scholarly journals Rod Phosphodiesterase-6 (PDE6) Catalytic Subunits Restore Cone Function in a Mouse Model Lacking Cone PDE6 Catalytic Subunit

2011 ◽  
Vol 286 (38) ◽  
pp. 33252-33259 ◽  
Author(s):  
Saravanan Kolandaivelu ◽  
Bo Chang ◽  
Visvanathan Ramamurthy
Keyword(s):  
Mouse Model ◽  
Catalytic Subunit ◽  
Catalytic Subunits ◽  
Cone Function

scholarly journals Protein Kinase A Distribution in Meningioma

Cancers ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 1686 ◽  
Author(s):  
Caretta ◽  
Denaro ◽  
D’Avella ◽  
Mucignat-Caretta
Keyword(s):  
Brain Tissue ◽  
Catalytic Subunit ◽  
Therapeutic Interventions ◽  
Normal Brain ◽  
Local Concentration ◽  
Correlation Pattern ◽  
Catalytic Subunits ◽  
Tissue Specimens ◽  
Pka Catalytic Subunit

Deregulation of intracellular signal transduction pathways is a hallmark of cancer cells, clearly differentiating them from healthy cells. Differential intracellular distribution of the cAMP-dependent protein kinases (PKA) was previously detected in cell cultures and in vivo in glioblastoma and medulloblastoma. Our goal is to extend this observation to meningioma, to explore possible differences among tumors of different origins and prospective outcomes. The distribution of regulatory and catalytic subunits of PKA has been examined in tissue specimens obtained during surgery from meningioma patients. PKA RI subunit appeared more evenly distributed throughout the cytoplasm, but it was clearly detectable only in some tumors. RII was present in discrete spots, presumably at high local concentration; these aggregates could also be visualized under equilibrium binding conditions with fluorescent 8-substituted cAMP analogues, at variance with normal brain tissue and other brain tumors. The PKA catalytic subunit showed exactly overlapping pattern to RII and in fixed sections could be visualized by fluorescent cAMP analogues. Gene expression analysis showed that the PKA catalytic subunit revealed a significant correlation pattern with genes involved in meningioma. Hence, meningioma patients show a distinctive distribution pattern of PKA regulatory and catalytic subunits, different from glioblastoma, medulloblastoma, and healthy brain tissue. These observations raise the possibility of exploiting the PKA intracellular pathway as a diagnostic tool and possible therapeutic interventions.

scholarly journals The Role of Protein Phosphatase 2A Catalytic Subunit Cα in Embryogenesis: Evidence from Sequence Analysis and Localization Studies

1999 ◽  
Vol 380 (9) ◽  
pp. 1117-1120 ◽  
Author(s):  
Jürgen Götz ◽  
Wilfried Kues
Keyword(s):  
Protein Phosphatase ◽  
Genomic Organization ◽  
Protein Phosphatase 2A ◽  
Catalytic Subunit ◽  
Regulatory Subunit ◽  
Catalytic Subunits ◽  
Regulatory Subunits ◽  
Phosphatase 2A ◽  
The Brain

AbstractProtein phosphatase 2A (PP2A) constitutes one of the major families of protein serine/threonine phosphatases found in all eukaryotic cells. PP2A holoenzymes are composed of a catalytic subunit complexed with a structural regulatory subunit of 65 kDa. These core subunits associate with regulatory subunits of various sizes to form different heterotrimers which have been purified and evaluated with regard to substrate specificity. In fully differentiated tissues PP2A expression levels are highest in the brain, however, relatively little is known about expression in the developing embryo.In order to determine the composition of PP2A catalytic subunits in the mouse, cDNAs were cloned and the genomic organization of PP2A Cα was determined.By a gene targeting approach in the mouse, we have previously shown that the absence of the major catalytic subunit of PP2A, Cα, resulted in embryonic lethality around embryonic day E6.5. No mesoderm was formed which implied that PP2A plays a crucial role in gastrulation.Here, we extended our studies and analyzed wildtype embryos for Cα expression at subsequent stages of development. After gastrulation is completed, we find high expression of Cα restricted to the neural folds, which suggests that PP2A plays an additional pivotal role in neurulation.

