Key Specifications Table
|Key Applications||Analytes Available||Detection Methods|
|Description||Ser/Thr Phosphatase Assay Kit 1 (K-R-pT-I-R-R)|
|Application||Ser/Thr Phosphatase Assay Kit 1used to detect PP2A activity by either dephosphorylation of the phosphopeptide.|
|Safety Information according to GHS|
|Storage and Shipping Information|
|Material Size||1 kit|
|Material Package||Kit capacity: 100 assays|
|Reference overview||Application||Pub Med ID|
|Antiproliferative mechanisms of action of the flavin dehydrogenase inhibitors diphenylene iodonium and di-2-thienyliodonium based on molecular profiling of the NCI-60 human tumor cell panel.|
James H Doroshow,Agnes Juhasz,Yun Ge,Susan Holbeck,Jiamo Lu,Smitha Antony,Yongzhong Wu,Guojian Jiang,Krishnendu Roy
Biochemical pharmacology 83 2012
Flavoprotein-dependent reactive oxygen species (ROS) play a critical role in cytokine-mediated signal transduction in normal tissues and tumor cells. The flavoenzyme inhibitors diphenylene iodonium (DPI) and di-2-thienyliodonium (DTI) have been used to inhibit membrane-bound, flavoprotein-containing NADPH oxidases, including epithelial and leukocyte NADPH oxidases (Nox1-5 and Duox 1 and 2). Recent evidence suggests that DPI can decrease tumor cell proliferation; however, the molecular mechanisms involved remain poorly defined. To explore the mechanisms underlying DPI- and DTI-related tumor cell growth delay, we examined growth inhibition patterns produced by both agents in the NCI-60 tumor panel, and determined expression levels of Nox gene family members across these cell lines. Possible molecular targets were predicted using the COMPARE program. DPI was more potent than DTI (GI(50): 10nM versus 10μM); DPI and DTI exposure produced unique patterns of growth inhibition when evaluated against the small molecule anticancer database of the National Cancer Institute. Growth inhibition profiling of DPI revealed a modest positive correlation with Nox1 levels; novel mechanisms of DPI and DTI action, including alterations in Stat, Erk1/2, and Akt pathways, were inferred by correlation with NCI-60 Affymetrix(®) array data. Exposure of HT-29 colon cancer cells, which express Nox1, to DPI and DTI confirmed their inhibitory effects on steady state ROS levels, and demonstrated decreased Stat, Erk1/2, and Akt signaling mediated by IL-4, IL-6, IL-13, and IL-22, possibly due to a concomitant increase in tumor cell phosphatase activity. These findings suggest that DPI and DTI may act therapeutically by altering ROS-related signal transduction.
|Transmigration of beta amyloid specific heavy chain antibody fragments across the in vitro blood-brain barrier.|
Rutgers KS, Nabuurs RJ, van den Berg SA, Schenk GJ, Rotman M, Verrips CT, van Duinen SG, Maat-Schieman ML, van Buchem MA, de Boer AG, van der Maarel SM
Previously selected amyloid beta recognizing heavy chain antibody fragments (VHH) affinity binders derived from the Camelid heavy chain antibody repertoire were tested for their propensity to cross the blood-brain barrier (BBB) using an established in vitro BBB co-culture system. Of all tested VHH, ni3A showed highest transmigration efficiency which is, in part, facilitated by a three amino acid substitutions in its N-terminal domain. Additional studies indicated that the mechanism of transcellular passage of ni3A is by active transport. As VHH ni3A combines the ability to recognize amyloid beta and to cross the BBB, it has potential as a tool for non-invasive in vivo imaging and as efficient local drug targeting moiety in patients suffering from cerebral amyloidosis such as Alzheimer\'s disease (AD) and cerebral amyloid angiopathy (CAA).Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.
|Failure of stress-induced downregulation of Bcl-2 contributes to apoptosis resistance in senescent human diploid fibroblasts.|
S J Ryu, Y S Oh, S C Park
Cell death and differentiation 14 1020-8 2007
We previously reported that senescent human diploid fibroblasts (HDFs) are resistant to apoptosis induced by H(2)O(2) and staurosporine. We report here that senescent HDFs are resistant to thapsigargin-induced apoptosis as well. These agonists caused the reductions in mitochondrial membrane potential (MMP) and in the apoptosis inhibitory protein (B-cell lymphoma) only in young HDFs but not in senescent HDFs. In addition, downregulation of Bcl-2 increased the sensitivity of senescent HDFs to apoptosis induction, suggesting the significant role of Bcl-2 in apoptosis resistance of the senescent HDFs. We further found that P-cAMP response element-binding protein (CREB), a positive regulator of Bcl-2, decreased in stress-induced apoptosis of young HDFs but not in senescent HDFs, and that Bcl-2 was markedly reduced in CREB small interfering RNA (siRNA), transfected senescent HDFs. In addition, activity of protein phosphatase 2A (PP2A), which dephosphorylates p-CREB, significantly increased in young HDFs but not in senescent HDFs treated with H(2)O(2), staurosporine or thapsigargin. Taken together, these results suggest that failure of stress-induced downregulation of Bcl-2 underlies resistance of senescent HDFs to apoptosis.
