Análisis de estabilidad global de los taludes del tajo

Protein phosphorylation is a major mechanism of enzymatic control and signal transduction in the cell and, although phosphorylation of serine and threonine residues make up 95% of the phosphorylated protein in a cell, tyrosine phosphorylation has been shown to play a critical role in signalling pathways from several B cell surface molecules, including the BCR (reviewed in Pleiinan et al. 1994). Triggering of the antigen specific receptors initiates a series of biochemical cascades, the earliest of which is the phosphorylation of proteins on tyrosine. The BCR does not have intrinsic tyrosine kinase activity and must therefore utilise the activity of recruited cytoplasmic tyrosine kinases. Studies using kinase inhibitors have shown that all known downstream events in signalling and biological responses are dependent on PTK activity.

unequivocally demonstrated by the identification of mutations in the BTK gene in XLA patients, suggesting a uniquely important role for this protein. The definition of this role in B cells, and in the other haematopoietic lineages in which Btk is expressed, however, has yet to be elucidated. The XLA defect appears to be limited to the B cell lineage, as XLA patients appear to have normal macrophage function, and seems to cause a block in B cell differentiation at the pre-B cell stage of development. The expression of the gene from the pro-B cell stage to the mature B cell stage, however, and its absence in fully differentiated plasma cells, suggests that Btk may function at more than one stage of development. As Btk is a non-receptor tyrosine kinase and has such a critical role in the development of B cells, it is highly likely that it functions in one or more B cell signalling pathways.

A number of papers have reported the beginnings of the investigation into Btk function. Cross-linking of surface IgM on B cells has been shown by three laboratories to result in the activation of Btk (de Weers et al. 1994a; Saouaf et al. 1994) in Daudi and Ramos cells (Burkitf s lymphoma cell lines) and in normal tonsillar B cells. Studies indicated that cross-linking of surface IgM resulted in a significant increase in Btk phosphorylation, predominantly on tyrosine residues, but also on serine and threonine residues (de Weers

et al. 1994a). An increase in Btk kinase activity was shown using immunoprecipitated Btk, suggesting that the increase in phosphorylation was, at least in part, due to autophosphorylation. This association of Btk with signalling pathways initiated at the BCR suggests a role for Btk in the response to antigenic stimulation of mature B cells. The XLA defect becomes evident, however, at the pre-B cell stage before the expression surface IgM. Pre-B cells express a small amount of ijl on the cell surface in association

with surrogate light chains Igof and Ig/?, as the pre-B cell receptor. The low level of expression of the pre-B cell receptor prevented the analysis of Btk activation on cross- linking of this receptor.

The structural basis by which signals from the BCR are transmitted in B cells has begun to be elucidated and is reviewed in Pleiman et al. (1994). The initial reactions are very complex, but have been shown definitively to involve PTK activation. Syk has been shown to associate with the short cytoplasmic tail of the ju chain of the BCR in resting

B cells. The Src family kinases Fyn, Lyn and Blk are thought to bind to the antigen recognition activation motif (ARAM) of Iga in resting cells via the first ten amino acids of the kinase molecule. On activation of the BCR, the ARAM motifs of Igce and Ig|8

become phosphorylated on tyrosine and the Src family kinases then bind in a phosphotyrosine dependent manner via their SH2 domains, with the concomitant release of the N terminal binding mechanism. The N terminal region of these kinases is then free to participate in other interactions. The SH3 domains of the Src family kinases Hck, Fyn and Lyn have been shown to bind to the proline rich sequences in the Tec homology domain of Btk. These interactions are specific and do not occur with the SH3 domains of other Src related tyrosine kinases (Cheng et a l 1994; Yu er al. 1994). This suggests a mechanism by which Btk could associate with Src family kinases that are known to associate with the BCR in resting B cells, linking Btk into the BCR signal transduction pathways.

The cross-linking of the high affinity IgE receptor (Fc, RI) on mast cells results in the phosphorylation on tyrosine of several PTKs, including Lyn, Yes, Src and Syk. Kawakami et a l (1994) have reported that Btk can be added to this list and that, after cross-linking, a fraction of Btk can be found at the plasma membrane. No direct association with the receptor itself could be detected. The same group have recently shown that the Btk PH domain is constitutively associated with PKC in mast cells (Yao

et a l 1994). The interaction results in Btk becoming phosphorylated on serine and downregulated. This suggests a role for Btk in a second signal transduction pathway.

Taken together, these results suggest a role for Btk in at least two surface-receptor coupled signal transduction pathways. Btk is probably not constitutively associated with membrane receptors, but may associate with another member of the Src family. However, no critical role has yet been assigned to Btk in pre-B cell development. Expression of the gene throughout B lineage development and in other haematopoietic cell lines, suggests that Btk may be involved in multiple signalling pathways at more than one stage of B cell development. The further study of proteins interacting with Btk in the cell will help in the elucidation of the role of this protein in signal transduction.