EVIDENCE FOR AN ASSOCIATION OF CD45 WITH 32000-33000-MW PHOSPHOPROTEINS ON MURINE T-LYMPHOCYTES AND B-LYMPHOCYTES

In human T cells CD45 is reported to associate with both cell surface and intracellular molecules including CD2, CD4/CD8, CD5, p56(lck) and p59(fyn). In this study the association of molecules with CD45 in muri ne T lymphocytes was explored using biotinylation, chemical cross-link ing, immunoprecipitation and P-32-labelling. Sodium dodecyl sulphate-p olyacrylamide gel electrophoresis (SDS-PAGE) analysis of CD45 monoclon al antibody (mAb) (S-450-15.2) immunoprecipitates from Triton X-100 ly sates of murine thymocytes that were surface biotinylated and treated with the chemical cross-linker 3,3'-dithio-bis(sulpho-succinimidylprop ionate (DTSSP) showed that CD45 can be chemically linked to molecules of 25 000-32 000, 42 000 and 60 000-70 000 MW. The CD45 mAb also co-pr ecipitated a prominent 32 000 MW molecule from digitonin lysates of su rface biotinylated murine thymocytes, splenocytes and D10 cells, but a weaker association was also detected on splenic B cells and on the mu rine B-cell lymphoma line A20. The results suggest that in these cells CD45 is associated with a 32 000 MW molecule which is exposed extrace llularly. Experiments in which thymocytes were biotinylated after perm eabilization with lysolecithin showed that additional molecules of 33 000, 55 000, 60 000 and 90 000 MW, presumably localized intracellularl y, also co-precipitated with CD45. Labelling of murine thymocytes or D 10 cells with (H3PO4)-P-32 in vivo, and of CD45 immunoprecipitates by in vitro kinase reaction, revealed that the 32 000-33 000 MW molecules are phosphoproteins. The relationship of these molecules with the 30 000-34 000 MW molecules previously reported to associate with CD45 in human T cells is not clear as a number of differences were observed. F irstly, the molecular weight of the CD45-associated 32 000-33 000 MW m olecule(s) on murine T cells and B cells is slightly lower than that o bserved in the human T-cell line Jurkat (34 000 MW). Secondly, phospho amino acid analysis after in vitro kinase labelling of CD45 immunoprec ipitates showed that the murine 32 000-33 000 MW molecules are phospho rylated exclusively on serines. Thirdly, although in vitro phosphoryla tion of the 32 000-33 000 MW molecules was inhibited by preincubation with either GTP-gamma-S or GDP-beta-S, the 32 000-33 000 MW CD45-assoc iated molecules did not bind P-32-GTP, GDP-agarose, or react with anti sera to a consensus sequence of G proteins. The crucial role of CD45 f or proper function of the T-cell receptor (TCR), suggests that the CD4 5-associated 32 000-33 000 MW molecules and kinases also may play a ro le in the signalling events leading to T-cell activation.