearts, in part, by decreased gene expression of molecules involved in SR Ca(2+) handling.     

A statistical investigation of amphiphilic properties of C-terminally anchored peptidases

A number of DD-peptidases have been reported to interact with the membrane via C-terminal amphiphilic α-helices, but experimental support for this rests with a few well-characterized cases. These show the C-terminal interactions of DD-carboxypeptidases to involve high levels of membrane penetration, DD-endopeptidases to involve membrane surface binding and class C penicillin-binding proteins to involve membrane binding with intermediate properties. Here, we have characterized C-terminal α-helices from each of these peptidase groups according to their amphiphilicity, as measured by mean <μH>, and the corresponding mean hydrophobicity, <H>. Regression and statistical analyses showed these properties to exhibit parallel negative linear relationships, which resulted from the spatial ordering of α-helix amino acid residues. Taken with the results of compositional and graphical analyses, our results suggest that the use of C-terminal α-helices may be a universal feature of the membrane anchoring for each of these groups of DD-peptidases. Moreover, to accommodate differences between these mechanisms, each group of C-terminal α-helices optimizes its structural amphiphilicity and hydrophobicity to fulfil its individual membrane-anchoring function. Our results also show that each anchor type analysed requires a similar overall balance between amphiphilicity for membrane interaction, which we propose is necessary to stabilize their initial membrane associations. In addition, we present a methodology for the prediction of C-terminal α-helical anchors from the classes of DD-peptidases analysed, based on a parallel linear model.     

The role of protein phosphatase-1 in the modulation of synaptic and structural plasticity

Synaptic plasticity is a phenomenon contributing to changes in the efficacy of neuronal transmission. These changes are widely believed to be a major cellular basis for learning and memory. Protein phosphorylation is a key biochemical process involved in synaptic plasticity that operates through a tight balance between the action of protein kinases and protein phosphatases (PPs). Although the majority of research in this field has concentrated primarily on protein kinases, the significant role of PPs is becoming increasingly apparent. This review examines one such phosphatase, PP1, and highlights recent advances in the understanding of its intervention in synaptic and structural plasticity and the mechanisms of learning and memory.     

A-KinaseAnchoringProteins: a key to selective activation of cAMP-responsive events?

The cAMP-dependent protein kinase (PKA) regulates a variety of diverse biochemical events through the phosphorylation of target proteins. Because PKA is a multifunctional enzyme with a broad substrate specificity, its compartmentalization may be a key regulatory event in controlling which particular target substrates are phosphorylated. In recent years it has been demonstrated that differential localization of the type II holoenzyme is directed through interaction of the regulatory subunit (RII) with a family ofA-KinaseAnchoringProteins (AKAPs). In this report, we review evidence for PKA compartmentalization and discuss the structural and functional properties of AKAPs.