Calyculin A and okadaic acid inhibit human platelet aggregation by blocking protein phosphatases types 1 and 2A

Nishikawa, Masakatsu; Toyoda, Hideki; Saito, Masahiro; Morita, Koichi; Tawara, Isao; Deguchi, Katsumi; Kuno, Toshiki; Shima, Hiroshi; Nagaos, Minako; Shirakawa, S

doi:10.1016/0898-6568(94)90061-2

Cited by 29 publications

(24 citation statements)

References 33 publications

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“…7), indicating the involvement of multiple signaling pathways. Other than MLCK (5) and MRLC phosphatase (50), phosphorylation of MRLC is also regulated by zipper-interacting protein kinase (21), citron kinase (51), death-associated protein kinase (9,12), and PP2A (10,22,41). The increase in pMRLC at the cell periphery in polyamine-depleted cells might be due to a complex signal transduction network involving multiple pathways.…”

Section: Discussionmentioning

confidence: 99%

Role of myosin regulatory light chain and Rac1 in the migration of polyamine-depleted intestinal epithelial cells

Ray

Guo

Patel

et al. 2007

American Journal of Physiology-Gastrointestinal and Liver Physi

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We have previously shown that polyamine depletion decreased migration, Rac activation, and protein serine threonine phosphatase 2A activity. We have also shown that polyamine depletion increased cortical F-actin and decreased lamellipodia and stress fibers. In this study, we used staurosporine (STS), a potent, cell-permeable, and broad-spectrum serine/ threonine kinase inhibitor, and studied migration. STS concentrations above 100 nM induced apoptosis. However, in polyamine-depleted cells, a lower concentration of STS (5 nM) increased attachment, spreading, Rac1 activation, and, subsequently, migration without causing apoptosis. STS-induced migration was completely prevented by a Rac1 inhibitor (NSC-23766) and dominant negative Rac1. These results imply that STS restores migration in polyamine-depleted cells through Rac1. The most important finding in this study was that polyamine depletion increased the association of phosphorylated myosin regulatory light chain (pThr 18 /Ser 19 -MRLC) at the cell periphery, which colocalized with thick cortical F-actin. Localization of pThr 18 -and pSer 19 -MRLC was found with stress fibers and nuclei, respectively. STS decreased the phosphorylation of cellular and peripheral pThr 18 -MRLC without any effect on nuclear pSer 19 -MRLC, dissolved thick cortical F-actin, and increased lamellipodia and stress fiber formation in polyamine-depleted cells. In control and polyaminedepleted cells, focal adhesion kinase (FAK) colocalized with stress fibers and the actin cortex, respectively. STS reorganized FAK, paxillin, and the cytoskeleton. These results suggest that polyamine depletion prevents the dephosphorylation of MRLC and thereby prevents the dynamic reorganization of the actin cytoskeleton and decreases lamellipodia formation resulting in the inhibition of migration.putrescine; ␣-difluoromethylornithine; F-actin; focal adhesion kinase; paxillin POLYAMINES [spermidine, spermine, and their diamine precursor putrescine (PUT)] are found in all tissues of almost all living species. These polyamines are organic, aliphatic, cationic amines. At physiological pH, they bind to proteins, DNA, RNA, and other negatively charged molecules to regulate diverse biological effects. The first rate-limiting step in polyamine synthesis is the production of PUT from the amino acid ornithine. This reaction is catalyzed by ornithine decarboxylase (ODC), which has one of the shortest half-lives of any mammalian enzyme and is present at extremely low levels in quiescent cells. Its activity is highly regulated to accommodate cellular function (6, 7). In our previous studies (17, 37, 46 -49), we showed that the early phase of mucosal healing due to cell migration requires polyamines and that polyamine depletion by means of treatment with ␣-difluoromethylornithine (DFMO), a specific inhibitor of ODC, inhibits migration in a rat stress ulcer model. Subsequently, we showed that the small intestinal epithelial crypt cell line IEC-6 could be used as an in vitro model to study the process of cell migration inv...

