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The role of SRC family protein tyrosine kinases and the GAB1 adapter protein in BCR-mediated activation of AKT Santos, Lorna

Abstract

Antibody production by B lymphocytes is important for the eradication of foreign pathogens and requires signaling by the BCR. Crosslinking of the BCR results in the activation of three main signaling pathways — the PI 3-kinase, MAP kinase and PLC-γ2 pathways. It is proposed that various combinations of protein tyrosine kinases and adapter proteins differentially regulate the three main signaling pathways following BCR engagement. A non-lymphoid cell system that stably expresses functional BCRs at the cell surface and expresses only one Src kinase (Fyn) was used to examine the role of the Gabl adapter protein and Src family members, Lyn, Blk and Lck, in the BCR-induced activation of the Akt pathway. It was determined that Syk kinase activity is required for amplifying and sustaining Akt pathway activation through the BCR. Lck has no effect on the activation of this pathway while Lyn and Blk both inhibit the activation of this pathway in response to BCR crosslinking. This inhibition correlated with the ability of Lyn and Blk to interact with the SHP-2 protein tyrosine phosphatase. Altering the plasma membrane localization of Lyn and Blk in lipid raft and non-rafts domains has no effect on the BCR-induced Akt phosphorylation. However, expression of a cytosolic mutant form of Blk in the AtT20 cells alleviates the inhibitory effect on Akt phosphorylation. Using the AtT20 cells, it was established that Gabl could inducibly translocate to the plasma membrane and become tyrosine phosphorylated in response to BCR crosslinking. Akt phosphorylation was 2-fold higher following BCR crosslinking in cell transfected with Gabl. The recruitment of Gabl to the plasma membrane, its inducible tyrosine phosphorylation, association with signaling molecules and increased Akt phosphorylation all required the PH domain of Gabl as well as the Syk kinase. Together, both parts of this thesis demonstrated that various BCR-activated PTKs and Gabl are involved in the regulation of Akt pathway downstream of the BCR. The Akt pathway could be modulated by the use of bona fide adapter proteins or PTKs acting as adapter proteins that recruit important signaling components to the plasma membrane where PI 3-kinase/Akt signaling takes place.

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