Mauno Vihinen¹, Pekka T. Mattsson^2,3 and C. I. Edvard Smith²

¹Department of Biosciences, Division of Biochemistry, P. O. Box 56, FIN-00014 University of Helsinki, Finland

²Center for BioTechnology, Department of Bioscience at Novum, Karolinska Institute, S-141 57 Huddinge and Department of Immunology, Microbiology, Pathology and Infectious Diseases (IMPI), Karolinska Institute, Huddinge University Hospital, S-141 86 Huddinge, Sweden

³Department of Biochemistry and Food Chemistry, University of Turku, Vatselankatu 2, Arcanum, FIN-20014 Turku, Finland

Received 11/24/96; Accepted 12/16/96; On-line 01/01/97

5. ACTIVATION OF BTK AND ITS CONNECTION TO SIGNAL TRANSDUCTION PATHWAYS

The Tec family of proteins appears to be involved in a vast array of signal transduction pathways. The signaling inadequancies in Xid mice suggest a pivotal role for Btk in lympho-hematopoietic growth and differentiation (16-21). The characteristics of the Tec family of PTKs are summarized in Table 1. Except for Bmx, which is expressed in bone marrow and endothelial cells, the members of the Tec family are mainly expressed in hematopoietic cell lineages. All Tec family kinases share the same organization consisting of PH, TH, SH3, SH2, and SH1 domains. The multiplicity of signals is guaranteed by different specificities and interplay of the domains of various proteins. A critical role for these domains in Btk associated signal transduction has been demonstrated by mutations found in XLA patients (28-31).

The crucial role of Btk in B cell differentiation has been studied by searching molecules regulating the activity of Btk and connecting it to various signal transduction pathways (32, 33). In Table 2, the known interactions of Btk and the regulation of Btk activity in B and mast cell signaling pathways are summarized. A gain-of-function mutation (Btk*) capable of transforming NIH3T3 cells has a single point mutation, E41K, in the PH domain (51). The Btk* exhibits increased tyrosine phosphorylation and a 5-fold enhancement in the membrane targeting (51). Although there is no evidence for direct interaction with G_ßgamma subunits or activation through the PH domain, Btk activity is stimulated by co-transfection with the subunits of heterotrimeric G protein (50).

Several PH domains have been found to associate with different phosphoinositides (56-65). It is possible that some PH domains serve as membrane-binding/associating units. Recently, PH domain of human ßISIGMAII spectrin was localized to the plasma membrane in vivo (66). Dbl PH domains may mediate cellular targeting to specific cytoskeletal locations (67). According to activation studies, also Btk is predominantly membrane-associated in cells (38). In additon, Btk PH domain interacts with both Ca²⁺-dependent and Ca²⁺-independent isoforms of protein kinase C (PKC) in mast cells resulting in inhibition of Btk (49). The cross-linking of IgE receptor (FcepsilonRI) has been shown to result in the activation of Btk (44). As the PKC plays important roles in many signal transduction pathways, including the FcepsilonRI signaling pathway (68), these results together with the membrane translocation of Btk and the activation of PLC-gamma (52) suggest Btk to function in membrane-proximal events following FcepsilonRI cross-linking (44).

The stimulation of antigen-specific B cell receptor (BCR) is intimately linked to the activation of three cytoplasmic tyrosine kinase families, namely the Src family, the Tec family and the Syk family (32). Time course -studies implicate temporal activation of these proteins. Src family kinases are activated first (5-10 seconds). This is followed by activation of Btk (2-5 minutes) and then Syk family of kinases (10-60 minutes) (36). This indicates a downstream role for Btk and Syk kinases in the signaling pathway which is initiated by the Src kinases. Recently, Btk activation was shown to correlate with the dose of Src family kinase activity (40).

The mechanism by which the Src kinases regulate Btk activity is not known in detail. The Blk, Fyn, Lyn and Hck may regulate Btk through an indirect mechanism, in which autophosphorylation of Btk Y551 is required for Btk activity (37). This observation is further supported by the interaction of Btk TH domain and Src family SH3 domains (43). In another study, Lyn was shown to activate Btk by transphosphorylating Y551 in the activation loop, after which Btk autophosphorylates at Y223 in the SH3 domain (38, 39), presumably affecting interactions with its partners. The identity of the partners of Btk during its activation and B cell differentiation is not yet known.

A number of activating signals lead to an increase in Btk activity and tyrosine phosphorylation. Btk and Tec are both stimulated via gp130 a receptor component of the interleukin-6 (IL-6) family of cytokines (48). Both kinases associate with gp130 in the absence of ligand (48), although the nature of the interacting domains remains to be determined. The growth factor IL-5 induces proliferation and differentiation of B cells by binding to receptor IL-5R and leads to the tyrosine phosphorylation of cellular proteins. IL-5 activation also stimulates JAK2 and Btk kinases (47).

Btk is involved in radiation-induced apoptosis in DT-40 lymphoma B cells. Btk, but not Lyn, Syk or Csk, mediates the radiation-induced apoptosis in a kinase domain-dependent manner (53). Recently, Xid B cells stimulated through surface IgM but not CD40 were shown to undergo apoptotic cell death (46). Cell viability correlates with the expression of bcl-x_L, a molecule which blocks apoptosis. bcl-x_L is suggested to be the first inducible protein downstream of Btk (46).

In Xid mice, serologic primary immune response is reduced due to substantially decreased number of memory B cells (55). The magnitude of the secondary response is not limited indicating that the reduced memory B cell number still exeeds a threshold value necessary for a normal secondary immune response (55).