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Section: Discussionmentioning

confidence: 99%

Role of myosin regulatory light chain and Rac1 in the migration of polyamine-depleted intestinal epithelial cells

Ray

Guo

Patel

et al. 2007

American Journal of Physiology-Gastrointestinal and Liver Physi

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“…Activities of PP1, PP2A, PP2B, and PP2C were determined using [ 32 P]-phosphorylated MLC of chicken gizzards as a substrate, as described elsewhere. 26,[33][34][35] PP1 and PP2A activities were measured in a reaction mixture (50L) containing 20 mM Tris-HCl, pH 7.4, 50 mM NaCl, 0.1 mM EGTA, 2 mM sodium vanadate Ϯ 10 nM okadaic acid Ϯ heat-stable phosphatase inhibitor-2, 4 mM phenylmethylsulfonyl fluoride, and 200 g/mL leupeptin. PP2A was completely inhibited with 10 nM okadaic acid, whereas PP1 at this concentration was little affected.…”

Section: In Vitro Cpi Phosphorylation By Pkc and Its Effect On Myosinmentioning

confidence: 99%

Protein kinase C–catalyzed phosphorylation of an inhibitory phosphoprotein of myosin phosphatase is involved in human platelet secretion

Watanabe

Kataoka

et al. 2001

Blood

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Protein kinase C (PKC)–potentiated inhibitory phosphoprotein of myosin phosphatase (CPI) was detected in human platelets. Like smooth muscle CPI-17, in vitro phosphorylation of platelet CPI by PKC inhibited the activity of myosin phosphatase containing the PP1δ catalytic subunit and the 130-kd myosin-binding subunit (MBS). Treatment of intact platelets with thrombin or the stable thromboxane A2 analog STA2 resulted in increased phosphorylation of both CPI and MBS at Thr-696, whereas phorbol myristate acetate (PMA) and the Ca++ ionophore ionomycin only induced CPI phosphorylation. PMA induced slow adenosine triphosphate (ATP) secretion of fura 2–loaded platelets with no change in cytosolic Ca++. The PMA-induced increase in CPI phosphorylation preceded phosphorylation of 20-kd myosin light chain (MLC20) at Ser-19 and ATP secretion. The PKC inhibitor, GF109203X, inhibited PMA-induced phosphorylation of CPI and MLC20 with similar IC50 values. These findings suggest that the activation of PKC by PMA induces MLC20phosphorylation by inhibiting myosin phosphatase through phosphorylation of CPI. STA2-induced MLC20phosphorylation was also diminished but not abolished by GF109203X, even at high concentrations that completely inhibited STA2-induced CPI phosphorylation. A combination of the Rho-kinase inhibitor Y-27632 and GF109203X led to a further decrease in STA2-induced MLC20 phosphorylation, mainly because of a significant inhibition of MBS phosphorylation at Thr-696. Inhibition of STA2-induced ATP release by Y-27632, GF109203X, or both appeared to correlate with the extent of MLC20 phosphorylation. Thus, CPI phosphorylation by PKC may participate in inhibiting myosin phosphatase, in addition to the Rho-kinase–mediated regulation of myosin phosphatase, during agonist-induced platelet secretion.

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“…The A subunit in the core dimer links multiple regulatory B subunits in a fashion that determines the substrate specificity, the subcellular location, and the catalytic activity of the phosphatase (10). Blockade by generic Ser/Thr phosphatase inhibitors like okadaic acid and calyculin A impair agonist-induced platelet aggregation, secretion (11)(12)(13), and ␣ IIb ␤ 3 outside-in signaling functions such as adhesion and spreading to immobilized fibrinogen. This act may be independent of ␤ 3 Thr 753 phosphorylation status (14,15); however, okadaic acid and calyculin A can inhibit multiple Ser/Thr phosphatases such as PP1, PP2A, and PP4 (16).…”

