Nucleotide Activation of the Ca-ATPase

Autry, Joseph M.; Rubin, John E.; Svensson, B. G.; Li, Ji; Thomas, David D.

doi:10.1074/jbc.m112.404434

Cited by 29 publications

(43 citation statements)

References 96 publications

(88 reference statements)

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“…This kinetic behavior for long time raised a debate about whether one or two ATP-binding sites existed for each SERCA monomer. Structural evidence supports the one-site hypothesis, and thus the second ATPase burst must result from a kinetic effect prompted by ATPbinding to the already phosphorylated catalytic portion [17,18].…”

supporting

confidence: 52%

Chelerythrine inhibits the sarco/endoplasmic reticulum Ca2+-ATPase and results in cell Ca2+ imbalance

Vieira

Oliveira

Valente

et al. 2015

Archives of Biochemistry and Biophysics

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supporting

confidence: 52%

Chelerythrine inhibits the sarco/endoplasmic reticulum Ca2+-ATPase and results in cell Ca2+ imbalance

Vieira

Oliveira

Valente

et al. 2015

Archives of Biochemistry and Biophysics

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“…FITC specifically reacts with Lys515 on SERCA, and the reaction with FITC is a measure of the structural integrity of the nucleotide-binding domain (N-domain) of SERCA [45]. FITC reaction with SERCA3b was analyzed using microsomes isolated from SERCA3b-overexpressing cells transfected with either empty vector or Bcl-2 encoding vector.…”

Section: Resultsmentioning

confidence: 99%

“…FITC is considered as a marker of the structural integrity of the N-domain of SERCA. FITC specifically labels Lys515 of SERCA, which is located close to the nucleotide binding site of the N-domain [35, 45]. Our FITC labeling data suggest that the N-domain of SERCA3b may be involved in the mechanism of Bcl-2/SERCA3b interaction.…”

Section: Discussionmentioning

confidence: 99%

Inhibition and conformational change of SERCA3b induced by Bcl-2

Hewarathna

Dremina

Schöneich

2017

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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An interaction of Bcl-2 with SERCA had been documented in vitro using the SERCA1a isoform isolated from rat skeletal muscle [Dremina, E. S., Sharov, V. S., Kumar, K., Azidi, A., Michaelis, E. K., Schöneich, C. (2004) Biochem. J. 383 (361–370)]. Here, we demonstrate the interaction of Bcl-2 with the SERCA3b isoform both in vitro and in cell culture. In vitro, the interaction of Bcl-2 with SERCA3b was studied using Bcl-2Δ21, a truncated form of human Bcl-2, and microsomes isolated from SERCA3b-overexpressing HEK-293 cells. For these experiments, SERCA3b was quantified by a combination of amino acid analysis and Western blotting. We observed that Bcl-2Δ21 both inactivates SERCA3b and co-immunoprecipitates with SERCA3b. The incubation with Bcl-2Δ21 changes the distribution of SERCA3b during sucrose density gradient centrifugation, likely as the result of Bcl-2Δ21-induced conformational change of SERCA3b. When SERCA3b-overexpressing HEK-293 cells were co-transfected with Bcl-2, Bcl-2-dependent SERCA3b inactivation was observed. In these cells, Bcl-2 interaction with SERCA3b was demonstrated by co-immunoprecipitation. Furthermore, overexpression of Bcl-2 reduced fluorescein isothiocyanate (FITC) labeling of SERCA3b. Together, our data provide evidence for the interaction of Bcl-2 with SERCA3b in vitro and in cell culture, and for Bcl-2-dependent conformational and functional changes of SERCA3b.

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“…Time-resolved Measurements-Time-resolved fluorescence decay measurements were taken by single photon counting (SPC-130-EM, Becker and Hickl) using a 485-nm subnanosecond pulse diode laser (PicoQuant), a 519-bandpass filter, and a PMH-100 photomultiplier module (Photonics Solutions) (46). Sensors contained the eGFP and mCherry FRET pair, and all experiments were performed with 50 -100 nM protein in PKC buffer containing 100 M ATP and 0.1 mg/ml of BSA.…”

Section: Methodsmentioning

confidence: 99%

Substrate Affinity Differentially Influences Protein Kinase C Regulation and Inhibitor Potency

Sommese¹,

Sivaramakrishnan

2016

Journal of Biological Chemistry

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The overlapping network of kinase-substrate interactions provides exquisite specificity in cell signaling pathways, but also presents challenges to our ability to understand the mechanistic basis of biological processes. Efforts to dissect kinase-substrate interactions have been particularly limited by their inherently transient nature. Here, we use a library of FRET sensors to monitor these transient complexes, specifically examining weak interactions between the catalytic domain of protein kinase C␣ and 14 substrate peptides. Combining results from this assay platform with those from standard kinase activity assays yields four novel insights into the kinase-substrate interaction. First, preferential binding of non-phosphorylated versus phosphorylated substrates leads to enhanced kinase-specific activity. Second, kinase-specific activity is inversely correlated with substrate binding affinity. Third, high affinity substrates can suppress phosphorylation of their low affinity counterparts. Finally, the substrate-competitive inhibitor bisindolylmaleimide I displaces low affinity substrates more potently leading to substrate selective inhibition of kinase activity. Overall, our approach complements existing structural and biophysical approaches to provide generalizable insights into the regulation of kinase activity.The canonical role of a protein kinase is to recognize, bind, and phosphorylate specific serine, threonine, or tyrosine residues on a substrate protein (1, 2). Given that kinases are one of the largest gene families in eukaryotes and are involved in nearly every cellular function (3), significant work has focused on understanding the mechanisms that dictate substrate-kinase pairings (4). Although the catalytic domains of eukaryotic kinases are structurally conserved (5, 6), the local environment around the substrate binding pocket of the kinase catalytic domain varies between kinases (7). This has led to the view that phosphorylation site recognition occurs through conserved residues flanking the phospho-residue on the substrate. Linear sequence motifs have now been identified for most kinases through a combination of structural analysis and peptide libraries (4, 8). Additionally, these phospho-sites are frequently found in unstructured regions of the substrate protein that may allow for more flexible accommodation in the kinase active site (9, 10). After recognizing the substrate, the catalytic domain then transfers a phosphate group from ATP to the phosphoresidue on the substrate. Phospho-transfer is followed by release of ADP and the phosphorylated substrate to prime the kinase for another catalytic cycle (11).Crystallographic and NMR approaches have provided detailed structural information on the catalytic domains of individual kinases in complex with a variety of nucleotide analogs and inhibitors (6, 12, 13). However, the lack of defined secondary structure surrounding the phospho-motif and the inherent transient nature of the interaction has limited efforts to dissect the different states of the ...

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Nucleotide Activation of the Ca-ATPase

Cited by 29 publications

References 96 publications

Chelerythrine inhibits the sarco/endoplasmic reticulum Ca2+-ATPase and results in cell Ca2+ imbalance

Chelerythrine inhibits the sarco/endoplasmic reticulum Ca2+-ATPase and results in cell Ca2+ imbalance

Inhibition and conformational change of SERCA3b induced by Bcl-2

Substrate Affinity Differentially Influences Protein Kinase C Regulation and Inhibitor Potency

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