Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

AlphaII-spectrin is an in vitro target for caspase-2, and its cleavage is regulated by calmodulin binding.

ABSTRACT: The spectrin-actin scaffold underlying the lipid bilayer is considered to participate in cell-shape stabilization and in the organization of specialized membrane subdomains. These structures are dynamic and likely to undergo frequent remodelling during changes in cell shape. Proteolysis of spectrin, which occurs during apoptosis, leads to destabilization of the scaffold. It is also one of the major processes involved in membrane remodelling. Spectrins, the main components of the membrane skeleton, are the targets for two important protease systems: m- and micro-calpains (Ca2+-activated proteases) and caspase-3 (activated during apoptosis). In this paper, we show that caspase-2 also targets spectrin in vitro, and we characterize Ca2+/calmodulin-dependent regulation of spectrin cleavage by caspases. Yeast two-hybrid screening reveals that the large isoform (1/L) of procaspase-2 specifically binds to alphaII-spectrin, while the short isoform does not. Like caspase-3, caspase-2 cleaves alphaII-spectrin in vitro at residue Asp-1185. This study emphasizes a role of executioner caspase for caspase-2. We also demonstrated that the executioner caspase-7 but not caspase-6 cleaves spectrin at residue Asp-1185 in vitro. This spectrin cleavage by caspases 2, 3 and 7 is inhibited by the Ca2+-dependent binding of calmodulin to spectrin. In contrast, calmodulin binding enhances spectrin cleavage by calpain at residue Tyr-1176. These results indicate that alphaII-spectrin cleavage is highly influenced by Ca2+ homoeostasis and calmodulin, which therefore represent potential regulators of the stability and the plasticity of the spectrin-based skeleton.

SUBMITTER: Rotter B

PROVIDER: S-EPMC1223933 | biostudies-other | 2004 Feb

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Similar Datasets

A conserved sequence in calmodulin regulated spectrin-associated protein 1 links its interaction with spectrin and calmodulin to neurite outgrowth.

Project description:Calmodulin regulated spectrin-associated protein 1 (CAMSAP1) is a vertebrate microtubule-binding protein, and a representative of a family of cytoskeletal proteins that arose with animals. We reported previously that the central region of the protein, which contains no recognized functional domain, inhibited neurite outgrowth when over-expressed in PC12 cells [Baines et al., Mol. Biol. Evol. 26 (2009), p. 2005]. The CKK domain (DUF1781) binds microtubules and defines the CAMSAP/ssp4 family of animal proteins (Baines et al. 2009). In the central region, three short well-conserved regions are characteristic of CAMSAP-family members. One of these, CAMSAP-conserved region 1 (CC1), bound to both ?II?1-spectrin and Ca(2+)/calmodulin in vitro. The binding of Ca(2+)/calmodulin inhibited spectrin binding. Transient expression of CC1 in PC12 cells inhibited neurite outgrowth. siRNA knockdown of CAMSAP1 inhibited neurite outgrowth in PC12 cells or primary cerebellar granule cells: this could be rescued in PC12 cells by wild-type CAMSAP1-enhanced green fluorescent protein, but not by a CC1 mutant. We conclude that CC1 represents a functional region of CAMSAP1, which links spectrin-binding to neurite outgrowth.

| S-EPMC4016758 | biostudies-literature

Brain spectrin binding to the NMDA receptor is regulated by phosphorylation, calcium and calmodulin.

Project description:The N-methyl-D-aspartate receptor (NMDA-R) and brain spectrin, a protein that links membrane proteins to the actin cytoskeleton, are major components of post-synaptic densities (PSDs). Since the activity of the NMDA-R channel is dependent on the integrity of actin and leads to calpain-mediated spectrin breakdown, we have investigated whether the actin-binding spectrin may interact directly with NMDA-Rs. Spectrin is reported here to interact selectively in vitro with the C-terminal cytoplasmic domains of the NR1a, NR2A and NR2B subunits of the NMDA-R but not with that of the AMPA receptor GluR1. Spectrin binds at NR2B sites distinct from those of alpha-actinin-2 and members of the PSD95/SAP90 family. The spectrin-NR2B interactions are antagonized by Ca2+ and fyn-mediated NR2B phosphorylation, but not by Ca2+/calmodulin (CaM) or by Ca2+/CaM-dependent protein kinase II-mediated NR2B phosphorylation. The spectrin-NR1 interactions are unaffected by Ca2+ but inhibited by CaM and by protein kinase A- and C-mediated phosphorylations of NR1. Finally, in rat synaptosomes, both spectrin and NR2B are loosened from membranes upon addition of physiological concentrations of calcium ions. The highly regulated linkage of the NMDA-R to spectrin may underlie the morphological changes that occur in neuronal dendrites concurrently with synaptic activity and plasticity.

| S-EPMC1170728 | biostudies-other

Caspase-3/7-mediated Cleavage of β2-spectrin is Required for Acetaminophen-induced Liver Damage.

