Hiroyuki Mano

Tec family of protein-tyrosine kinases: an overview of their structure and function.

The Tec family is a recently emerging subfamily of non-receptor protein-tyrosine kinases (PTKs) represented by its first member, Tec. This family is composed of five members, namely Tec, Btk. Itk/Emt/Tsk, Bmx and Txk/Rlk. The most characteristic feature of this family is the presence of a pleckstrin homology (PH) domain in their protein structure. The PH domain is known to bind phosphoinositides; on this basis, Tec family PTKs may act as merge points of phosphotyrosine-mediated and phospholipid-mediated signaling systems. Many Tec family proteins are abundantly expressed in hematopoietic tissues, and are presumed to play important roles in the growth and differentiation processes of blood cells. Supporting this, mutations in the Btk gene cause X chromosome-linked agammaglobulinemia (XLA) in humans and X chromosome-linked immunodeficiency (Xid) in mice, indicating that Btk activity is indispensable for B-cell ontogeny. In addition, Tec family kinases have been shown to be involved in the intracellular signaling mechanisms of cytokine receptors, lymphocyte surface antigens, heterotrimeric G-protein-coupled receptors and integrin molecules. Efforts are being made to identify molecules which interact with Tec kinases to transfer Tec-mediated signals in vivo. Candidates for such second messengers include PLC-gamma2, guanine nucleotide exchange factors for RhoA and TFII-I/BAP-135. This review summarizes current knowledge concerning the input and output factors affecting the Tec kinases.

Tec/Bmx non-receptor tyrosine kinases are involved in regulation of Rho and serum response factor by Gα12/13

A transient transfection system was used to identify regulators and effectors for Tec and Bmx, members of the Tec non‐receptor tyrosine kinase family. We found that Tec and Bmx activate serum response factor (SRF), in synergy with constitutively active α subunits of the G12 family of GTP‐binding proteins, in transiently transfected NIH 3T3 cells. The SRF activation is sensitive to C3, suggesting the involvement of Rho. The kinase and Tec homology (TH) domains of the kinases are required for SRF activation. In addition, kinase‐deficient mutants of Bmx are able to inhibit Gα13‐ and Gα12‐induced SRF activation, and to suppress thrombin‐induced SRF activation in cells lacking Gαq/11, where thrombins effect is mediated by G12/13 proteins. Moreover, expression of Gα12 and Gα13 stimulates autophosphorylation and transphosphorylation activities of Tec. Thus, the evidence indicates that Tec kinases are involved in Gα12/13‐induced, Rho‐mediated activation of SRF. Furthermore, Src, which was previously shown to activate kinase activities of Tec kinases, activates SRF predominantly in Rho‐independent pathways in 3T3 cells, as shown by the fact that C3 did not block Src‐mediated SRF activation. However, the Rho‐dependent pathway becomes significant when Tec is overexpressed.

Tec protein-tyrosine kinase is an effector molecule of Lyn protein-tyrosine kinase.

The Tec family is a recently emerging subfamily among nonreceptor type protein‐tyrosine kinases (PTKs) consisting of Tec, Txk, Btk, Bmx, and Itk/Tsk/Emt. They have a long amino‐terminal unique region containing a pleckstrin homology domain and a Tec‐homology domain. We could previously show that, through the Tec‐homology domain, Tec is bound to Lyn kinase both in vitro and in vivo. Because Tec is coexpressed with Lyn in many hematopoietic cell types, it has been intriguing to investigate the biological role of the Tec‐Lyn association. Here we demonstrate that Lyn can phospho‐rylate tyrosine residues of the Tec protein, and thereby activate Tec in 3T3 fibroblasts. However, coexpression of Tec has little effect on the phospho‐tyrosine‐contents of Lyn. By using the in vitro kinase assay and the yeast system, we could prove that the Tec protein is a direct substrate of the Lyn kinase both in vitro and in vivo. From this evidence we conclude that Tec acts downstream of Lyn in intracellular signaling pathways. This is a novel case where one PTK is phosphorylated and regulated by an‐other.—Mano, H., Yamashita, Y., Miyazato, A., Mi‐ura, Y., Ozawa, K. Tec protein‐tyrosine kinase is an effector molecule of Lyn protein‐tyrosine kinase. FASEB J. 10, 637‐642 (1996)

