Calyculin A reveals serine/threonine phosphatase protein phosphatase 1 as a regulatory nodal point in canonical signal transducer and activator of transcription 3 signaling of human microvascular endothelial cells.
ABSTRACT Vascular inflammation is initiated by stimuli acting on endothelial cells. A clinical feature of vascular inflammation is increased circulating interleukin 6 (IL-6) type cytokines such as leukemia inhibitory factor (LIF), but their role in vascular inflammation is not fully defined. IL-6 type cytokines activate transcription factor signal transducer and activator of transcription 3 (STAT3), which has a key role in inflammation and the innate immune response. Canonical STAT3 gene induction is due to phosphorylation of (1) Y705, leading to STAT3 dimerization and DNA binding and (2) S727, enhancing homodimerization and DNA binding by recruiting p300/CBP. We asked whether enhancing S727 STAT3 phosphorylation using the protein phosphatase 1 (PP1) inhibitor, calyculin A, would enhance LIF-induced gene expression in human microvascular endothelial cells (HMEC-1). Cotreatment with calyculin A and LIF markedly increased STAT3 S727 phosphorylation, without affecting the increase in the nuclear fraction of STAT3 phosphorylated on Y705. PP2A inhibitors, okadaic acid and fostriecin, did not enhance STAT3 S727 phosphorylation. Surprisingly, calyculin A eliminated LIF-induced gene expression: (1) calyculin A reduced binding of nuclear extracts to a STAT3 consensus site, thereby reducing the overall level of binding observed with LIF; and (2) calyculin A caused p300/CBP phosphorylation, thus resulting in reduced acetylation activity and degradation. Together, these findings reveal a pivotal role of a protein serine/threonine phosphatases that is likely PP1 in HMEC in controlling STAT3 transcriptional activity.
Full-textDOI: · Available from: Mazen Kurdi, Aug 13, 2015
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- "Sequence homologies of PP1, C subunit of PP2A and PP2B create difficulties to study inhibition of one phosphatase. Calyculin A, isolated from the marine sponge Discodermia calyx, is a combined large phosphates inhibitor which mainly acts on PP1 and PP2A (Ishihara et al., 1989; Resjo et al., 1999; Janssens and Goris, 2001; Edelson and Brautigan, 2012; Heimfarth et al., 2012; Zgheib et al., 2012). The effects of PP1 can only be attributed to PP1 after deduction of the PP2A specific effects solved by the use of OKA inhibition which has a negligible effect on PP1 activity. "
ABSTRACT: Tau phosphorylation is regulated by a balance between tau kinase and phosphatase activities. Disruption of this equilibrium was suggested to be at the origin of abnormal tau phosphorylation and thereby that might contributes to tau aggregation. Thus, understanding the regulation modes of tau dephosphorylation is of high interest in determining the possible causes at the origin of the formation of tau aggregates and to elaborate protection strategies to cope with these lesions in AD. Among the possible and relatively specific interventions that reverse tau phosphorylation is the stimulation of certain tau phosphatases. Here, we reviewed tau protein phosphatases, their physiological roles and regulation, their involvement in tau phosphorylation and the relevance to AD. We also reviewed the most common compounds acting on each tau phosphatase including PP2A.Ageing research reviews 07/2012; 12(1):39-49. DOI:10.1016/j.arr.2012.06.008 · 7.63 Impact Factor
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ABSTRACT: Multiple lines of evidence suggest that the transcription factor STAT3 is linked to a protective and reparative response in the heart. Thus, increasing duration or intensity of STAT3 activation ought to minimize damage and improve heart function under conditions of stress. Two recent studies using genetic mouse models, however, report findings that appear to refute this proposition. Unfortunately, studies often approach the question of the role of STAT3 in the heart from the perspective that all STAT3 signaling is equivalent, particularly when it comes to signaling by IL-6 type cytokines, which share the gp130 signaling protein. Moreover, STAT3 activation is typically equated with phosphorylation of a critical tyrosine residue. Yet, STAT3 transcriptional behavior is subject to modulation by serine phosphorylation, acetylation, and redox status of the cell. Unphosphorylated STAT3 is implicated in gene induction as well. Thus, how STAT3 is activated and also what other signaling events are occurring at the same time is likely to impact on the outcome ultimately linked to STAT3. Notably STAT3 may serve as a scaffold protein allowing it to interact with other singling pathways. In this context, canonical gp130 cytokine signaling may function to integrate STAT3 signaling with a protective PI3K/AKT signaling network via mutual involvement of JAK tyrosine kinases. Differences in the extent of integration may occur between those cytokines that signal through gp130 homodimers and those through heterodimers of gp130 with a receptor α chain. Signal integration may have importance not only for deciding the particular gene profile linked to STAT3, but for the newly described mitochondrial stabilization role of STAT3 as well. In addition, disruption of integrated gp130-related STAT3 signaling may occur under conditions of oxidative stress, which negatively impacts on JAK catalytic activity. For these reasons, understanding the importance of STAT3 signaling to heart function requires a greater appreciation of the plasticity of this transcription factor in the context in which it is investigated.04/2012; 1(2):101-10. DOI:10.4161/jkst.19776
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ABSTRACT: We previously showed that oxidative stress inhibits leukemia inhibitory factor (LIF) signaling by targeting JAK1, and the catalytic domains of JAK 1 and 2 have a cysteine-based redox switch. Thus, we postulated that the NO sibling and thiophylic compound, nitroxyl (HNO), would inhibit LIF-induced JAK-STAT3 activation. Pretreatment of human microvascular endothelial cells (HMEC-1) or neonatal rat cardiomyocytes with the HNO donors Angeli's salt or nitrosocyclohexyl acetate (NCA) inhibited LIF-induced STAT3 activation. NCA pretreatment also blocked the induction of downstream inflammatory genes (e.g. intercellular adhesion molecule 1, CCAAT/enhancer binding protein delta). The related 1-nitrosocyclohexyl pivalate (NCP; not a nitroxyl donor) was equally effective in inhibiting STAT3 activation, suggesting that these compounds act as thiolate targeting electrophiles. The JAK1 redox switch is likely not a target of acyloxy nitroso compounds, as NCA had no effect on JAK1 catalytic activity and only modestly affected JAK1-induced phosphorylation of the LIF receptor. However, pretreatment of recombinant human STAT3 with NCA or NCP reduced labeling of free sulfhydryl residues. We show that NCP in the presence of diamide enhanced STAT3 glutathionylation and dimerization in adult mouse cardiac myocytes and altered STAT3 under non-reducing conditions. Finally, we show that monomeric STAT3 levels are decreased in the Gαq model of heart failure in a redox-sensitive manner. Altogether, our evidence indicates that STAT3 has redox-sensitive cysteines that regulate its activation and are targeted by HNO donors and acyloxy nitroso compounds. These findings raise the possibility of new therapeutic strategies to target STAT3 signaling via a redox-dependent manner, particularly in the context of cardiac and non-cardiac diseases with prominent pro-inflammatory signaling.PLoS ONE 08/2012; 7(8):e43313. DOI:10.1371/journal.pone.0043313 · 3.53 Impact Factor