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Abstract
To further understand the functional interactions between CD45 and p56(lck) in T-cells, we stably reconstituted their expression in a nonlymphoid system. The results of our analyses demonstrated that CD45 could dephosphorylate tyrosine 505 of p56(lck) in NIH 3T3 fibroblasts. As is the case for T-cells, removal of the unique domain of p56(lck) interfered with dephosphorylation of tyrosine 505 in fibroblasts, further stressing the importance of this region in the interactions between CD45 and p56(lck). The ability of CD45 to dephosphorylate tyrosine 505 in NIH 3T3 cells was also greatly influenced by the catalytic activity of p56(lck). Indeed, whereas CD45 provoked dephosphorylation of kinase-defective Lck molecules in this system, it failed to stably dephosphorylate kinase-active p56(lck) polypeptides. Finally, our studies showed that CD45 was also able to inhibit the oncogenic potential of a constitutively activated version of p56(lck) in NIH 3T3 cells. This effect did not require the Lck unique domain and apparently resulted from selective dephosphorylation of substrates of activated p56(lck) in fibroblasts. In addition to providing insights into the nature and regulation of the interactions between CD45 and p56(lck) in T-cells, these results indicated that CD45 clearly has the capacity to both positively and negatively regulate p56(lck)-mediated functions in vivo.
Highlights
P56lck is a lymphocyte-specific member of the Src family of tyrosine-protein kinases (Refs. 1–3; reviewed in Refs. 4 and 5)
To ensure that full-length CD45 polypeptides were expressed in these cells, CD45 was immunoprecipitated with monoclonal antibody (mAb) M1.89.18.7 and subsequently immunoblotted with a polyclonal rabbit anti-CD45 serum
The results of our experiments showed that expression of the R0 isoform of CD45 in NIH 3T3 fibroblasts caused a ϳ5-fold decrease in the extent of tyrosine 505 phosphorylation of kinase-defective (Arg273) p56lck molecules
Summary
P56lck is a lymphocyte-specific member of the Src family of tyrosine-protein kinases (Refs. 1–3; reviewed in Refs. 4 and 5). Contrary to Lck polypeptides expressed in NIH 3T3 cells, those isolated from T-lymphocytes are poorly phosphorylated at tyrosine 505 [23] This difference is seemingly consequent to the action of CD45, a transmembrane protein-tyrosine phosphatase selectively expressed in nucleated hemopoietic cells (for a review, see Ref. 24). This notion is supported by the observation that Lck polypeptides immunoprecipitated from CD45-deficient T-cells exhibited a marked (8 –10-fold) increase in tyrosine 505 occupancy, when compared with p56lck molecules recovered from their CD45-positive counterparts [25,26,27,28,29].
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