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The role of the tyrosines in the carboxyl tail of connexin43 in regulating cytoskeletal rearrangements in B-Lymphocytes

University of British Columbia

Cellular processes requiring cytoskeletal rearrangements such as adhesion, spreading, immune synapse formation, and migration are crucial for normal B-lymphocyte (B cell) development and for immune responses. Our lab has shown that the gap junction protein connexin43 (Cx43) is both necessary and sufficient for promoting B cell adhesion, B cell receptor (BCR)-mediated spreading, B cell motility and migration. The carboxyl tail (CT) of Cx43 is important for these effects and the hypothesis is that specific regions of the CT contain sites important for interactions that can influence these processes. To identify these regions, a series of deletions and point mutations in the CT of Cx43 were made. These mutated proteins were expressed in mouse B cell lines and examined for localization, cell surface expression and for their effect on supporting cell spreading in response to BCR-signaling, the latter assay being used as one ‘read-out’ for cytoskeletal rearrangements. With regard to BCR-mediated cell spreading, the data in this thesis from the Cx43 deletions showed that the amino acids in the Cx43 CT between 246 and 307 are the most important, and that amino acids between 307 and 382 are less critical. In addition, since one of the most important proximal signaling events after BCR signaling is the activation of different protein tyrosine kinases, the effects of different tyrosine mutations in the Cx43 CT were examined. Three point mutations at tyrosine (Y) 247, 265 and 267 to phenylalanine (F), and a double mutant, Y247F/Y265F, were made and their effect on BCR-mediated spreading assessed. All of these tyrosine mutations in the Cx43 CT were found to impede BCR-mediated cell spreading. These findings have helped to better define the region of the Cx43 CT critical for regulating cytoskeletal rearrangements in response to BCR signaling. Future studies will identify other important residues of the CT, with the goal to identify the importance of these sites. These results will provide novel insights into understanding basic B lymphocyte responses that contribute to immune function, and can lead to identifying potential targets for the development of therapeutics to treat immune diseases.

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2015-04-20

Attribution-NonCommercial-NoDerivs 2.5 Canada

http://hdl.handle.net/2429/52884

Cell and Developmental Biology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/2.5/ca/