The CD45 family of transmembrane protein tyrosine phosphatases (PTPases) plays a key role in T cell activation by regulating the activity of critical protein tyrosine kinases (PTKs) and their phosphorylated substrates. However, the exact nature of this regulation, and the identity of additional in vivo substrates remains uncertain. CD45 is composed of multiple isoforms which differ only in their extracellular domains. These are differentially distributed on subsets of T cells having distinct functions. It has been proposed that the CD45 extracellular domains differentially regulate signaling through the cytoplasmic domain. However, the actual role of the individual isoforms remains unclear. Such studies have been hampered by the previous inability to individually express different CD45 isoforms in the same cell line. The principal investigator has developed a unique model whereby endogenous CD45 expression in the Jurkat human T cell leukemia line has been specifically blocked by transfection of a CD45-antisense gene. Antisense transfected cells lacking CD45 were then reconstituted to uniquely express either the largest or smallest human CD45 isoform. The applicant has found isoform-specific differences in IL-2 secretion and in the tyrosine phosphorylation of several key signaling molecules. These findings indicate preferential regulation of TCR-generated signaling pathways by these two isoforms. Using this model system, the investigator seeks to extend these findings and determine the role of CD45 and its isoforms in regulating T cell activation.
In Aim 1, additional Jurkat derivatives, uniquely expressing the CD45(0) and CD45(ABC) isoforms in the context of a cloned Ag-specific TCR will be created, to see if the current observations can be reproduced in an Ag-specific system. Single isoform transfectants will be compared in experiments designed to determine the role of individual CD45 isoforms in regulating the activity and phosphorylation of PTKs, particular critical substrates, and IL-2 secretion.
In Aim 2, the role of each of the two cytoplasmic CD45 PTPase domains in activation signaling and substrate recruitment in vivo will be determined by examining anti-sense transfectants reconstituted with defined PTPase mutants. In addition, the potential role of tyrosine phosphorylation of CD45 in signaling will be determined using specific Tyr to Phe point mutants. To evaluate possible differential utilization or regulation of these PTPase domains, these mutants will be expressed in the context of two different CD45 isoforms.
In Aim 3, the function of the remaining three human CD45 isoforms in regulating IL-2 production, PTK activity, and early tyrosine phosphorylation events will be determined.