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confidence: 99%

Protein Phosphatase 2A Negatively Regulates Integrin αIIbβ3 Signaling

Gushiken¹,

Patel

Liu³

et al. 2008

Journal of Biological Chemistry

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Integrin ␣ IIb ␤ 3 activation is critical for platelet physiology and is controlled by signal transduction through kinases and phosphatases. Compared with kinases, a role for phosphatases in platelet integrin ␣ IIb ␤ 3 signaling is less understood. We report that the catalytic subunit of protein phosphatase 2A (PP2Ac) associates constitutively with the integrin ␣ IIb ␤ 3 in resting platelets and in human embryonal kidney 293 cells expressing ␣ IIb ␤ 3 . The membrane proximal KVGFFKR sequence within the cytoplasmic domain of integrin ␣ IIb is sufficient to support a direct interaction with PP2Ac. Fibrinogen binding to ␣ IIb ␤ 3 during platelet adhesion decreased integrinassociated PP2A activity and increased the phosphorylation of a PP2A substrate, vasodilator associated phosphoprotein. Overexpression of PP2Ac ␣ in 293 cells decreased ␣ IIb ␤ 3 -mediated adhesion to immobilized fibrinogen. Conversely, small interference RNA mediated knockdown of endogenous PP2Ac ␣ expression in 293 cells, enhanced extracellular signal-regulated kinase (ERK1/2) and p38 activation, and accelerated ␣ IIb ␤ 3 adhesion to fibrinogen and von Willebrand factor. Inhibition of ERK1/2, but not p38 activation, abolished the increased adhesiveness of PP2Ac ␣ -depleted 293 cells to fibrinogen. Furthermore, knockdown of PP2A c␣ expression in bone marrow-derived murine megakaryocytes increased soluble fibrinogen binding induced by protease-activated receptor 4-activating peptide. These studies demonstrate that PP2Ac ␣ can negatively regulate integrin ␣ IIb ␤ 3 signaling by suppressing the ERK1/2 signaling pathway.Integrin cytoplasmic tails are devoid of any intrinsic catalytic activity. Nevertheless, integrins can transmit bidirectional signals across the plasma membrane of a cell and regulate several cellular processes, such as, adhesion, migration, and apoptosis. In the context of the major platelet integrin ␣ IIb ␤ 3 , emerging evidence indicates that cytoplasmic tails act as a molecular scaffold for intracellular enzymes and for both cytoskeletal and adaptor proteins and can either positively or negatively regulate signaling (1). For example, during an agonist-mediated insideout signaling process, talin interacts with the integrin ␤ 3 tail and induces integrin ␣ IIb ␤ 3 activation (2), whereas calcium and integrin-binding protein 1 binds to the ␣ IIb tail and negatively regulates ␣ IIb ␤ 3 activation (3).Subsequent binding of fibrinogen to the activated ␣ IIb ␤ 3 integrin initiates an outside-in signaling process that regulates platelet function. Outside-in signaling can be mediated by intricate interplay of a set of proteins that associate constitutively with the integrin and by others that either associate or dissociate with the integrin in response to fibrinogen binding. For instance, c-Src associates constitutively to the ␤ 3 tail (4). Fibrinogen binding to ␣ IIb ␤ 3 induces association of protein tyrosine phosphatase 1B, spleen tyrosine kinase, and protein kinase C␤ to the ␤ 3 tail (4 -6) and calcium and integrin-binding protein 1 to t...

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Calyculin A and okadaic acid inhibit human platelet aggregation by blocking protein phosphatases types 1 and 2A

Cited by 29 publications

References 33 publications

Role of myosin regulatory light chain and Rac1 in the migration of polyamine-depleted intestinal epithelial cells

Role of myosin regulatory light chain and Rac1 in the migration of polyamine-depleted intestinal epithelial cells

Protein kinase C–catalyzed phosphorylation of an inhibitory phosphoprotein of myosin phosphatase is involved in human platelet secretion

Protein Phosphatase 2A Negatively Regulates Integrin αIIbβ3 Signaling

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