Differential Association of CD45 Isoforms with CD4 and CD8 Regulates the Actions of Specific Pools of p56lck Tyrosine Kinase in T Cell Antigen Receptor Signal Transduction

Abstract

An investigation into the role of CD45 isoforms in T cell antigen receptor signal transduction was carried out by transfecting CD45-negative CD4؉ CD8 ؉ HPB-ALL T cells with the CD45R0, CD45RBC, and CD45RABC isoforms. Fluorescence resonance energy transfer analysis showed that the CD45R0 isoform, but not the CD45RBC or CD45RABC isoforms, was found as homodimers and also preferentially associated with CD4 and CD8 at the cell-surface. A comparison was therefore made of T cell antigen receptor signaling between sub-clones expressing either CD45R0 or CD45RBC. Under basal conditions CD4-associated p56 lck tyrosine kinase activity and cellular protein tyrosine phosphorylation levels were higher in the CD45R0 ؉ than in the CD45RBC ؉ sub-clones. The CD45 tyrosine phosphatase regulates the threshold of T cell antigen receptor (TCR) 1 signaling by modulating the actions of the p56 lck and p59 fyn tyrosine kinases (1-4). The p56 lck kinase associates with the CD4 and CD8 co-receptors and initiates TCR signaling cascades by phosphorylation of immunoreceptor tyrosine-based activation motifs located in the TCRand CD3-⑀ chains (5, 6). In CD45 Ϫ/Ϫ mice T cell development is severely affected (7,8), and elevated TCR signaling thresholds are marked by dysfunctional p56 lck , together with defective TCR-chain phosphorylation and ZAP-70 recruitment (9). CD45 exerts a positive effect on p56 lck actions by de-phosphorylating the inhibitory C-terminal pTyr-505 (10 -13), but can also negatively regulate the kinase by dephosphorylating the pTyr-394 autophosphorylation site (14, 15). The actions of CD45 on p56 lck in CD4 ϩ T cells are directed selectively at the CD4-associated pool of the kinase (16). The dominant effect of CD45 in the T-lineage appears to be at pTyr-505, since T cell development in CD45 Ϫ/Ϫ mice can be largely restored by backcrossing to mice expressing the mutant lck Y505F -active transgene (17,18).Alternative splicing generates up to eight different CD45 isoforms (19,20) of which five are expressed at significant levels in T cells (21). All T cells express more than one CD45 isoform, and differential isoform expression is tightly controlled during thymic development and the activation of mature T cells (reviewed in Ref.1). However, the molecular consequences of differential CD45 isoform expression for TCR signaling, if any, are not yet well understood. Reconstitution of a murine CD45 Ϫ cell line with CD45 isoforms suggested that the CD45R0 isoform (lacking the A, B, and C exon-encoded segments of the ectodomain) promoted greater interleukin-2 secretion upon engagement of the TCR with the cognate major histocompatibility complex-peptide as compared with other isoforms (22). Co-capping experiments in these cells revealed preferential CD4-CD45R0 association (23). However, more detailed capping and co-immunoprecipitation studies indicated a basal association of CD45R0 with the TCR independent of CD4 expression and suggested that co-capping of CD4 with CD45R0 was mediated by this prior CD45R0-TCR association (24). Neverthel...

Topography of plasma membrane microdomains and its consequences for mast cell signaling

“…Unbound antibodies were removed by washing the cells twice in BSS-0.1% BSA, and the cells were measured immediately by FACSCalibur (Becton Dickinson) to determine FRET efficiency between FITC-and TRITC-conjugated antibodies as described [30]. Donor fluorescence of double-labeled samples was compared with that of samples where the acceptor antibody was replaced by non-labeled antibody to compensate for any competition between the donor and acceptor antibodies.…”

Changes in the Role of the CD45 Protein Tyrosine Phosphatase in Regulating Lck Tyrosine Phosphorylation during Thymic Development

“…In contrast, the heavily glycosylated large m.w. CD45 isoforms may have relatively reduced access to lipid raft-associated Lck, and/or relatively diminished access to non-coreceptor-associated Lck, consistent with a reduced role for CD45 at the DN stage (20,21,29,31).…”

“…The regulated expression of distinct CD45 isoforms during T cell development and activation has suggested a role for the CD45 ectodomain in regulating both phosphatase activity and access of substrates to CD45. Cell lines transfected with distinct CD45 isoforms or variable size ectodomains, demonstrate differential ability to homodimerize, to associate with CD4 and/or the CD3/TCR complex, and exhibit differences in membrane microdomain compartmentalization (20,21,29,31,50). At the DN stage, thymocytes express a mixed pattern of mostly high m.w.…”

“…CD4 and CD8 coreceptor both associate with Lck and may alter access of Lck to both CD45 and Csk, the main regulators of Lck-Y505 phosphorylation. Indeed, previous reports have suggested that CD45 is closely associated with CD4, which may promote CD45 access to CD4-associated Lck (20,28,29). Alternatively, the different roles of CD45 in regulating Lck tyrosine phosphorylation during thymocyte maturation may be an intrinsic characteristic of cells at different stages of thymocyte development, and may be regulated independently of coreceptor expression.…”

Selecting the right fluorophores and flow cytometer for fluorescence resonance energy transfer measurements

“…The most apparent manifestation of the FRET process is the decrease of donor fluorescence in the presence of an acceptor in close vicinity (1-10 nm), which is accompanied by an increase of fluorescence of the acceptor (if the acceptor is a fluorescent molecule). The FRET phenomenon has been adapted for microscopy (3)(4)(5)(6) and flow cytometry (7)(8)(9) and several biological structures have been successfully investigated: receptors involved in immune response (10)(11)(12), growth factor receptors on tumor cells (13)(14)(15), determination of protein conformation (16,17), and lipid microdomain structures (9,18,19). Several reviews are also available for biomedical and clinical applications of FRET (8,18,(20)(21)(22)(23)(24).…”