Project description:UnlabelledThe ubiquitously expressed β2-spectrin (β2SP, SPTBN1) is the most common non-erythrocytic member of the β-spectrin gene family. Loss of β2-spectrin leads to defects in liver development, and its haploinsufficiency spontaneously leads to chronic liver disease and the eventual development of hepatocellular cancer. However, the specific role of β2-spectrin in liver homeostasis remains to be elucidated. Here, we reported that β2-spectrin was cleaved by caspase-3/7 upon treatment with acetaminophen which is the main cause of acute liver injury. Blockage of β2-spectrin cleavage robustly attenuated β2-spectrin-specific functions, including regulation of the cell cycle, apoptosis, and transcription. Cleaved fragments of β2-spectrin were physiologically active, and the N- and C-terminal fragments retained discrete interaction partners and activity in transcriptional regulation and apoptosis, respectively. Cleavage of β2-spectrin facilitated the redistribution of the resulting fragments under conditions of liver damage induced by acetaminophen. In contrast, downregulation of β2-spectrin led to resistance to acetaminophen-induced cytotoxicity, and its insufficiency in the liver promoted suppression of acetaminophen-induced liver damage and enhancement of liver regeneration.Conclusionsβ2-Spectrin, a TGF-β mediator and signaling molecule, is cleaved and activated by caspase-3/7, consequently enhancing apoptosis and transcriptional control to determine cell fate upon liver damage. These findings have extended our knowledge on the spectrum of β2-spectrin functions from a scaffolding protein to a target and transmitter of TGF-β in liver damage.

| S-EPMC4737674 | biostudies-literature

Progranulin mediates caspase-dependent cleavage of TAR DNA binding protein-43.

Project description:TAR DNA binding protein-43 (TDP-43) is the pathologic substrate of neuronal and glial inclusions in frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTDL-U) and in amyotrophic lateral sclerosis (ALS). Mutations in the progranulin gene (PGRN) have been shown to cause familial FTLD-U. The relationship between progranulin and TDP-43 and their respective roles in neurodegeneration is unknown. We report that progranulin mediates proteolytic cleavage of TDP-43 to generate approximately 35 and approximately 25 kDa species. Suppression of PGRN expression with small interfering RNA leads to caspase-dependent accumulation of TDP-43 fragments that can be inhibited with caspase inhibitor treatment. Cells treated with staurosporine also induced caspase-dependent cleavage and redistribution of TDP-43 from its nuclear localization to cytoplasm. Altered cleavage and redistribution of TDP-43 in cell culture models are similar to findings in FTLD-U and ALS. The results suggest that abnormal metabolism of TDP-43 mediated by progranulin may play a pivotal role in neurodegeneration.

| S-EPMC6673167 | biostudies-literature

Calmodulin-binding proteins and calmodulin-regulated enzymes in dog pancreas.

Project description:Calmodulin was isolated and purified to homogeneity from dog pancreas. Highly purified subcellular fractions were prepared from dog pancreas by zonal sucrose-density ultracentrifugation and assayed for their ability to bind 125I-calmodulin in vitro. Proteins contained in these fractions were also examined for binding of 125I-calmodulin after their separation by polyacrylamide-gel electrophoresis in SDS. Calmodulin-binding proteins were detected in all subcellular fractions except the zymogen granule and zymogen-granule membrane fractions. One calmodulin-binding protein (Mr 240,000), observed in a washed smooth-microsomal fraction, has properties similar to those of alpha-fodrin. The postribosomal-supernatant fraction contained three prominent calmodulin-binding proteins, with apparent Mr values of 62,000, 50,000 and 40,000. Calmodulin-binding proteins, prepared from a postmicrosomal-supernatant fraction by Ca2+-dependent affinity chromatography on immobilized calmodulin, exhibited calmodulin-dependent phosphodiesterase, protein phosphatase and protein kinase activities. In the presence of Ca2+ and calmodulin, phosphorylation of smooth-muscle myosin light chain and brain synapsin and autophosphorylation of a Mr-50,000 protein were observed. Analysis of the protein composition of the preparation by SDS/polyacrylamide-gel electrophoresis revealed a major protein of Mr 50,000 which bound 125I-calmodulin. This protein shares characteristics with the calmodulin-dependent multifunctional protein kinase (kinase II) recently observed to have a widespread distribution. The possible role of calmodulin-binding proteins and calmodulin-regulated enzymes in the regulation of exocrine pancreatic protein synthesis and secretion is discussed.

