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Regulation of Rap2 in B lymphocytes

University of British Columbia

Ras and members of the Ras-related family of small GTP-binding proteins are involved in many biological functions. These include regulation of cell growth, differentiation, and activation; actin cytoskeleton rearrangement, membrane trafficking, vesicle trafficking and nuclear transport. These GTP-binding proteins cycle between an inactive GDP-bound form or an active GTP-bound form which can bind downstream effectors. Rap1A, Rap1B, Rap2A, and Rap2B proteins share approximately 50% sequence identity with Ras. In addition, Rap proteins share a similar effector region as Ras, but have different flanking amino acids. Despite the similarities, the functions of Rap1 and Rap2 have not been completely elucidated although recent studies suggest that both Rap proteins may be involved in cell migration and adhesion. Previous work has shown that Rap1 is activated by many receptors. In our laboratory, we showed that Rap1 was activated by BCR cross-linking and CXCR4 stimulation. Hence, the first aim of this thesis was to determine i f these receptors also activated Rap2. This is because Rap2 may or may not have the same function as Rapl in B lymphocytes. The second objective was to uncover how BCR cross-linking and CXCR4 activate Rap proteins. A PLC inhibitor was used to elucidate the mechanism of this Rap activation signalling pathway. Finally, the third goal was to develop loss of function approaches for blocking Rap1 and Rap2 activation. Expression of the Rap-specific GAP proteins, RapGapII and Spa-1, was used to inhibit activation of Rap1 and Rap2 in B cell lines. These experiments have resulted in three major findings. First I showed that anti-Ig antibodies, stromal cell-derived factor-1 (SDF-1), and phorbol 12,13-dibutyrate (PdBu) all activate Rap2 in B cells with activation kinetics similar to that for Rap1. Second, I showed that the BCR activates Rap2 via PLC-y, and Rap2 activation can be inhibited by 1-[6-[[17β-3- methoxyestra-l,3,5(10)-trien-17-yl]amino]hexyl]-lH-pyrrole-2,5dione (U73122). Finally, I showed that RapGapII and Spa-1 differentially inhibit the activation of Rap 1 and Rap2.

Thesis/Dissertation

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2009-09-16

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http://hdl.handle.net/2429/12843

Microbiology and Immunology

University of British Columbia

UBCV

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