ANÁLISIS DEL MERCADO

The protein named Bruton’s tyrosine kinase (Btk) is a member o f the Btk/Tec family o f non-receptor tyrosine kinases (NRTKs). Upon cell stimulation, the recruitment o f NRTKs to the plasma membrane and activated signalling complexes occurs without direct interactions with cell surface receptors. The Btk/Tec family proteins consist o f five distinct structural domains (Tsukada et al., 1993). Like the Src family NRTKs, the Btk/Tec proteins contain a catalytic SHI kinase domain, and also the non-catalytic SH2 and SH3 domains. However, in addition the Btk/Tec protein exhibits a number o f distinct structural features which distinguish these two families o f NRTKs and suggest ways in which the mechanisms o f regulation o f the two families may differ. As an exemplar o f the Btk/Tec family, Btk lacks the N-terminal myristoylation signal which targets Src to the plasma membrane. Btk also lacks the C- terminal tyrosine residue (Y527) which is crucial for Src regulation. In contrast, the N-terminal region o f Btk/Tec family kinases contains a PH domain and a Tec homology (TH) domain (Vetrie et al., 1993), (Rawlings et al., 1993) {Figure 4-1). It is interesting to speculate that, since PH domains are considered to act as general membrane localisation devices (Hemmings, 1997), there may exist an evolutionary relationship between the Src and Btk/Tec family NRTKs in which the N-terminal Src myristoylation signal was functionally replaced by the Btk PH domain, or vice-versa.

Although the functions o f Btk are not clearly determined, its physiological importance has been known for some time. The loss o f Btk activity results in the human immunodeficiency called X-linked agammaglobulinaemia (XLA), which is estimated to affect 1 in 150 000 males (Mattsson et al., 1996), and also in murine X- linked immunodeficiency (Xid) (Thomas et al., 1993). Btk is crucial for B-cell development and proliferation at the transition between the pre-B-cell stage and later B-cell stages. Mutations in Btk can therefore result in a decreased number o f mature B-cells, resulting in the increased susceptibility to bacterial infections which characterises XLA and Xid. Different mutations detected in the Btk genes o f XLA patients have been found to occur in positions relating to each o f the identified

The following supplements the description o f the functions Btk given opposite. Upon activation, the B-ceU receptor (BCR) stimulates the Src-related tyrosine kinases Lyn, Fyn and BIk, which is rapidly followed by the activation o f Btk and the Syk tyrosine kinasek It is has been shown^ that the Src-related kinases phosphorylate Btk on residue Y551, possibly allowing a conformational

exchange which enhances autophosphorylation on Btk-Ygz^.

The consequences o f Btk activation have not been weU defined, although some insight has recently been gained concerning the downstream targets o f Btk. For example, the BAP-135 protein was shown to be associated with Btk in vivo. Moreover, BAP-135 is a substrate for Btk-mediated tyrosine phosphorylation in vitrd'. Indeed, BAP-135 was shown to be transiendy phosphorylated on tyrosine in response to BCR cross-hnking, suggesting that BAP-135 lies downstream o f Btk in a signalling pathway originating with the BCR. Flowever, the function o f BAP-135 is currendy unclear.

Further insight into the role o f Btk has come from evidence obtained from the analysis o f BCR signalling in the chicken (lymphoma) B cell Hne DT40, suggesting the involvement o f Btk in phosphoHpase-dependent signalling^. In DT40 cells, disruption o f the Btk gene locus led to reduced presence o f phospholipase C (PLC)-y2 products, i.e. there was reduced D-myo-inositol-1,4,5-

trisphosphate ( I P3) generation and Ca^^ mobilisation in response to BCR cross-hnking. There was also a reduction in the level o f BCR-induced tyrosine phosphorylation o f PLC-7 2. In contrast, BCR-

induced activation o f Ras/Raf kinase cascade was unperturbed in Btk-deficient cells, suggesting that the differences observed occur through a specific Btk-dependent pathway, rather than via a general disruption o f cell signalling.

Btk is also thought to have a role in supporting radiation-induced apoptosis. While this process is known to require intact PH, SH2 and kinase domains o f Btk, the precise function o f Btk in this context is unknown^.

1. Saouaf, S.J., Mahajan, S., Rowley, R.B., et al. (1994). Temporal differences in the activation of 3 classes of non-transmembrane protein-tyrosine kinases following B-cell antigen receptor surface engagement. Broceedings of the 'National A.cademy of Sciences (USA.), 91, 9524-9528.

2. Rawlings, D.J., Scharenberg, A.M., Park, H., et al. (1996). Activation of Btk by a phosphorylation mechanism initiated by Src family kinases. Science, 271, 822-825.

3. Yang, W.Y., & Desiderio, S., (1997). BAP-135, a target for Btk in response to B-cell receptor engagement. Proceedings of the National Academy of Sciences (USA), 94, 604-609.

4. Takata, M., & Kurosaki, T., (1996). A role for Btk in B-ceU antigen receptor-mediated activation of phosphohpase Q-fl. Journal of Experimental Medicine, 184, 31-40.

5. Uckun, F.M., Waddick, K.G., Mahajan, S., (1996). Btk as a mediator of radiation-induced apoptosis in DT40 lymphoma B cells. Science, 273,1096-1100.

structural domains o f the Btk protein (Vihinen et al., 1996). Interest in XLA and Btk has prompted the compilation o f an extensive database, called BTKBase, in which all the known Btk mutations have been recorded (Vihinen et al., 1996). It is o f particular interest that a number o f XLA-causing mutations fall within the Btk PH domain. To date, Btk is the only known protein in which PH domain mutations are known to cause disease. This observation has importance not only in the context o f XLA, but also in relation to understanding the physiological role o f the PH domain in general.

The function o f Btk has been studied from both genetic and biochemical perspectives. Btk has been implicated as a component o f several intracellular signal transduction pathways, including those utilised by the B-cell antigen receptor (De Weers et al., 1994) and the interleukin 5-receptor (Sato et al., 1994). A number o f downstream effectors required for Btk-mediated signalling have been identified. For example, the activity o f a gain-of-fiinction Btk mutant (called Btk*) requires the Src- family kinases such as Lyn, Fyn and Hck, which bind through their SH3 domains to the Btk proline-rich region (Afar et al., 1996). The gain-of-fiinction o f Btk* is correlated with increased translocation o f Btk to the plasma membrane and increased tyrosine phosphorylation on Btk residue Y551 (Rawlings et al., 1996). The isolated, wild-type Btk PH domain binds to D - I P ^ , and also to D - I P4 with a Kg=40 nM.

However, for the Btk* PH domain, which contains the mutation E41 to K41, interactions with inositol polyphosphates were demonstrated to be twice as strong as for the wild-type PH domain (Fukuda et al., 1996). These observations suggest that the Btk PH domain may be required for recruitment to the plasma membrane via binding to phosphoinositides (with head groups similar to inositol polyphosphates) such as Ptdlns (3,4,5)P3. Upon phosphoinositide binding, downstream signalling events could be organised through interactions with Src-family kinases. However, a direct interaction with Ptdlns (3,4,5)P3 has not been reported and questions still

remain concerning the function and regulation o f Btk.

Correction’, additional information on Btk function is provided on the opposite page.