| S-EPMC1147483 | biostudies-other

Phosphorylation-dependent interaction of SATB1 and PIAS1 directs SUMO-regulated caspase cleavage of SATB1.

Project description:Special AT-rich sequence-binding protein 1 (SATB1) is a tissue-restricted genome organizer that provides a key link between DNA loop organization, chromatin modification/remodeling, and transcription factor association at matrix attachment regions (MARs). The SUMO E3 ligase PIAS1 enhances SUMO conjugation to SATB1 lysine-744, and this modification regulates caspase-6 mediated cleavage of SATB1 at promyelocytic leukemia nuclear bodies (PML NBs). Since this regulated caspase cleavage occurs on only a subset of SATB1, and the products are relatively stable, proteolysis likely mediates cellular processes other than programmed cell death. However, the mechanism for the spatial and temporal regulation of SATB1 sumoylation and caspase cleavage is not known. Here we report that these processes are controlled by SATB1 phosphorylation; specifically, PIAS1 interaction with SATB1 is inhibited by phosphorylation. Mutagenesis studies identified interaction of the PIAS SAP (scaffold attachment factor-A/B/acinus/PIAS) motif with SATB1 N-terminal sequences. Notably, phosphorylation of SATB1 at threonine-188 regulates its interaction with PIAS1. Sequences near this phosphorylation site, LXXLL (residues 193 to 197), appear to be conserved among a subset of SUMO substrate proteins. Thus, this motif may be commonly involved in interaction with the PIAS SAP, and phosphorylation may similarly inhibit some of these substrates by preventing their interaction with the ligase.

| S-EPMC2876529 | biostudies-literature

Quantitative studies of caspase-3 catalyzed αII-spectrin breakdown.

Project description:Under various physiological and patho-physiological conditions, spectrin breakdown reactions generate several spectrin breakdown products (SBDPs)-in particular SBDPs of 150 kDa (SBDP150) and 120 kDa (SBDP120). Recently, numerous studies have shown that reactions leading to SBDPs are physiologically relevant, well regulated, and complex. Yet molecular studies on the mechanism of the SBDP formation are comparatively scarce. We have designed basic systems to allow us to follow the breakdown of αII-spectrin model proteins by caspase-3 in detail with gel electrophoresis, fluorescence and mass spectrometry methods. Amongst the predicted and reported sites, our results show that caspase-3 cleaves after residues D1185 and D1478, but not after residues D888, D1340 and D1475. We also found that the cleavage at these two sites is independent of each other. It may be possible to inhibit one site without affecting the other site. Cleavage after residue D1185 in intact αII-spectrin leads to SBDP150, and cleavage after D1478 site leads to SBDP120. Our results also show that the cleavage after the D1185 residue is unusually efficient, with a kcat/KM value of 40,000 M(-1) s(-1), and the cleavage after the D1478 site is more similar to most of the other reported caspase-3 substrates, with a kcat/KM value of 3000 M(-1) s(-1). We believe that this study lays out a methodology and foundation to study caspase-3 catalyzed spectrin breakdown to provide quantitative information. Molecular understanding may lead to better understanding of brain injuries and more precise and specific biomarker development.

| S-EPMC3786445 | biostudies-literature

An unbiased proteomic screen reveals caspase cleavage is positively and negatively regulated by substrate phosphorylation.

