The CD45 tyrosine phosphatase regulates phosphotyrosine homeostasis and its loss reveals a novel pattern of late T cell receptor-induced Ca2+ oscillations.

PAG/Cbp (hereafter named PAG) is a transmembrane adaptor molecule found in lipid rafts. In resting human T cells, PAG is tyrosine phosphorylated and associated with Csk, an inhibitor of Src-related protein tyrosine kinases. These modifications are rapidly lost in response to T-cell receptor (TCR) stimulation. Overexpression of PAG was reported to inhibit TCR-mediated responses in Jurkat T cells. Herein, we have examined the physiological relevance and the mechanism of PAG-mediated inhibition in T cells. Our studies showed that PAG tyrosine phosphorylation and association with Csk are suppressed in response to activation of normal mouse T cells. By expressing wild-type and phosphorylation-defective (dominant-negative) PAG polypeptides in these cells, we found that the inhibitory effect of PAG is dependent on its capacity to be tyrosine phosphorylated and to associate with Csk. PAG-mediated inhibition was accompanied by a repression of proximal TCR signaling and was rescued by expression of a constitutively activated Src-related kinase, implying that it is due to an inactivation of Src kinases by PAG-associated Csk. We also attempted to identify the protein tyrosine phosphatases (PTPs) responsible for dephosphorylating PAG in T cells. Through cell fractionation studies and analyses of genetically modified mice, we established that PTPs such as PEP and SHP-1 are unlikely to be involved in the dephosphorylation of PAG in T cells. However, the transmembrane PTP CD45 seems to play an important role in this process. Taken together, these data provide firm evidence that PAG is a bona fide negative regulator of T-cell activation as a result of its capacity to recruit Csk. They also suggest that the inhibitory function of PAG in T cells is suppressed by CD45. Lastly, they support the idea that dephosphorylation of proteins on tyrosine residues is critical for the initiation of T-cell activation.

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“…The CD45-negative YAC-1 variant, YACN1 (36), was provided by Jonathan Ashwell, National Institutes of Health, Bethesda, Md.…”

Section: Cellsmentioning

confidence: 99%

Phosphorylation-Dependent Regulation of T-Cell Activation by PAG/Cbp, a Lipid Raft-Associated Transmembrane Adaptor

Davidson

Bakinowski

Thomas

et al. 2003

Molecular and Cellular Biology

180

185

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“…It has been demonstrated that CD45 activates src family tyrosine kinase by dephosphorylating the COOH-terminal tyrosine residues on negative regulatory sites of p56 lck [36], or interacts with the z chain [37]. The CD45 ¹ T cell line failed to proliferate and produce IL-2 in response to antigen [38][39][40], but proliferative capacity and IL-2 production were restored after transfection with cDNA encoding CD45 or truncated CD45 that lacked extracellular domains [41][42][43][44]. This indicates that PTPase activity has a key role in the regulatory function of CD45 in transmembrane signalling [45].…”

Section: Discussionmentioning

confidence: 99%

Reduced protein tyrosine phosphatase (PTPase) activity of CD45 on peripheral blood lymphocytes in patients with systemic lupus erythematosus (SLE)

Takeuchi

Pang

Amano

et al. 1997

Clinical and Experimental Immunology

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SUMMARYTo disclose the mechanism of aberrant function of peripheral blood lymphocytes (PBL) in SLE, we focused on the catalytic function of CD45, and determined the CD45 PTPase activity in SLE patients. The sample population consisted of 32 SLE patients with different disease activity. PTPase activity of cell lysates immunoprecipitated by anti-CD45 MoAb was assayed against phosphotyrosine analogue PNPP, followed by measuring the release of para-nitro phenol at 410 nm. CD45 PTPase activity of PBL was significantly decreased in SLE patients, compared with that of normal controls and patients with systemic sclerosis (964 Ϯ 265, 1202 Ϯ 172, 1210 Ϯ 125, respectively; SLE versus normal, P < 0 . 05). It was correlated with SLE Disease Activity Index (SLEDAI) score (r ¼ 0 . 597, P ¼ 0 . 0006), but not with the dose of prednisolone (r ¼ 0 . 214, P ¼ 0 . 2657), indicating that CD45 PTPase activity became reduced when the disease was active, but it was not affected by prednisolone. Moreover, it was not corrected by in vitro culture with or without stimulation. The expression of CD45 on PBL was comparable between normal and SLE, raising a possibility that it may be due to aberrant regulation of catalytic function of CD45 in SLE. Given the evidence that tyrosine phosphorylation of cellular proteins by tyrosine kinases and phosphatases is one of the key biochemical events in the signal transduction pathway, the decreased CD45 PTPase activity in SLE may account for the defective signal transduction via TCR/CD3, leading to dysregulated effector function of the lymphocytes.

