Selección de la celda Peltier

A lthough protein dom ains have individual binding properties and a degree of functional independence, it is necessary to understand how these features are combined and co-ordinated w ithin the whole molecule. Several recent papers

CHAPTER 7___________________________________________________________ CONCLUSIONS

have highlighted the im portance of intram olecular interactions betw een dom ains in Itk an d in the Src family kinases, Src and Hck

It has been previously show n that both the SH2 and SH3 dom ains of c-Src are necessary for its repression by Csk (Superti-furga et aL, 1993). The recent crystal structures of Src (Xu et aL, 1997) and Hck (Sicheri et al., 1997) show how these dom ains combine in the w hole molecule to m aintain the repressed state. In addition to the SH2 dom ain binding intram olecularly to the

phosphorylated C-term inal regulatory tyrosine residue (section 1.5.2.1), the SH3 dom ain binds to a linker region betw een the SH2 and kinase dom ains th at adopts a poly-proline type II helical conformation. Together these

interactions stabilise the repressed state by displacing a catalytically essential a-helix from the active site. Furtherm ore, binding of the SH3 dom ain ligand, Nef, to Hck dram atically increases its kinase activity (Moarefi et al., 1997), suggesting that the intram olecular SH3 interaction is disrupted and the non­ repressed, active state stabilised. These findings suggest a role for inter- m olecular SH3 binding in activation of tyrosine kinases.

A recom binant protein derived from the Btk/Tec m ember, Itk, th at contains its SH3 an d TH sequences exhibits decreased binding capacity for SH3 its dom ain ligands, Grb2 and Sam68 (Ham ilton A ndreotti et al., 1997). NMR studies show th at the SH3 dom ain binds the TH region intram olecularly, suggesting that this m ay hinder access of the SH3 dom ain to binding sites w ithin other

proteins. An earlier study of the Btk TH region show ed th at it did not bind an individually expressed Btk SH3 dom ain in vitro although it did bind the SH3 dom ains of Src family m em bers (Cheng et al., 1994). This suggests that an intram olecular association w ithin Btk maybe weak and displaced by SH3 ligands such as WASP w ith higher affinities. Also, addition of purified SH3 dom ain ligands such as WASP to Btk kinase assays m ay help determ ine if ligand binding leads to activation of Btk. However, addition of increasing concentrations of polyproline to Btk im m unoprecipitates (predicted to disrupt putative T H /SH 3 dom ain interactions had no effect on kinase activity (data

CHAPTER 7___________________________________________________________ CONCLUSIONS

n o t shown). H ow these potential intram olecular interactions relate to regulation of Btk in vivo will require further study and w ould be greatly facilitated by a crystal structure of Btk

A nother outstanding question concerns the identity of Btk's in vivo substrate.

Btk negative DT40 cells show a decrease in the tyrosine phosphorylation of PLCy but in com parison to Lyn and Syk knockouts show little change in the gross phosphorylation pattern of cellular proteins following BCR cross-linking (Takata and Kurosaki, 1996). This suggests that Btk activity m aybe required for a specific phosphorylation event, rather than for the assem bly of the initial BCR associated complex m ediated by Src kinases and Syk (section 1.6).

7.5

FUTURE PROSPECTS

The aim of the w ork presented in this thesis w as to elucidate Btk's role in B cell signal transduction by isolating candidate ligands for its protein domains. The identification of IP4 /P IP3 as a ligand for the PH dom ain and of c-Cbl and WASP as ligands for the SH3 dom ain have provided clues to Btk's function described above. Further inform ation could be obtained by expressing m utant form s of Btk in XLA patient B cell lines and exam ining the effects on the

reconstitution of various signals such as calcium flux. Should in vivo ligands of w ild type Btk be identified, im m unoprécipitation and immunofluorescence studies of m u tan t form s (for example SH3 or PH dom ain m utants) m ay provide know ledge of the im portant m olecular lesions th at occur in XLA. The large variation in the severity of disease suffered by XLA patients points to the influence of several factors in addition to Btk. This idea is supported by w ork in this laboratory that has show n that Btk protein is not detectable in m ononuclear haem atopoietic cells of m ost XLA patients studied, irrespective of their phenotype (H. Caspar, submitted). These findings call into question the validity of putative genotype/ phenotype com parisons extrapolated from genetic data and dem and th at the involvem ent of other determ inants of the

CHAPTER 7___________________________________________________________ CONCLUSIONS

disease be investigated. In order to ascertain the effect of the genetic

background on Btk's function, Btk negative (or m utant e.g. xid) mice could be crossed w ith mice having various backgrounds, including knockout mice lacking genes know n to encode potential lig an d s/reg u lato rs of Btk. The effects on the xid phenotype could then be evaluated.

Recent w ork has show n that correction of the xid defect occurred in transgenic mice th at contained the hum an Btk gene controlled either by the class 11 major histocom patibility complex Ea locus control region (Drabek et al., 1997) or 340 kilobases of genomic DNA containing the Btk control region (R. H endriks, personal communication). The creation of transgenic mice containing m utant form s of Btk will provide a m odel system in which both physiological and m olecular readouts will allow m any aspects of Btk function to be studied. In the long term, reconstitution of the haem atopoietic lineages of severely im m une com prom ised mice m ay provide a m odel system to investigate the role of Btk in B cell development. Im m unodeficient mice such as NOD /SCID mice can be succesfully engrafted w ith hum an bone m arrow (Lapidot et al., 1992; Larochelle et al., 1995) and provide a surrogate blood system in which hu m an haem atopoiesis can be studied. For example, XLA bone m arrow could be transfected w ith w ild type and m utant Btk constructs and the effects on B cell reconstitution examined using this model. In view of the w ork discussed above, such an approach will require the introduction of large constructs (such as artificial chromosomes) that contain the appropriate prom oter sequences. Once Btk's functions and interactions w ith other haem atopoietic com ponents are understood, therapies for XLA based u pon gene delivery m ay be

attem pted.