Turnover and regulation of Na-K-ATPase in HeLa cells

1981 ◽  
Vol 241 (5) ◽  
pp. C173-C183 ◽  
Author(s):  
L. R. Pollack ◽  
E. H. Tate ◽  
J. S. Cook
Keyword(s):  
Transit Time ◽  
Hela Cells ◽  
Surface Density ◽  
Specific Binding ◽  
Surface Membrane ◽  
Catalytic Subunit ◽  
Cell Compartment ◽  
Catalytic Subunits ◽  
Synthetic Rate ◽  
Glycoside Intoxication

HeLa cells in log growth have 10(6) surface Na-K-ATPase molecules as estimated by the specific binding of [3H]-ouabain. Studies utilizing ouabain as a label show that the ligand is internalized at a rate corresponding to the turnover of three sets of Na-K-ATPase enzymes per generation. The label is taken up exclusively into a particulate cell compartment where it is codistributed with beta-hexosaminidase, identifying the internal compartment as lysosomal. Turnover is an important parameter in the recovery of the cells from glycoside intoxication. The unmetabolized glycoside is subsequently released by exocytosis. 13C-density-labeled Na-K-ATPase has been identified by specific phosphorylation of its catalytic subunit with [32P]ATP or [33P]ATP, and the rate of turnover of the density label is shown to be the same as the internalization of the ouabain-labeled site. There is a transit time of about 4 h from the onset of synthesis of the catalytic subunit to its insertion in the surface membrane; 2,800 catalytic subunits are synthesized per minute per cell, and 2,100 are turned over K+-starved cells respond to the stress in 24-30 h with modulation of the surface density of Na-K-ATPase the synthetic rate remains constant; the number of functional enzymes per cell is controlled by change in the rate constant for turnover.

scholarly journals Kinetics and thermodynamics of activation by pretreatment with guanosine 5′-[βγ-imido]triphosphate of smooth-muscle adenylate cyclase

1982 ◽  
Vol 205 (2) ◽  
pp. 249-255 ◽  
Author(s):  
J. Frederick Krall ◽  
Steven C. Leshon ◽  
Stanley G. Korenman
Keyword(s):  
Smooth Muscle ◽  
Adenylate Cyclase ◽  
Order Rate Constant ◽  
Catalytic Subunit ◽  
Appreciable Effect ◽  
Dependent Manner ◽  
Temperature Dependent ◽  
Catalytic Subunits ◽  
Pretreatment Temperature

Catalytic subunits (C) of uterine smooth-muscle adenylate cyclase were activated (C*) by incubating the enzyme with the GTP analogue guanosine 5′-[βγ-imido]triphosphate (p[NH]ppG), followed by treatment with GTP and washing at 2°C. Activation (C→C*) proceeded in a time- and temperature-dependent manner as disclosed by subsequent assay of the pretreated particles at 37°C. The properties of the activated subunits were a function of the pretreatment temperature and not those of the enzyme assay performed at 37°C. Over the range 6–24°C, activation by pretreatment with p[NH]ppG followed simple Michaelis–Menten kinetics, and increase in temperature increased the concentration of catalytic subunits in the C* state and decreased Km for the guanosine nucleotide. Characterization of the temperature-dependent effects of pretreatment with p[NH]ppG suggested that activation of the catalytic subunit at the temperature in situ (37°C) was moderately endergonic (ΔH0 ∼8kJ·mol−1) and accompanied by an increase in entropy (ΔS0 ∼146J·mol−1·K−1). The β-adrenergic catecholamine receptor, reflected by isoproterenol's effect on activation by pretreatment with p[NH]ppG, increased the concentration of catalytic subunits in the C* state but had an insignificant (P>0.05) effect on the Km at every temperature. This result suggested that formation of the receptor–hormone complex produced an increase in the first-order rate constant without an appreciable effect on the actual catalytic-subunit activation step. The primary function of the β-adrenergic catecholamine receptor under these conditions appeared to be regulation of the concentration of activation sites available for binding of p[NH]ppG.

scholarly journals Plasmin alters the activity and quaternary structure of human plasma carboxypeptidase N

