REGULATION OF INOSITOL LIPIDS BY THE T CELL MOLECULE CD5

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CD5, a lymphocyte-specific cell-surface molecule, can provide the accessory signals necessary to permit antigen receptor (CD3/Ti)-mediated activation of resting peripheral T lymphocytes. Signal transduction by CD3/Ti involves the hydrolysis of membrane polyphosphoinositides (PPI) with the generation of inositol phosphates (InsPs) and diacylglycerol. We observe that perturbation of CD5 substantially augments CD3/Ti- mediated generations of InsPs and allows an otherwise transient InsP response to be sustained. In the absence of other stimuli, perturbation of CD5 does not change InsP levels but does have selective effects on phosphatidylinositol phosphate (PIP) and bisphosphate (PIP2). These observations suggest that CD5 may increase the activity of either the PI kinase, or the PIP kinase, thereby increasing the levels of PPI and augmenting CD3/Ti- induced generation of InsPs. To test this hypothesis, the effects of stimulating CD5 and CD3/Ti, individually and in combination, on the mass of PI, PIP, PIP2, and diacylglycerol will be examined. Using membranes prepared from T cells, we will determine whether stimulation of CD5 enhances the incorporation of 32p from (32p) ATP into PIP and PIP2 and whether guanine nucleotides can regulate this process. The possibilities that CD5 is physically associated with a PI/PIP kinase and that CD5 can functionally interact with PI/PIP kinases will be examined using immunoprecitated CD5 and partially purified PI and PIP kinase preparations. By transfecting non-lymphoid cells with the CD5 gene, we will determine whether CD5 can regulate PPI levels in non-lymphoid cells and whether CD5 can augment InsP generation by receptors other than CD3/Ti. Because the the cytoplasmic domain of CD5 contains three tyrosine residues, we will determine whether tyrosine phosphorylation of CD5 occurs during T cell activation. If so, phenylalanine will be substituted for tyrosine, and the consequences of this substitution on the ability of CD5 to regulate PPI will be determined. The general aims of these studies are to understand the molecular mechanisms by which CD5 regulates PIP and PIP2 and to provide insights into the regulation of inositol phospholipid metabolism during T cell activation.