Calyculin A–induced actin phosphorylation and depolymerization in renal epithelial cells

Gu, Lian‐Quan; Zhang, Hui; Chen, Qi; Chen, Jing

doi:10.1002/cm.10099

Cited by 15 publications

(11 citation statements)

References 39 publications

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“…Another category of proteins identified as differentially phosphorylated are the cytoskeleton proteins actin and vimentin. Both are known to be phosphorylated [48], [49], [50], [51], [52] in conjunction with cytoskeleton reorganization, which may conceivably occur during differentiation [53]. In particular, serine-phosphorylation of vimentin by Rho-associated kinase is involved in the agonist-induced neurite retraction of neuronal cells in NB [50]; these data are in accordance with our observation of a a diminution of phosphorylation levels following differentiation in NB leading to neurite spreading.…”

Section: Discussionsupporting

confidence: 91%

Identification of Phosphoproteins as Possible Differentiation Markers in All-Trans-Retinoic Acid-Treated Neuroblastoma Cells

Mandili

Marini

Carta³

et al. 2011

PLoS ONE

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BackgroundNeuroblastic tumors account for 9–10% of pediatric tumors and neuroblastoma (NB) is the first cause of death in pre-school age children. NB is classified in four stages, depending on the extent of spreading. A fifth type of NB, so-called stage 4S (S for special), includes patients with metastatic tumors but with an overall survival that approximates 75% at five years. In most of these cases, the tumor regresses spontaneously and regression is probably associated with delayed neuroblast cell differentiation.Methodology/Principal FindingsIn order to identify new early markers to follow and predict this process for diagnostic and therapeutics intents, we mimicked the differentiation process treating NB cell line SJ-NK-P with all-trans-retinoic acid (ATRA) at different times; therefore the cell proteomic pattern by mass spectrometry and the phosphoproteomic pattern by a 2-DE approach coupled with anti-phosphoserine and anti-phosphotyrosine western blotting were studied.Conclusions/SignificanceProteomic analysis identified only two proteins whose expression was significantly different in treated cells versus control cells: nucleoside diphosphate kinase A (NDKA) and reticulocalbin-1 (RCN1), which were both downregulated after 9 days of ATRA treatment. However, phosphoproteomic analysis identified 8 proteins that were differentially serine-phosphorylated and 3 that were differentially tyrosine-phosphorylated after ATRA treatment. All proteins were significantly regulated (at least 0.5-fold down-regulated). Our results suggest that differentially phosphorylated proteins could be considered as more promising markers of differentiation for NB than differentially expressed proteins.

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Section: Discussionsupporting

confidence: 91%

Identification of Phosphoproteins as Possible Differentiation Markers in All-Trans-Retinoic Acid-Treated Neuroblastoma Cells

Mandili

Marini

Carta³

et al. 2011

PLoS ONE

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“…For example, recent studies indicate that CalyA treatment induces focal adhesion assembly and tyrosine phosphorylation of specific protein substrates in fibroblasts [Leopoldt et al, 2001] and causes actin phos-phorylation-dependent redistributions in renal epithelia [Gu et al, 2003]. We are currently attempting to identify the coelomocyte proteins that are phosphorylated during drug treatment and we have also begun to examine tyrosine phosphorylation patterns in these cells.…”

Section: Caveats Of Calya Treatment and Summary Of Resultsmentioning

confidence: 97%

Actin‐based centripetal flow: Phosphatase inhibition by calyculin‐A alters flow pattern, actin organization, and actomyosin distribution