2005 ◽  
Vol 388 (1) ◽  
pp. 81-91 ◽  
Author(s):  
Mercy O. QUAGRAINE ◽  
Fulong TAN ◽  
Hironori TAMEI ◽  
Ervin G. ERDÖS ◽  
Randal A. SKIDGEL
Keyword(s):  
Quaternary Structure ◽  
Gel Filtration ◽  
Catalytic Subunit ◽  
Surface Proteins ◽  
Covalent Bonds ◽  
Catalytic Subunits ◽  
Regulatory Subunits ◽  
C Terminus ◽  
Carboxypeptidase N ◽  
Structure And Activity

Human CPN (carboxypeptidase N) is a tetrameric plasma enzyme containing two glycosylated 83 kDa non-catalytic/regulatory subunits that carry and protect two active catalytic subunits. Because CPN can regulate the level of plasminogen binding to cell surface proteins, we investigated how plasmin cleaves CPN and the consequences. The products of hydrolysis were analysed by activity assays, Western blotting, gel filtration and sequencing. When incubated with intact CPN tetramer, plasmin rapidly cleaved the 83 kDa subunit at the Arg457–Ser458 bond near the C-terminus to produce fragments of 72 and 13 kDa, thereby releasing an active 142 kDa heterodimer, and also cleaved the active subunit, decreasing its size from 55 kDa to 48 kDa. Further evidence for the heterodimeric form of CPN was obtained by re-complexing the non-catalytic 72 kDa fragment with recombinant catalytic subunit or by immunoprecipitation of the catalytic subunit after plasmin treatment of CPN using an antibody specific for the 83 kDa subunit. Upon longer incubation, plasmin cleaved the catalytic subunit at Arg218–Arg219 to generate fragments of 27 kDa and 21 kDa, held together by non-covalent bonds, that were more active than the native enzyme. These data show that plasmin can alter CPN structure and activity, and that the C-terminal 13 kDa fragment of the CPN 83 kDa subunit is a docking peptide that is necessary to maintain the stable active tetrameric form of human CPN in plasma.

scholarly journals Protein Kinase A Regulates MYC Protein through Transcriptional and Post-translational Mechanisms in a Catalytic Subunit Isoform-specific Manner

2013 ◽  
Vol 288 (20) ◽  
pp. 14158-14169 ◽  
Author(s):  
Achuth Padmanabhan ◽  
Xiang Li ◽  
Charles J. Bieberich
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Term Effect ◽  
Catalytic Subunit ◽  
Short Term ◽  
Catalytic Subunits ◽  
Multiple Substrates ◽  
Pka Catalytic Subunit ◽  
Kinase A ◽  
In Cells

MYC levels are tightly regulated in cells, and deregulation is associated with many cancers. In this report, we describe the existence of a MYC-protein kinase A (PKA)-polo-like kinase 1 (PLK1) signaling loop in cells. We report that sequential MYC phosphorylation by PKA and PLK1 protects MYC from proteasome-mediated degradation. Interestingly, short term pan-PKA inhibition diminishes MYC level, whereas prolonged PKA catalytic subunit α (PKACα) knockdown, but not PKA catalytic subunit β (PKACβ) knockdown, increases MYC. We show that the short term effect of pan-PKA inhibition on MYC is post-translational and the PKACα-specific long term effect on MYC is transcriptional. These data also reveal distinct functional roles among PKA catalytic isoforms in MYC regulation. We attribute this effect to differential phosphorylation selectivity among PKA catalytic subunits, which we demonstrate for multiple substrates. Further, we also show that MYC up-regulates PKACβ, transcriptionally forming a proximate positive feedback loop. These results establish PKA as a regulator of MYC and highlight the distinct biological roles of the different PKA catalytic subunits.

scholarly journals Langevin Dynamics Simulation of AKAP-PKA Complex: Re-Envisioning the Local Concentration Mechanism for Directing PKA Phosphorylation

2017 ◽  
Author(s):  
Marc Rigatti ◽  
Paul J. Michalski ◽  
Kimberly L. Dodge-Kafka ◽  
Ion I. Moraru
Keyword(s):  
Molecular Mechanism ◽  
Characteristic Time ◽  
Langevin Dynamics ◽  
Catalytic Subunit ◽  
Dynamics Simulation ◽  
Regulatory Subunit ◽  
Signaling System ◽  
Average Characteristic ◽  
Catalytic Subunits ◽  
Regulatory Subunits

