Protein Phosphatase 2C Inactivates F-actin Binding of Human Platelet Moesin

Hishiya, Akinori; Ohnishi, Motoko; Tamura, Shinji; Nakamura, Fumihiko

doi:10.1074/jbc.274.38.26705

Cited by 47 publications

(35 citation statements)

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“…Unfolding of the molecule into an active conformation occurs following binding to phosphoinositides and phosphorylation of the C-terminal threonine (T558 in moesin, T567 in ezrin, T564 in radixin) (Fievet et al, 2004). The open molecules bind with various membrane proteins (Tsukita et al, 1994;Bretscher et al, 1997;Reczek et al, 1997;Simons et al, 1998;Yonemura et al, 1998) at the N-terminal region and F-actin through the C-terminal domain Hishiya et al, 1999;Pearson et al, 2000). As osteosarcoma cells in culture express high levels of ezrin and phosphorylated ERM, it was possible that the high levels of phosphorylated ERM seen in single cells that arrived in the lung following tail vein injection was merely a residual effect of their growth in vitro.…”

Section: Discussionmentioning

confidence: 99%

The actin-cytoskeleton linker protein ezrin is regulated during osteosarcoma metastasis by PKC

et al. 2008

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Ezrin is a member of the ERM (ezrin, radixin, moesin) protein family and links F-actin to the cell membrane following phosphorylation. Ezrin has been associated with tumor progression and metastasis in several cancers including the pediatric solid tumors, osteosarcoma and rhabdomyosarcoma. In this study, we were surprised to find that ezrin was not constitutively phosphorylated but rather was dynamically regulated during metastatic progression in osteosarcoma. Metastatic osteosarcoma cells expressed phosphorylated ERM early after their arrival in the lung, and then late in progression, only at the invasive front of larger metastatic lesions. To pursue mechanisms for this regulation, we found that inhibitors of PKC (protein kinase C) blocked phosphorylation of ezrin, and that ezrin coimmunoprecipitated in cells with PKCa, PKCi and PKCc. Furthermore, phosphorylated forms of ezrin and PKC had identical expression patterns at the invasive front of pulmonary metastatic lesions in murine and human patient samples. Finally, we showed that the promigratory effects of PKC were linked to ezrin phosphorylation. These data are the first to suggest a dynamic regulation of ezrin phosphorylation during metastasis and to connect the PKC family members with this regulation.

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Section: Discussionmentioning

confidence: 99%

The actin-cytoskeleton linker protein ezrin is regulated during osteosarcoma metastasis by PKC

et al. 2008

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“…Phosphorylation at T567 in ezrin (the homologous sites are T564 in radixin and T558 in moesin) has received a great deal of attention because this phosphorylation event is believed to open up the NH 2 terminal-to-COOH terminal (N-C) binding of ezrin, tranforming ezrin into an active state with accessible domains for binding to membrane and F-actin. In vitro experiments with platelet moesin have indicated that T558 phosphorylation is necessary for binding to F-actin (16,23). The increased affinity to F-actin is not likely due to the phosphate group itself, because COOH terminal radixin fragments bind to F-actin with similar efficiencies, whether phosphorylated on T564 or not (22).…”

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confidence: 98%

High turnover of ezrin T567 phosphorylation: conformation, activity, and cellular function

Zhu¹,

Zhou²,

Mettler³

et al. 2007

American Journal of Physiology-Cell Physiology

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In its dormant state, the membrane cytoskeletal linker protein ezrin takes on a NH2 terminal-to-COOH terminal (N-C) binding conformation. In vitro evidence suggests that eliminating the N-C binding conformation by Thr567 phosphorylation leads to ezrin activation. Here, we found for resting gastric parietal cells that the levels of ezrin phosphorylation on Thr567 are low and can be increased to a small extent (∼40%) by stimulating secretion via the cAMP pathway. Treatment of cells with protein phosphatase inhibitors led to a rapid, dramatic increase in Thr567 phosphorylation by 400% over resting levels, prompting the hypothesis that ezrin activity is regulated by turnover of phosphorylation on Thr567. In vitro and in vivo fluorescence resonance energy transfer analysis demonstrated that Thr567 phosphorylation opens the N-C interaction. However, even in the closed conformation, ezrin localizes to membranes by an exposed NH2 terminal binding site. Importantly, the opened phosphorylated form of ezrin more readily cosediments with F-actin and binds more tightly to membrane than the closed forms. Furthermore, fluorescence recovery after photobleaching analysis in live cells showed that the Thr567Asp mutant had longer recovery times than the wild type or the Thr567Ala mutant, indicating the Thr567-phosphorylated form of ezrin is tightly associated with F-actin and the membrane, restricting normal activity. These data demonstrate and emphasize the functional importance of reversible phosphorylation of ezrin on F-actin binding. A novel model is proposed whereby ezrin and closely associated kinase and phosphatase proteins represent a motor complex to maintain a dynamic relationship between the varying membrane surface area and filamentous actin length.

