Dissection of Lymphocyte Activation: Defining a Role for PI-3 Kinase

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Doctoral Dissertation


Graduate School of Biomedical Sciences, Department of Cell Biology


Lymphocyte Activation; Phosphatidylinositols; Phosphotransferases; Academic Dissertations


This dissertation was intended to identify potential roles for phosphatidylinositol-3 kinase (PI-3 kinase) in the responses of lymphocytes to activation. To understand what functions PI-3 kinase is performing in lymphocytes, experiments were performed to identify proteins that will stably associate with the p85 subunit of PI-3 kinase. Co-precipitation revealed an activation dependent association of p85 with two different phosphotyrosine containing proteins. One protein, pp36-38, is a membrane protein that interacts with PI-3 kinase, PLCγ1, and Grb2/S0S. The other associated protein was identified as the proto-oncogene c-Cbl. The interaction of p85 with cbl was shown to be mediated through the SH2 domains of p85. More importantly, the interactions of p85 with p36-38 and cbl were found to be specific for p85 isoforms. Although the SH2 domains of the α and β isoforms are highly similar in amino acid sequence, they are shown to establish distinct protein interactions in intact cells. Experiments on the cbl/PI-3 kinase complex revealed a stimulation dependent translocation into membrane and insoluble/cytoskeletal fractions of wild type, but not mutant cells. The movement of cbl did not require tyrosine phosphorylation or PI-3 kinase activity. The cbl/PI-3 kinase complex was greatly enhanced in the membrane fraction in contrast to the cytosol, where the largest concentration of cbl can be found. In addition, these complexes were found to form at the membrane in the absence of the tyrosine kinase, p56lck.


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