AbstractThe second messenger cAMP and its effector cAMP-dependent protein kinase A (PKA) constitute a ubiquitous cell signaling system. In its inactive state PKA is composed of two regulatory subunits that dimerize, and two catalytic subunits that are inhibited by the regulatory subunits. Activation of the catalytic subunits occurs upon binding of two molecules of cAMP to each regulatory subunit. Although many receptor types existing within the same cell may use this signaling system, compartmentation of signaling is thought to occur due to A-Kinase Anchoring Proteins (AKAPs), which act to co-localize PKA with specific substrates. However, the molecular mechanism allowing AKAPs to direct PKA phosphorylation to a particular substrate remained elusive, as prior evidence suggested that the catalytic subunit, which is highly diffusible, is released after cAMP binding to the regulatory subunit. Recent evidence from Smith et al. suggests that in the cell, the catalytic subunit may in fact not be released from the AKAP complex [1, 2]. They further demonstrated that alterations in the structure of the PKA regulatory subunit tether affect substrate phosphorylation. We use a novel computational software based on Langevin dynamics, SpringSaLaD, to simulate the AKAP-PKA complex in order to determine a molecular mechanism for the changes in phosphorylation seen with alteration in tether length and flexibility, and to demonstrate whether or not AKAPs can effectively direct PKA phosphorylation to a particular substrate upon release of the catalytic subunit from the complex. We find that short and flexible tethers contribute to a decrease in the average characteristic time of binding, allowing the catalytic subunit to spend more time in a bound state with the substrate, which yields faster characteristic times of phosphorylation. We further demonstrate that release of the catalytic subunit from the AKAP complex abrogates the effect of tethering, with characteristic times of phosphorylation similar to non-AKAP bound PKA. The data demonstrates that AKAPs likely do not release the catalytic subunit in directing PKA phosphorylation to AKAP bound substrates. In combination with the changes in characteristic time of phosphorylation which are driven by tether structure, this work indicates that the purpose of AKAPs may be to increase the efficiency of phosphorylation of particular AKAP substrates.

scholarly journals Native mass spectrometry identifies the HybG chaperone as carrier of the Fe(CN)2CO group during maturation of E. coli [NiFe]-hydrogenase 2

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Christian Arlt ◽  
Kerstin Nutschan ◽  
Alexander Haase ◽  
Christian Ihling ◽  
Dirk Tänzler ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Native Mass Spectrometry ◽  
Cysteine Residue ◽  
Catalytic Subunit ◽  
Accessory Proteins ◽  
Catalytic Subunits ◽  
E Coli ◽  
Client Proteins ◽  
Covalent Modifications ◽  
Hydrogenase 2

Abstract[NiFe]-hydrogenases activate dihydrogen. Like all [NiFe]-hydrogenases, hydrogenase 2 of Escherichia coli has a bimetallic NiFe(CN)2CO cofactor in its catalytic subunit. Biosynthesis of the Fe(CN)2CO group of the [NiFe]-cofactor occurs on a distinct scaffold complex comprising the HybG and HypD accessory proteins. HybG is a member of the HypC-family of chaperones that confers specificity towards immature hydrogenase catalytic subunits during transfer of the Fe(CN)2CO group. Using native mass spectrometry of an anaerobically isolated HybG–HypD complex we show that HybG carries the Fe(CN)2CO group. Our results also reveal that only HybG, but not HypD, interacts with the apo-form of the catalytic subunit. Finally, HybG was shown to have two distinct, and apparently CO2-related, covalent modifications that depended on the presence of the N-terminal cysteine residue on the protein, possibly representing intermediates during Fe(CN)2CO group biosynthesis. Together, these findings suggest that the HybG chaperone is involved in both biosynthesis and delivery of the Fe(CN)2CO group to its target protein. HybG is thus suggested to shuttle between the assembly complex and the apo-catalytic subunit. This study provides new insights into our understanding of how organometallic cofactor components are assembled on a scaffold complex and transferred to their client proteins.

Sign in / Sign up

Export Citation Format

Share Document