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“…Recent studies have shown that phosphorylation of the moesin, ezrin, and radixin family of proteins is required for mediation of actin cross-linking to the PM (22,23). To induce phosphorylation-dependent association of the actinbinding proteins with the PM we treated human platelets with calyculin A, a serine/threonine phosphatase inhibitor that inhibits protein phosphatases 1 and 2 (11,23).…”

Section: Effect Of Cyt D and Jp On Preactivated Store-mediated Ca 2ϩmentioning

confidence: 99%

A Role for the Actin Cytoskeleton in the Initiation and Maintenance of Store-mediated Calcium Entry in Human Platelets

Rosado¹,

Jenner

Sage

2000

Journal of Biological Chemistry

192

189

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The nature of the mechanism underlying store-mediated Ca 2؉ entry has been investigated in human platelets through a combination of cytoskeletal modifications. Inhibition of actin polymerization by cytochalasin D or latrunculin A had a biphasic time-dependent effect on Ca 2؉ entry, showing an initial potentiation followed by inhibition of Ca 2؉ entry. Moreover, addition of these agents after induction of store-mediated Ca 2؉ entry inhibited the Ca 2؉ influx mechanism. Jasplakinolide, which reorganizes actin filaments into a tight cortical layer adjacent to the plasma membrane, prevented activation of store-mediated Ca 2؉ entry but did not modify this process after its activation. In addition, jasplakinolide prevented cytochalasin D-induced inhibition of store-mediated Ca 2؉ entry. Calyculin A, an inhibitor of protein serine/threonine phosphatases 1 and 2 which activates translocation of existing F-actin to the cell periphery without inducing actin polymerization, also prevented activation of store-mediated Ca 2؉ entry. Finally, inhibition of vesicular transport with brefeldin A inhibited activation of store-mediated Ca 2؉ entry but did not alter this mechanism once initiated. These data suggest that store-mediated Ca 2؉ entry in platelets may be mediated by a reversible trafficking and coupling of the endoplasmic reticulum with the plasma membrane, which shows close parallels to the events mediating secretion.In many cell types, including human platelets, depletion of the intracellular Ca 2ϩ stores induces entry of Ca 2ϩ across the plasma membrane (PM) 1 (1). However, the mechanism by which the filling state of stores is communicated to the PM remains unclear. Hypotheses have considered both indirect and direct coupling mechanisms. Indirect coupling assumes the existence of a diffusible messenger generated by the storage organelles such as cyclic GMP (2), cytochrome P-450 metabolites (3), tyrosine kinases (4, 5), or release of a calcium influx factor from depleted stores (6); however, no messenger molecule has been identified. Alternatively, direct coupling (conformational coupling) proposes a physical interaction between the endoplasmic reticulum (ER) and the PM and considers that some calcium sensitivity may reside on the InsP 3 receptor, which is thought to be responsible for transmitting information from the ER to the PM (7). Consistent with this model, some studies indicate that Ca 2ϩ entry is closely localized to sites where InsP 3 evokes emptying of the Ca 2ϩ stores and that Ca 2ϩ entry is unlikely to be activated by a diffusible molecule (8 -10).A different model suggests that Ca 2ϩ channels or membranebound activator molecules are exocytotically incorporated into the PM upon store depletion (10). This hypothesis is compatible with the conformational coupling model only if it is assumed that the link between the ER and the PM is mechanically weak before store depletion and strong afterward.Recently, a secretion-like coupling model has been proposed by Patterson et al. (11). This mechanism involves a phys...

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Protein Phosphatase 2C Inactivates F-actin Binding of Human Platelet Moesin

Cited by 47 publications

References 47 publications

The actin-cytoskeleton linker protein ezrin is regulated during osteosarcoma metastasis by PKC

The actin-cytoskeleton linker protein ezrin is regulated during osteosarcoma metastasis by PKC

High turnover of ezrin T567 phosphorylation: conformation, activity, and cellular function

A Role for the Actin Cytoskeleton in the Initiation and Maintenance of Store-mediated Calcium Entry in Human Platelets

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