Tec is involved in G protein-coupled receptor- and integrin-mediated signalings in human blood platelets

Abstract

Tec is a non-receptor type tyrosine kinase which is tyrosine phosphorylated and activated upon stimulation of hematopoietic cells with various cytokines. The role of Tec in G protein-coupled receptor-and integrin-mediated signalings has not been elucidated. We therefore investigated the regulation of Tec in human blood platelets. Tec was rapidly tyrosine phosphorylated in response to platelet agonists which activate G proteincoupled receptors such as thromboxane A 2 analog (U46619), thrombin, and thrombin receptor activating peptide (TRAP). TRAP-induced phosphorylation in Tec was signi®cantly reduced under the conditions which abrogate ®brinogen binding to GP IIb ± IIIa and subsequent platelet aggregation. However, TRAP induced signi®cant levels of the phosphorylation even under these conditions and also in thrombasthenic platelets which lack functional GP IIb ± IIIa molecules, suggesting that activation of G-protein-coupled receptor causes the phosphorylation. To clarify whether integrin engagement by itself causes the phosphorylation in Tec, we examined the state of the phosphorylation in platelets activated by integrin engagement. Platelet adhesion to immobilized ®brinogen or collagen induced signi®cant levels of the phosphorylation. Furthermore, Tec was translocated to cytoskeleton in response to TRAP in a manner dependent on platelet aggregation, suggesting that Tec can be a component of integrin-mediated signalings. These results collectively indicate that Tec is involved in G protein-coupled receptor-and integrin-mediated signalings in human blood platelets.

Gα12 activates Rho GTPase through tyrosine-phosphorylated leukemia-associated RhoGEF

“…The exact mechanism of G␣12 to regulate Tec kinase is currently unclear. However, it is interesting to note that thrombin, which can activate the G␣12͞13 pathway, has also been reported to activate Tec in platelets (17). It is possible that activated G␣12 may recruit Tec …”

“…LARG (1-1543), ⌬PDZ-LARG (307-1543), ⌬N-LARG (617-1543), PDZ-RhoGEF, p115RhoGEF, Tec (1-629), and kinase domain-deleted Tec (Tec-KD) (1-358) were subcloned into pcDNA-myc vector with N-terminal myc-tag. cDNAs for Tec lacking TH domain (⌬TH-Tec) and the constitutively active form of Tec (mHTec), which has N-terminal myristoylation signal, were subcloned into pSR␣ mammalian expression vector (17,19). cDNAs encoding the constitutively active G␣12 (G␣12Q229L) and G␣13 (G␣13Q226L) were subcloned into pCMV5 vector.…”

“…RhoGEF with or without G␣12͞13 was incubated with Tec in the kinase buffer at 30°C for 20 min. The reactions were terminated by adding SDS͞PAGE sample buffer, and the samples were separated by SDS͞PAGE, followed by immunoblotting using anti-Tec antibody (17) or antiphosphotyrosine antibody PY20 (Zymed).…”

“…Tec kinases form a family of nonreceptor tyrosine kinases that share pleckstrin homology and Tec homology (TH) domains at the N-terminal region (16). These kinases are activated by various stimuli, including ligands for G protein-coupled receptors (17). However, their regulatory functions in cells remain unclear.…”

Molecular cloning of a docking protein, BRDG1, that acts downstream of the Tec tyrosine kinase

“…The NH 2 -terminal region of Btk, encompassing the PH domain and a portion of the TH domain, is able to bind directly to the ␣ subunit of heterotrimeric GTP-binding proteins (17), suggesting that Tec family kinases also might act downstream of such G proteins. Indeed, in human platelets, stimulation of the thrombin receptor, a transmembrane receptor coupled to G proteins, induces activation of Tec (18). Furthermore, integrin stimulation has been shown to regulate Tec activity (18).…”