Project description:Post-translational modifications of proteins regulate diverse cellular functions, with mounting evidence suggesting that hierarchical cross-talk between distinct modifications may fine-tune cellular responses. For example, in apoptosis, caspases promote cell death via cleavage of key structural and enzymatic proteins that in some instances is inhibited by phosphorylation near the scissile bond. In this study, we systematically investigated how protein phosphorylation affects susceptibility to caspase cleavage using an N-terminomic strategy, namely, a modified terminal amino isotopic labeling of substrates (TAILS) workflow, to identify proteins for which caspase-catalyzed cleavage is modulated by phosphatase treatment. We validated the effects of phosphorylation on three of the identified proteins and found that Yap1 and Golgin-160 exhibit decreased cleavage when phosphorylated, whereas cleavage of MST3 was promoted by phosphorylation. Furthermore, using synthetic peptides we systematically examined the influence of phosphoserine throughout the entirety of caspase-3, -7, and -8 recognition motifs and observed a general inhibitory effect of phosphorylation even at residues considered outside the classical consensus motif. Overall, our work demonstrates a role for phosphorylation in controlling caspase-mediated cleavage and shows that N-terminomic strategies can be tailored to study cross-talk between phosphorylation and proteolysis.

| S-EPMC4014278 | biostudies-literature

Tyrosine phosphorylation regulates alpha II spectrin cleavage by calpain.

Project description:Spectrins, components of the membrane skeleton, are implicated in various cellular functions. Understanding the diversity of these functions requires better characterization of the interacting domains of spectrins, such as the SH3 domain. Yeast two-hybrid screening of a kidney cDNA library revealed that the SH3 domain of alpha II-spectrin binds specifically isoform A of low-molecular-weight phosphotyrosine phosphatase (LMW-PTP). The alpha II-spectrin SH3 domain does not interact with LMW-PTP B or C nor does LMW-PTP A interact with the alpha I-spectrin SH3 domain. The interaction of spectrin with LMW-PTP A led us to look for a tyrosine-phosphorylated residue in alpha II-spectrin. Western blotting showed that alpha II-spectrin is tyrosine phosphorylated in vivo. Using mutagenesis on recombinant peptides, we identified the residue Y1176 located in the calpain cleavage site of alpha II-spectrin, near the SH3 domain, as an in vitro substrate for Src kinase and LMW-PTP A. This Y1176 residue is also an in vivo target for kinases and phosphatases in COS cells. Phosphorylation of this residue decreases spectrin sensitivity to calpain in vitro. Similarly, the presence of phosphatase inhibitors in cell culture is associated with the absence of spectrin cleavage products. This suggests that the Y1176 phosphorylation state could modulate spectrin cleavage by calpain and may play an important role during membrane skeleton remodeling.

| S-EPMC133798 | biostudies-literature

Effect of Ca2+ on the promiscuous target-protein binding of calmodulin.

Project description:Calmodulin (CaM) is a calcium sensing protein that regulates the function of a large number of proteins, thus playing a crucial part in many cell signaling pathways. CaM has the ability to bind more than 300 different target peptides in a Ca2+-dependent manner, mainly through the exposure of hydrophobic residues. How CaM can bind a large number of targets while retaining some selectivity is a fascinating open question. Here, we explore the mechanism of CaM selective promiscuity for selected target proteins. Analyzing enhanced sampling molecular dynamics simulations of Ca2+-bound and Ca2+-free CaM via spectral clustering has allowed us to identify distinct conformational states, characterized by interhelical angles, secondary structure determinants and the solvent exposure of specific residues. We searched for indicators of conformational selection by mapping solvent exposure of residues in these conformational states to contacts in structures of CaM/target peptide complexes. We thereby identified CaM states involved in various binding classes arranged along a depth binding gradient. Binding Ca2+ modifies the accessible hydrophobic surface of the two lobes and allows for deeper binding. Apo CaM indeed shows shallow binding involving predominantly polar and charged residues. Furthermore, binding to the C-terminal lobe of CaM appears selective and involves specific conformational states that can facilitate deep binding to target proteins, while binding to the N-terminal lobe appears to happen through a more flexible mechanism. Thus the long-ranged electrostatic interactions of the charged residues of the N-terminal lobe of CaM may initiate binding, while the short-ranged interactions of hydrophobic residues in the C-terminal lobe of CaM may account for selectivity. This work furthers our understanding of the mechanism of CaM binding and selectivity to different target proteins and paves the way towards a comprehensive model of CaM selectivity.

| S-EPMC5898786 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data