Biochemical basis of B cell dysfunction in Lyn kinase deficient mice

B lymphocytes constitutes an important arm of the immune system, and their response to antigen is largely dependent upon signal transduction through the B cell receptor (BCR). Such a potent receptor, however, needs to be further balanced by positive and negative regulators to prevent harmful effects that may arise from inappropriate stimulation. Src family protein tyrosine kinase Lyn is involved in both positive and negative regulation, since the both gain-of-function Lyn and loss-of-function Lyn mutations caused autoimmunity in mice. The exact signalling pathway(s) regulated by Lyn in B cells, however, are still not clear. Work presented in this thesis attempts to elucidate the biochemical mechanisms that underline the double-edged nature of Lyn in BCR signalling. CD19 is an important B cell co-receptor that helps to set the threshold for BCR signalling. Although CD19 expression level is same in lyn and lyn B cells, it was difficult to solubilize CD19 from lyn mice by TX-100 detergent, compared with wild-type mice. Since lipid rafts are defined as a TX-100 insoluble domain of the plasma membrane, the possibility that CD19 was constitutively located in this signalling platform in Lyn-deficient B cells would explain the hyper-responsiveness of lyn B cells. Therefore the membrane localization of CD19 in the absence of Lyn was examined carefully. Lipid rafts were found to possess all the necessary proposed properties as signalling platforms, such as containing the Src kinase family member Lyn and excluding the receptor phosphatase CD45. The BCR was rapidly translocated into the lipid raft domain after BCR cross-linking. In the resting state, however, CD19 was found to be outside lipid rafts in both wild type and lyn B cells. Given a normal membrane distribution of CD19 in the absence of Lyn, the biochemical consequences of BCR ligation in mouse B cells deficient in either Lyn or CD19 was examined for evidence of interaction or co-dependence between these molecules. In contrast to published results, the extent of CD19 phosphorylation following BCR ligation was unaffected by the absence of Lyn yet was dependent on other Src-family PTKs as it was inhibited fully by pp2, a Src-family specific inhibitor. Consistent with the normal phosphorylation of CD19 in lyn B cells, the recruitment of phosphoinositide-3 kinase to CD19 and the ability of CD19 to enhance both intracellular calcium flux and Erk1/2 activation after co-ligation with the BCR were intact in the absence of Lyn. Similarly, unique functions of Lyn were found to be independent of CD19. The increase in Lyn kinase activity following BCR ligation and the inhibition of BCR-mediated calcium flux by CD22 were both normal in CD19 B cells. Collectively, these data argue that the unique functions of Lyn do not require CD19 and that the signal amplification mediated by CD19 is independent of Lyn. Therefore, the roles of Lyn and CD19 following BCR ligation are independent and opposing, one being primarily inhibitory and the other stimulatory. The opposite phenotype of Lyn-deficient B cells to that of PI3K-deficient B cells prompted us to investigate the possibility that Lyn works through PI3K. We found that many aspects of the hyper-sensitive phenotype of Lyn-deficient primary B cells in response to BCR ligation, such as Erk MAP kinase activation, calcium elevation and hyper-proliferation were attenuated by removal of PI3K activity, suggesting an elevated PI3K pathway in the absence of Lyn. Furthermore, the specific activity of PI3K from lyn primary B cells was found to be higher both before and after BCRstimulation, indicating the inhibition of PI3K activity by Lyn. In a search for the factors that cause the activation of PI3K in primary B cells, we found the BCRinduced phosphorylation of a PI3K associated protein was inhibited by pp2, a Src family PTK inhibitor, but elevated in Lyn-deficient primary B cells. Moreover, the Src family PTK activity was demonstrated to be required for the activation of PI3K in both wild type and Lyn-deficient primary B cells. On the basis of these data we propose a novel inhibitive mechanism of Lyn in BCR signalling acting on PI3K.