Leukocyte activity is controlled by balancing activating and inhibitory signals in order to generate effective immune responses while avoiding tissue damage and autoimmunity. This project examines an important regulatory circuit in B cells that involves the lipid phosphatase SHIP. Localization, phosphorylation, and binding to interaction partners are important factors in the regulation of this protein, however knowledge of their inter-relationships is incomplete. Here, we examine the influence of two interaction partners on the molecular behaviour and function of SHIP. First, we examine in detail how the inhibitory receptor FcγRIIB controls SHIP localization dynamics. We have observed that co-engagement of FcγRIIB along with the B cell receptor in A20 cells does not influence the magnitude or kinetics of SHIP-EGFP recruitment to the membrane as assessed by confocal microscopy, however it does alter mobility at the cell periphery as measured by fluorescence recovery after photobleaching. Next, we probe the influence of a novel binding partner, the adaptor protein Nck. We have demonstrated an interaction between SHIP and Nck by both Biacore affinity analysis and pull-down assays. Functional relevance will be addressed through mutagenesis experiments. With these aims we hope to enhance our understanding of an interaction that is already recognized as relevant and investigate the function of a novel interaction that occurs at a previously uncharacterized regulatory site.
- Copyright © 2013 by The American Association of Immunologists, Inc.