Extracellular ligation-dependent CD45RB enzymatic activity negatively regulates lipid raft signal transduction

Abstract

CD45 is the most prominent membrane protein on lymphocytes. The function and regulation of this protein tyrosine phosphatase remain largely obscure, mainly because of the lack of a known ligand, and it still remains unknown whether such tyrosine phosphatases are subject to extracellular control at all. We report that an anti-CD45RB antibody (Ab) that prevents rejection and induces tolerance activates CD45RB tyrosine phosphatase enzymatic activity in T lymphocytes, allowing us to directly monitor the effects of increased CD45RB activity on signal transduction. Using both kinase substrate peptide arrays as well as conventional biochemistry, we also provide evidence of the various kinases involved in bringing about the inhibitory effect of this Ab on CD3-induced T-cell receptor signaling. Furthermore, we report that activated CD45RB translocates to lipid rafts and interferes with lipid raft localization and activation state of CD45 substrate Lck. Thus, these findings indeed prove that CD45 is subject to extracellular control and also define a novel mechanism by which receptor tyrosine phosphatases control lymphocyte biology and provide further insight into the intracellular signaling pathways effected by anti-CD45RB mono- IntroductionProtein phosphorylation plays a crucial role in regulating almost every aspect of cellular physiology, tyrosine phosphorylation even more so, as it is extensively used only in eukaryotes acting as a critical control point for important cellular processes, such as communication between and within cells, proliferation or differentiation, cell death, motility, and transportation of molecules into or out of cells. [1][2][3] The phosphorylation status of these control points is tightly regulated by the antagonistic activities of protein kinases and protein phosphatases. Abnormal functioning of protein kinases and phosphatases is implicated in various human diseases, ranging from cancer to immune deficiencies. 4 Strikingly, whereas the action of many different protein kinases is now fairly well understood for a large variety of biologic events, the role of most protein phosphatases remains unclear.CD45 was one of the first protein tyrosine phosphatases found to be expressed on all nucleated hematopoietic cells as a major high molecular weight leukocyte cell surface antigen. [5][6][7][8] Structural analysis of the CD45 cytoplasmic region has shown that the intracellular tail of CD45 contains 2 homologous domains. 7 The first membrane, proximal phosphatase domain, has phosphatase activity, whereas the second domain is thought to be required for proper folding and substrate recruitment. 9 Six different isoforms, varying in molecular weight (180-220 kDa), antigenicity, and glycosylation have been identified and characterized. 10 Different isoforms may exert different functions in cellular physiology; however, this issue is still under debate. Little evidence for clear functional differences between isoforms has emerged from experiments using transgenic mice expressing single isoforms of CD45 on...

Transient antibody targeting of CD45RC induces transplant tolerance and potent antigen-specific regulatory T cells

“…We determined that the anti-rat CD45RC OX22 induced tolerance and showed high killing activity in vitro. This is reminiscent to anti-CD45RB mAb effects, since comparison of two different anti-CD45RB mAb showed that the one effective for prolonging allograft survival was also more effective in killing cells through apoptosis (43,(56)(57)(58). A chimeric mAb binding to both CD45RB and CD45RO has been described to prevent islet allograft rejection in mice through depletion of memory T cells (57 This, together with the increased suppressive activity of Tregs when adoptively transferred or used in vitro, indicates that their function is likely increased through activation on alloantigens.…”

Profiling the changes in signaling pathways in ascorbic acid/β‐glycerophosphate‐induced osteoblastic differentiation

“…A kinase peptide substrate array consisting of 1,024 peptides with specific phosphorylation sites was used to comprehensively evaluate the kinome during osteoblastic differentiation, as previously described [Diks et al, 2004;Lö wenberg et al, 2005;Tuynman et al, 2008;Parikh et al, 2009]. After treatment with AA/b-GP for 7 days, the cells were washed in ice-cold phosphatebuffered saline (PBS) and harvested in lysis buffer (Pierce Biotechnology, Inc.) supplemented with protease and phosphatase inhibitors (Roche).…”

Kinase Activity Profiling of Gram-Negative Pneumonia

“…Data were exported to a spreadsheet for further analysis. The raw data were normalized to the 90th percentile, as described previously (15)(16)(17)(18). From this data, the heat map in Figure 1 was constructed in R 2.12.0 (R Foundation for Statistical Computing, Vienna, Austria) using the heatmap.2 function of the gplots library, making use of the hclust function set using complete-linkage.…”

“…Figure 2B depicts signaling events centered by SRC (spots 52, 590 and 736). The SRC kinases protooncogene tyrosineprotein kinase Fyn (FYN) (spots 196 and 318) and leukocyte-specific protein tyrosine kinase (LCK) (spots 59 and 457) display enhanced activity after 24 and 48 h respectively (17,31,32). SRC-inhibiting kinase c-src tyrosine kinase (CSK) (spot 836) (33) activity was diminished after 6 and 24 h, intermediate between CSK and SRC, v-yes-1 Yamaguchi sarcoma viral-related oncogene homolog (LYN) (spot 11) (34) activity was enhanced after 48 h. These signals toward SRC balance out and result in overall activation of SRC kinase.…”