Henson

Kolnik

Fried

et al. 2003

Cell Motil. Cytoskeleton

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Previous studies have suggested that the actin-based centripetal flow process in sea urchin coelomocytes is the result of a two-part mechanism, actin polymerization at the cell edge coupled with actomyosin contraction at the cell center. In the present study, we have extended the testing of this two-part model by attempting to stimulate actomyosin contraction via treatment of coelomocytes with the phosphatase inhibitor Calyculin A (CalyA). The effects of this drug were studied using digitally-enhanced video microscopy of living cells combined with immunofluorescent localization and scanning electron microscopy. Under the influence of CalyA, the coelomocyte actin cytoskeleton undergoes a radical reorganization from a dense network to one displaying an array of tangential arcs and radial rivulets in which actin and the Arp2/3 complex concentrate. In addition, the structure and dynamics of the cell center are transformed due to the accumulation of actin and membrane in this region and the constriction of the central actomyosin ring. Physiological evidence of an increase in actomyosin-based contractility following CalyA treatment was demonstrated in experiments in which cells generated tears in their cell centers in response to the drug. Western blotting and immunofluorescent localization with antibodies against the phosphorylated form of the myosin regulatory light chain (MRLC) suggested that the demonstrated constriction of actomyosin distribution was the result of CalyA-induced phosphorylation of MRLC. Overall, the results suggest that there is significant cross talk between the two underlying mechanisms of actin polymerization and actomyosin contraction, and indicate that changes in actomyosin tension may be translated into alterations in the structural organization of the actin cytoskeleton.

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“…1996; Shtrahman et al. 2005); the PP2A inhibitor calyculin induces actin serine phosphorylation, depolymerization, filament redistribution and disassembly of F‐actin in renal epithelial cells (Gu et al. 2003).…”

Section: Discussionmentioning

confidence: 99%

“…Two spots separated on the pH axis of 2D gels were identified by MS as actin ( Fig. 8h) and probably correspond to actin and phospho-actin (Gu et al 2003).…”

Section: Phosphoproteomics Of Lfdmentioning

confidence: 99%

Calcineurin and cytoskeleton in low‐frequency depression

Silverman‐Gavrila

Charlton

2009

Journal of Neurochemistry

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kinase C; PMA, phorbol 12-myristate13-acetate; PP1, protein phosphatase 1; PP2A, protein phosphatase 2A; PP2B, protein phosphatase 2B; Rp-cAMPS, adenosine 3¢,5¢-cyclic monophosphorothioate Rp-isomer triethylammonium salt; SDS, sodium dodecyl sulphate. AbstractTransmitter release at high probability phasic synapses of crayfish neuromuscular junctions depresses by over 50% in 60 min when stimulated at 0.2 Hz. Inhibition of the protein phosphatase calcineurin by intracellular pre-synaptic injection of autoinhibitory peptide inhibited low-frequency depression (LFD) and resulted in facilitation of transmitter release. Since this inhibitor had no major effects when injected into the postsynaptic cell, only pre-synaptic calcineurin activity is necessary for LFD. To examine changes in phosphoproteins during LFD we performed a phosphoproteomic screen on proteins extracted from motor axons and nerve terminals after LFD induction or treatment with various drugs that affect kinase and phosphatase activity. Proteins separated by PAGE were stained with phospho-specific/total protein ratio stains (Pro-Q Diamond/SYPRO Ruby) to identify protein bands for analysis by mass spectrometry. Phosphorylation of actin and tubulin decreased during LFD, but increased when calcineurin was blocked. Tubulin and phosphoactin immunoreactivity in presynaptic terminals were also reduced after LFD. The actin depolymerizing drugs cytochalasin and latrunculin and the microtubule stabilizer taxol inhibited LFD. Therefore, dephosphorylation of pre-synaptic actin and tubulin and consequent changes in the cytoskeleton may regulate LFD. LFD is unlike long-term depression found in mammalian synapses because the latter requires in most instances postsynaptic calcineurin activity.Thus, this simpler invertebrate synapse discloses a novel pre-synaptic depression mechanism.

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Calyculin A–induced actin phosphorylation and depolymerization in renal epithelial cells

Cited by 15 publications

References 39 publications

Identification of Phosphoproteins as Possible Differentiation Markers in All-Trans-Retinoic Acid-Treated Neuroblastoma Cells

Identification of Phosphoproteins as Possible Differentiation Markers in All-Trans-Retinoic Acid-Treated Neuroblastoma Cells

Actin‐based centripetal flow: Phosphatase inhibition by calyculin‐A alters flow pattern, actin organization, and actomyosin distribution

Calcineurin and cytoskeleton in low‐frequency depression

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