Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC99
Bruton’s tyrosine kinase participates in the regulation of STIM 1 by tyrosine phosphorylation during SOCE in human platelets.
E. Lopez1, A. Berna-Erro1, G. M. Salido1, J. A. Rosado1, P. C. Redondo1
1. Physiology, University of Extremadura, C
Stromal interaction molecule 1 (STIM 1) is a key protein for conducting store-operated Ca2+ entry (SOCE) in human platelets that can be phosphorylated on Ser/Thr- and Pro-residues, as recently reported (1,2). In contrast, Tyr-phosphorylation of STIM1 remains poorly described (2), and is the aim of the present study. Human platelets were obtained from healthy donors, according to the Declaration of Helsinki, as previously described elswhere (3). Human platelets were stimulated with thapsigargin (TG; 200 nM), and then automatically fixed at different time points using a Quench flow system. Subsequently, STIM 1 immunoprecipitation and Western blotting using a specific anti-phosphor-tyrosine antibody (4G10) was performed. Treatment of dimethyl BAPTA-loaded human platelets suspended in a free Ca2+-medium (100 μM of EGTA was added) with TG evoked a maximum STIM1 Tyr-phosphorylation after 2.0 ± 0.5 s of stimulation. STIM1 located in the plasma membrane presented a similar Tyr-phosphorylation pattern. Incubation of human platelets for 10 min with LFM-A13 (10 μM), a specific Bruton’s tyrosine kinase (Btk) inhibitor (4), prevented TG-induced STIM1 Tyr-phosphorylation as well as coupling between STIM1 and Orai1, which has been presented as a key step for conducting SOCE in human platelets (n= 4-6)(5). Finally, we have used EGS-crosslinker in order to analyze whether Tyr-phosphorylation is required for STIM1 multimerization. Surprisingly, our results indicate that in resting platelets STIM1 is already multimerized. STIM1-multimerization was significantly enhanced by stimulation of human platelets with TG, independently of changes in the cytosolic Ca2+ concentration. Treatment of human platelets with 10 µM LFM-A13 for 10 min was unable to prevent TG-evoked STIM1 multimerization (n=4). Altogether, our results indicate that Btk-dependent STIM1 Tyr-phosphorylation is required for STIM1 complexing with others elements that participate in SOCE, but not for the STIM1 multimerization.
Where applicable, experiments conform with Society ethical requirements