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“…In some CD45-cell lines (e.g., Jurkat), baseline tyrosine phosphorylation is normal, but the normal enhancement that usually follows TCR perturbation does not occur (21). In CD45-YAC-1 T cells, there is marked spontaneous tyrosine hyperphosphorylation, but no further increases are induced after TCR cross-linking (44). The reason for the differences between cell lines is unknown but presumably reflects differences in other biologically active molecules that may vary between them.…”

mentioning

confidence: 95%

“…Transformed and spontaneously proliferating T-cell lines that lose CD45 expression no longer manifest increases in phosphatidylinositol hydrolysis or normal rapid increases in [Ca']i when their TCRs are perturbed (22). An atypical Ca2' response is maintained, however, as ligation of TCRs on CD45-T cells results in delayed, asynchronous Ca2+ oscillations due to periodic release of Ca2+ from intracellular stores (44). There are also defects in signaling for tyrosine phosphorylation.…”

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confidence: 98%

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CD45 tyrosine phosphatase activity and membrane anchoring are required for T-cell antigen receptor signaling.

Niklinska¹,

Hou²,

June³

et al. 1994

Mol. Cell. Biol.

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T cells that lack the CD45 transmembrane tyrosine phosphatase have a variety of T-cell receptor (TCR) signaling defects that are corrected by reexpression of wild-type CD45 or its intracytoplasmic domains. In this study, a chimeric molecule containing the myristylation sequence of Src and the intracellular portion of CD45, previously shown to restore function in CD45-T cells, was mutagenized to determine if membrane-associated CD45 tyrosine phosphatase activity is required to restore TCR-mediated signaling in CD45-T cells. Abolition of enzymatic activity by substitution of a serine for a critical cysteine in the first catalytic domain resulted in failure of this molecule to restore TCR signaling. Another mutation, in which a single amino acid substitution destroyed the myristylation site, resulted in failure of the chimeric molecule to partition to the plasma membrane. Although expressed at high levels and enzymatically active, this form of intracellular CD45 also failed to restore normal signaling in CD45-T cells. These findings strongly suggest that CD45's function in TCR signaling requires its proximity to membrane-associated tyrosine phosphatase substrates.T-cell activation via the T-cell antigen receptor (TCR) is accompanied by increases in tyrosine phosphorylation of many proteins. The known substrates for activation-induced tyrosine kinases include 4 and other chains of the TCR (30, 34), phospholipase C yl (26), the ZAP-70 tyrosine kinase (6), the clathrin-binding valosin-containing protein (10, 29), Shc (31), Vav (4), and the cytoskeletal-associated protein ezrin (11). The importance of tyrosine phosphorylation as a primary event in T-cell activation is supported by several observations. First, tyrosine phosphorylation after TCR perturbation precedes other signals (17). Second, drugs that inhibit tyrosine kinase activation also prevent TCR-induced increases in intracellular Ca2+ concentration ([Ca2+]1) and hydrolysis of phosphatidylinositol bisphosphate (18,25). Third, T cells that lack the Lck or Fyn member of the Src family of tyrosine kinases develop abnormally and have aberrant TCR signaling properties (1,37,38).While much attention has focused on the role of tyrosine kinases in modulating the state of intracellular protein phosphorylation, it has become clear the tyrosine phosphatases play an equally critical role. Of particular interest is CD45, a transmembrane tyrosine phosphatase expressed by the large majority of bone marrow-derived cells. Jurkat), baseline tyrosine phosphorylation is normal, but the normal enhancement that usually follows TCR perturbation does not occur (21). In CD45-YAC-1 T cells, there is marked spontaneous tyrosine hyperphosphorylation, but no further increases are induced after TCR cross-linking (44). The reason for the differences between cell lines is unknown but presumably reflects differences in other biologically active molecules that may vary between them. Mice in which CD45 has been eliminated by homologous recombination also manifest a variety of lymphocyte abnor...

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The CD45 tyrosine phosphatase regulates phosphotyrosine homeostasis and its loss reveals a novel pattern of late T cell receptor-induced Ca2+ oscillations.

Cited by 73 publications

References 44 publications

Phosphorylation-Dependent Regulation of T-Cell Activation by PAG/Cbp, a Lipid Raft-Associated Transmembrane Adaptor

Phosphorylation-Dependent Regulation of T-Cell Activation by PAG/Cbp, a Lipid Raft-Associated Transmembrane Adaptor

Reduced protein tyrosine phosphatase (PTPase) activity of CD45 on peripheral blood lymphocytes in patients with systemic lupus erythematosus (SLE)

CD45 tyrosine phosphatase activity and membrane anchoring are required for T-cell antigen receptor signaling.

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