|FMRP phosphorylation reveals an immediate-early signaling pathway triggered by group I mGluR and mediated by PP2A.|
Usha Narayanan, Vijayalaxmi Nalavadi, Mika Nakamoto, David C Pallas, Stephanie Ceman, Gary J Bassell, Stephen T Warren
The Journal of neuroscience : the official journal of the Society for Neuroscience 27 14349-57 2007
Fragile X syndrome is a common form of inherited mental retardation and is caused by loss of fragile X mental retardation protein (FMRP), a selective RNA-binding protein that influences the translation of target messages. Here, we identify protein phosphatase 2A (PP2A) as an FMRP phosphatase and report rapid FMRP dephosphorylation after immediate group I metabotropic glutamate receptor (mGluR) stimulation (1 min) in neurons caused by enhanced PP2A enzymatic activity. In contrast, extended mGluR activation (1-5 min) resulted in mammalian target of rapamycin (mTOR)-mediated PP2A suppression and FMRP rephosphorylation. These activity-dependent changes in FMRP phosphorylation were also observed in dendrites and showed a temporal correlation with the translational profile of select FMRP target transcripts. Collectively, these data reveal an immediate-early signaling pathway linking group I mGluR activity to rapid FMRP phosphorylation dynamics mediated by mTOR and PP2A.
|Specific beta1 integrin site selectively regulates Akt/protein kinase B signaling via local activation of protein phosphatase 2A.|
Pankov, Roumen, et al.
J. Biol. Chem., 278: 18671-81 (2003) 2003
Integrin transmembrane receptors generate multiple signals, but how they mediate specific signaling is not clear. Here we test the hypothesis that particular sequences along the beta(1) integrin cytoplasmic domain may exist that are intimately related to specific integrin-mediated signaling pathways. Using systematic alanine mutagenesis of amino acids conserved between different beta integrin cytoplasmic domains, we identified the tryptophan residue at position 775 of human beta(1) integrin as specific and necessary for integrin-mediated protein kinase B/Akt survival signaling. Stable expression of a beta(1) integrin mutated at this amino acid in GD25 beta(1)-null cells resulted in reduction of Akt phosphorylation at both Ser(473) and Thr(308) activation sites. As a consequence, the cells were substantially more sensitive to serum starvation-induced apoptosis when compared with cells expressing wild type beta(1) integrin. This inactivation of Akt resulted from increased dephosphorylation by a localized active population of protein phosphatase 2A. Both Akt and protein phosphatase 2A were present in beta(1) integrin-organized cytoplasmic complexes, but the activity of this phosphatase was 2.5 times higher in the complexes organized by the mutant integrin. The mutation of Trp(775) specifically affected Akt signaling, without effects on other integrin-activated pathways including phosphoinositide 3-kinase, MAPK, JNK, and p38 nor did it influence activation of the integrin-responsive kinases focal adhesion kinase and Src. The identification of Trp(775) as a specific site for integrin-mediated Akt signaling supports the concept of specificity of signaling along the integrin cytoplasmic domain.
|Reduced expression of the regulatory A subunit of serine/threonine protein phosphatase 2A in human breast cancer MCF-7 cells|
Suzuki, K. and Takahashi, K.
Int J Oncol, 23:1263-8 (2003) 2003
|N-Ethylmaleimide inhibits platelet-derived growth factor BB-stimulated Akt phosphorylation via activation of protein phosphatase 2A.|
Yellaturu, CR; Bhanoori, M; Neeli, I; Rao, GN
The Journal of biological chemistry 277 40148-55 2002
The redox state plays an important role in gene regulation. Thiols maintain the intracellular redox homeostasis. To understand the role of thiols in redox signaling, we have studied the effect of thiol alkylation on platelet-derived growth factor-BB (PDGF-BB)-induced cell survival events in vascular smooth muscle cells. PDGF-BB stimulated Akt phosphorylation predominantly at Ser-473. N-Ethylmaleimide (NEM), a thiol alkylating agent, blocked PDGF-BB-induced Akt phosphorylation without affecting its upstream phosphatidylinositol 3-kinase (PI3K). On the other hand, LY294002 and wortmannin, specific inhibitors of PI3K, prevented PDGF-BB-induced phosphorylation of Akt and its downstream effector molecules, p70S6K, ribosomal protein S6, 4E-BP1, and eIF4E. NEM also abrogated the phosphorylation of p70S6K, ribosomal protein S6, 4E-BP1, and eIF4E induced by PDGF-BB, suggesting that thiol alkylation interferes with the PI3K/Akt pathway at the level of Akt. In addition, NEM blocked PDGF-BB-induced phosphorylation of BAD and forkhead transcription factor FKHR-L1, and these events correlated with increased apoptosis. NEM alone and in concert with PDGF-BB increased reactive oxygen species (ROS) production and protein phosphatase 2A (PP2A) activity in VSMC. The inhibition of PDGF-BB-induced Akt phosphorylation by NEM was completely reversed by PP2A inhibitors fostriecin and okadaic acid, ceramide synthase inhibitor fumonisin B1, and ROS scavenger N-acetylcysteine (NAC). NAC also attenuated the apoptosis induced by NEM, alone or in combination with PDGF-BB. Together, these findings demonstrate for the first time that PP2A mediates thiol alkylation-dependent redox regulation of Akt and cell survival.
|Serine (threonine) phosphatase(s) acting on cAMP-dependent phosphoproteins in mammalian mitochondria.|
Signorile A, Sardanelli AM, Nuzzi R, Papa S.
FEBS letters 512 91-4 2001
Immunochemical and functional evidence showing the existence in the inner membrane and matrix fraction of mammalian mitochondria of serine/threonine phosphatases acting on cAMP-dependent phosphoproteins is presented. Mg(2+)-dependent Ca(2+)-inhibitable PP2C phosphatase, associated to the inner membrane, dephosphorylates the 18 kDa (NDUFS4 gene) of complex I.
|The serine phosphatases PP1 and PP2A associate with and activate the actin-binding protein cofilin in human T lymphocytes|
Ambach, A., et al
Eur J Immunol, 30:3422-31 (2000) 2000
|Regulation of protein phosphatase 2A activity by caspase-3 during apoptosis|
Santoro, M. F., et al
J Biol Chem, 273:13119-28 (1998) 1998