Ca2+-independent activation of Bruton's tyrosine kinase is required for store-mediated Ca2+ entry in human platelets

doi:10.1016/j.cellsig.2004.11.019

Cellular Signalling

Volume 17, Issue 8, August 2005, Pages 1011-1021

https://doi.org/10.1016/j.cellsig.2004.11.019 Get rights and content

Abstract

Store-mediated Ca²⁺ entry (SMCE), which is rapidly activated by depletion of the intracellular Ca²⁺ stores, is a major mechanism for Ca²⁺ influx. Several studies have involved tyrosine kinases in the activation of SMCE, such as pp60^src, although at present those involved in the early activation steps are unknown. Here we report the involvement of Bruton's tyrosine kinase (Btk) in the early stages of SMCE in human platelets. Cell treatment with thrombin or thapsigargin (TG) plus ionomycin (Iono) results in rapid activation of Btk, which was independent of rise in intracellular Ca²⁺ concentration ([Ca²⁺]_i) but dependent on H₂O₂ generation. Platelet treatment with Btk inhibitors, LFM-A13 or terreic acid, significantly reduced TG+Iono- and thrombin-evoked SMCE. Btk was rapidly activated by addition of low concentrations of H₂O₂, whose effect on Ca²⁺ entry was prevented by Btk inhibitors. Our results indicate that pp60^src and Btk co-immunoprecipitate after platelet stimulation with TG+Iono, thrombin or H₂O₂. In addition, we have found that LFM-A13 impaired actin filament reorganization after store depletion and agonist-induced activation of pp60^src, while the inhibitor of pp60^src, a protein that requires actin reorganization for its activation, did not modify Btk activation, suggesting that Btk is upstream of pp60^src. We propose a role for Btk in the early steps of activation of SMCE in human platelets.

Introduction

Stimulation of platelets and other cells with agonists results in an increase in the intracellular Ca²⁺ concentration ([Ca²⁺]_i), which consist of two components: release of Ca²⁺ from internal stores and Ca²⁺ entry through plasma membrane (PM) channels [1]. In non-excitable cells, the main mechanism for Ca²⁺ influx is store-mediated Ca²⁺ entry (SMCE), where the filling state of the intracellular Ca²⁺ stores regulates the entry of Ca²⁺ across the PM [2]. However, the exact mechanism that underlies SMCE is not well understood. Hypotheses have considered both indirect and conformational coupling mechanisms. Indirect coupling supports the existence of a diffusible messenger generated by the Ca²⁺ pools upon store depletion. Alternatively, the conformational coupling proposes a physical interaction between the endoplasmic reticulum (ER) and the PM [3]. Recently, a secretion-like conformational coupling model has been proposed in several cell types including human platelets [4], [5], [6] that is based on a reversible coupling of the ER with the PM modulated by the actin cytoskeleton, as it does in secretion.

This model integrates some of the elements of the indirect and conformational coupling models, so that messenger molecules such as small GTPases, cytochrome P450 metabolites and tyrosine kinase proteins have been shown to interact with the actin cytoskeleton to activate Ca²⁺ entry [6], [7], [8], [9]. A role for tyrosine kinases in the regulation of SMCE has been demonstrated on the basis of the correlation between store depletion and the increase in phosphotyrosine levels, as well as the effects of different tyrosine kinase inhibitors, such as methyl 2,5-dihydroxycinnamate and genistein, on agonist- and TG-evoked Ca²⁺ entry [10], [11], [12]. Tyrosine kinases have been shown to be a component of the cytoskeleton-dependent signaling complex involved in the activation of SMCE [13]. To our knowledge, the only tyrosine kinase that has been demonstrated to be involved in SMCE is the cytosolic protein pp60^src [6], [13], [14]. We have previously shown that pp60^src associates with the newly polymerised actin cytoskeleton and then activates upon Ca²⁺ store depletion, a process that is required for the activation of SMCE [6], [13]. Therefore, since actin reorganization is needed for the activation of pp60^src this protein is expected to participate in the final steps of Ca²⁺ entry.

Bruton's tyrosine kinase (Btk) is an emerging cytosolic tyrosine kinase involved in signal transduction processes initiated by the occupation of a variety of surface receptors [15]. Btk is present in significant amount in human platelets and has been shown to be a component of cytoskeleton-related signaling complexes [16]. In the present study we sought to expand our understanding of the involvement of tyrosine kinases in SMCE in platelets. We report here that depletion of the intracellular Ca²⁺ stores evokes rapid Ca²⁺-independent activation of Btk and that this kinase is involved in the activation of SMCE in platelets, probably as a upstream modulator of pp60^src.

Section snippets

Materials

Fura-2 acetoxymethyl ester (fura-2/AM) and calcein were from Molecular Probes (Leiden, The Netherlands). Apyrase (grade VII), aspirin, bovine serum albumin, thrombin, phenylmethylsulfonyl fluoride, leupeptin, benzamidine, paraformaldehyde, fluorescein isothiocyanate-labeled phalloidin, nonidet P-40, thapsigargin (TG) and ionomycin (Iono) were from Sigma (Madrid, Spain). LFM-A13 and terreic acid were from Calbiochem (Madrid, Spain). Anti-phospho-Btk (Y-223) antibody was from Cell Signaling

Store depletion by TG+Iono or thrombin activates Btk in normal and dimethyl BAPTA-loaded human platelets

The activation of Btk was analysed by Western blotting using a rabbit monoclonal phosphospecific anti-Btk antibody that only detects Btk autophosphorylated at the tyrosine residue 223, which has been shown to be the full activated form of Btk [19], [20]. Treatment of human platelets in a Ca²⁺-free medium with 1 μM TG, a specific inhibitor of the endomembrane Ca²⁺-ATPase (SERCA) [21], plus a low concentration (50 nM) of Iono, required to induce extensive depletion of the intracellular Ca²⁺

Discussion

Platelet shows a substantial increase in protein tyrosine phosphorylation upon their activation, suggesting an important role for tyrosine kinases in platelet physiology. These proteins potentiate agonist-evoked Ca²⁺ signals, since loss of their activity contribute to the reduction in agonist-evoked elevations in [Ca²⁺]_i [10], [28]. The involvement of tyrosine phosphorylation in SMCE has been demonstrated in a number of cell types, including pancreatic acinar cells [29], brain microglia [30],

Acknowledgments

We thank Mercedes Gómez Blázquez for her technical assistance. P.C.R. is supported by a DGESIC fellowship (BFI2001-0624). N.B.A. held a fellowship from the Tunisian Ministry of High Education, Science, Research and Technology. This work was supported by MCyT-DGI grant BFI2001-0624.

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¹: These authors contributed equally to this work.

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Cellular Signalling

Abstract

Introduction

Section snippets

Materials

Store depletion by TG+Iono or thrombin activates Btk in normal and dimethyl BAPTA-loaded human platelets

Discussion

Acknowledgments

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