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

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 protein-coupled receptors such as thromboxane A2 analog (U46619), thrombin, and thrombin receptor activating peptide (TRAP). TRAP-induced phosphorylation in Tec was significantly reduced under the conditions which abrogate fibrinogen binding to GP IIb–IIIa and subsequent platelet aggregation. However, TRAP induced significant 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 fibrinogen or collagen induced significant 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.

Grb10/GrbIR as an in vivo substrate of Tec tyrosine kinase

Tec is a member of the recently emerging subfamily among nonreceptor protein‐tyrosine kinases (PTKs). Although many members of this family have been shown to be involved in a wide range of cytokine‐mediated signalling systems, the molecular mechanism by which they exert in vivo effects remains obscure. To gain insights into the downstream pathways of Tec, we here looked for Tec‐interacting proteins (TIPs) by using the yeast two‐hybrid screening.

Mediation by the Protein-tyrosine Kinase Tec of Signaling between the B Cell Antigen Receptor and Dok-1

A variety of growth factor receptors induce the tyrosine phosphorylation of a nonreceptor protein-tyrosine kinase Tec as well as that of a Tec-binding protein of 62 kDa. Given the similarity in properties between this 62-kDa protein and p62Dok-1, the possibility that these two proteins are identical was investigated. Overexpression of a constitutively active form of Tec in a pro-B cell line induced the hyperphosphorylation of endogenous Dok-1. Tec also associated with Dok-1 in a phosphorylation-dependent manner in 293 cells. Tec mediated marked phosphorylation of Dok-1 both in vivo and in vitro, and this effect required both the Tec homology and Src homology 2 domains of Tec in addition to its kinase activity. Expression of Dok-1 in 293 cells induced inhibition of Ras activity, suggesting that Dok-1 is a negative regulator of Ras. In the immature B cell line Ramos, cross-linking of the B cell antigen receptor (BCR) resulted in tyrosine phosphorylation of Dok-1, and this effect was markedly inhibited by expression of dominant negative mutants of Tec. Furthermore, overexpression of Dok-1 inhibited activation of the c-fos promoter induced by stimulation of the BCR. These results suggest that Tec is an important mediator of signaling from the BCR to Dok-1.

HIGH EXPRESSION OF THE TEC GENE PRODUCT IN MURINE TESTICULAR GERM CELLS AND ERYTHROBLASTS

Tec is a novel non‐receptor‐type protein tyroslne kinase that was originally Identified from a murine liver cDNA library. While the function of Tec remains unknown, it was shown recently that two Tec‐related kinases are involved directly in the growth and differentiation of bone marrow stem cells. As the localization of Tec protein has not been reported yet, immunohlstochemical and immunochemlcal studies of various murine organs were conducted in the present study to clarify which cells express this kinase protein. An intense immunohistologic reaction was observed in neonatal and adult testicular germ cells, and neonatal and fetal hepatic erythroblasts. In addition, a clear immunostaining was noted in neonatal and adult tubal epithelial cells, hepatocytes, basal cells of the non‐glandular stomach, foveolar epithelium of the glandular stomach, sebaceous cells of the skin and fetal cartilage. The immune reaction of germ cells and erythroblasts was observed in the cell membrane, although this protein does not have a transmembrane domain. Supportive western blotting of testis, adult liver, spleen and heart of adult C.B‐17 mice with the use of anti‐Tec antibody demonstrated a heavy 70kDa band in the liver and testis, and a much weaker, small band in the heart and spleen. These results suggest that Tec protein has a specific role in testicular germ cells and erythroblasts.