Molecular biology of Brouton´s tyrosine kinase

___________________________________________________5 LIST OF PUBLICATIONS _________________________________________6 LIST OF ABBREVIATIONS________________________________________7 INTRODUCTION ________________________________________________9 X-linked agammaglobulinemia (XLA)_____________________________9 X-linked immunodeficiency (Xid) _______________________________10 The BTK gene _______________________________________________11 The Btk protein ______________________________________________13 The Btk PH domain__________________________________________14 The TH domain _____________________________________________15 The SH3 domain____________________________________________15 The SH2 domain____________________________________________16 The catalytic domain_________________________________________16 Btk homologue in other species _______________________________17 Kinases ____________________________________________________17 Btk in signal transduction_____________________________________17 Activators _________________________________________________17 Activation mechanism________________________________________18 The BCR signalosome _______________________________________19 Effects of calcium ___________________________________________20 Regulation of transcription ____________________________________20 Cytoskeletal regulation _______________________________________21 Downregulation of Btk activity__________________________________22 AIMS OF THE PRESENT STUDY__________________________________23 COMMENTS ON METHODOLOGY ________________________________24 Circular dichroism analysis ___________________________________24 Transfections _______________________________________________24 Green fluorescent protein _____________________________________24 Cell proliferation assay _______________________________________25 RESULTS AND DISCUSSION ____________________________________26 Paper I _____________________________________________________26 Paper II_____________________________________________________27 Paper III ____________________________________________________29 Paper IV ____________________________________________________30 CLOSING REMARKS ___________________________________________32 ACKNOWLEDGEMENTS ________________________________________33 REFERENCES_________________________________________________35 Molecular biology of Bruton’s tyrosine kinase 5 ABSTRACT X-linked agammaglobulinemia (XLA) is caused by an arrest in the B lymphocyte differentiation. XLA patients lack mature B-cells and subsequently antibody production. The defective gene causing XLA is the BTK gene, which codes for the protein Bruton’s tyrosine kinase (Btk), a cytoplasmic enzyme related to the Src family of kinases. Btk is only expressed in the hematopoietic cells, except for T lymphocytes and plasma cells. In an attempt to investigate the function of Btk, we have studied how mutations and other proteins affect the activity, phosphorylation and localization of Btk. XLA causing mutations in the Src homology 2 (SH2) domain of Btk expressed in E. coli were found to disrupt the binding to phosphotyrosine, indicating a necessary function for the Btk protein. Only the mutant directly involved in the binding did not significantly deviate structurally from the native SH2 domain. Btk can phosphorylate its own tyrosine 223 in the SH3 domain. We found that the site Y223 could also be phosphorylated by c-Abl. Btk and c-Abl did also colocalize and interact in cells. Phosphorylation of Y223 has been suggested to downregulate the activity of Btk. Moreover, we found that the substrate specificity for Btk and the protein family members Itk, Bmx and Tec are different. Btk preferentially phosphorylates the SH3 domain of Btk and Bmx, while Itk phosphorylated Itk and Btk SH3 domains, whereas Tec could phosphorylate the SH3 domain of Btk and Tec, but also with reduced efficiency Bmx and Itk. The phosphorylation sites in the SH3 domains were also mapped. Tec was phosphorylated on a conserved tyrosine, which differs from the conserved Y223, and was the site of phosphorylation in the other family members. A region in the PH domain binds phospholipids in the cell membrane. Changing negative charged amino acids to positive induce microspikes structures on the cell membrane of the cells. D43R and the previously reported gain-of-function, E41K, showed an increased cell proliferation. These mutations also made IL-5 dependent cells proliferate in the absence of IL-5, indicating a transforming potential. In summary, these studies show some important functions of the Btk protein that are necessary for B-cell development and illustrate the complexity of these mechanisms.