Ca2+-independent activation of Bruton's tyrosine kinase is required for store-mediated Ca2+ entry in human platelets.
Store-mediated Ca(2+) entry (SMCE), which is rapidly activated by depletion of the intracellular Ca(2+) stores, is a major mechanism for Ca(2+) 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 Ca(2+) concentration ([Ca(2+)](i)) but dependent on H(2)O(2) 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 H(2)O(2), whose effect on Ca(2+) entry was prevented by Btk inhibitors. Our results indicate that pp60(src) and Btk co-immunoprecipitate after platelet stimulation with TG+Iono, thrombin or H(2)O(2). 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.
Redondo
Pedro C
PC
Department of Physiology, University of Extremadura, Spain.
Ben-Amor
Nidhal
N
Salido
Ginés M
GM
Bartegi
Aghleb
A
Pariente
José A
JA
Rosado
Juan A
JA
D000199:Actins / Q000378:metabolism; D000077329:Agammaglobulinaemia Tyrosine Kinase; D001792:Blood Platelets / Q000201:enzymology* / Q000378:metabolism; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002470:Cell Survival; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004789:Enzyme Activation; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D047468:Immunoprecipitation; D015759:Ionomycin / Q000494:pharmacology; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000737:chemistry*; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism; D019284:Thapsigargin / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism; D013997:Time Factors
D000199:Actins; D004533:Egtazic Acid; D015759:Ionomycin; D019284:Thapsigargin; D006861:Hydrogen Peroxide; D011505:Protein-Tyrosine Kinases; D000077329:Agammaglobulinaemia Tyrosine Kinase; C000625949:BTK protein, human; D016392:Proto-Oncogene Proteins pp60(c-src); D013917:Thrombin; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium