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

Niklinska, B B; Hou, Damon; June, Carl H.; Weissman, Allan M.; Ashwell, Jonathan D.

doi:10.1128/mcb.14.12.8078

Cited by 12 publications

(5 citation statements)

References 42 publications

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“…The YAC-1 T-lymphoma cell line and its CD45 Ϫ variant N1 have been previously described (49). GA2.1 and CS269.2 cells were derived from N1 cells by transfection with cDNA encoding myr-iCD45 containing a Gly-23Ala or a Cys-2693Ser substitution, respectively (34).…”

Section: Methodsmentioning

confidence: 99%

Mutational Analysis of Lck in CD45-Negative T Cells: Dominant Role of Tyrosine 394 Phosphorylation in Kinase Activity

D’Oro

Sakaguchi

Appella

et al. 1996

Molecular and Cellular Biology

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The CD45 tyrosine phosphatase has been reported to activate the src family tyrosine kinases Lck and Fyn by dephosphorylating regulatory COOH-terminal tyrosine residues 505 and 528, respectively. However, recent studies with CD45 ؊ T-cell lines have found that despite the fact that Lck and Fyn were constitutively hyperphosphorylated, the tyrosine kinase activity of both enzymes was actually increased. In the present study, phosphoamino acid analysis revealed that the increased phosphorylation of Lck in CD45؊ YAC-1 T cells was restricted to tyrosine residues. To understand the relationship between tyrosine phosphorylation and Lck kinase activity, CD45؊ YAC-1 cells were transfected with forms of Lck in which tyrosines whose phosphorylation is thought to regulate enzyme activity (Tyr-192, Tyr-394, Tyr-505, or both Tyr-394 and Tyr-505) were replaced with phenylalanine. While the Y-to-F mutation at position 192 (192-Y3F) had little effect, the 505-Y3F mutation increased enzymatic activity. In contrast, the 394-Y3F mutation decreased the kinase activity to very low levels, an effect that the double mutation, 394-Y3F and 505Y3F, could not reverse. Phosphopeptide analysis of tryptic digests of Lck from CD45؊ YAC-1 cells revealed that it is hyperphosphorylated on two tyrosine residues, Tyr-505 and, to a lesser extent, Tyr-394. The purified and enzymatically active intracellular portion of CD45 dephosphorylated Lck Tyr-394 in vitro. These results demonstrate that in addition to Tyr-505, CD45 can dephosphorylate Tyr-394, and that in the absence of CD45 the hyperphosphorylation of Tyr-394 can cause an increase in the kinase activity of Lck despite the inhibitory hyperphosphorylation of Tyr-505. Therefore, Lck kinase activity is determined by the balance of activating and inhibitory tyrosine phosphorylations that are, in turn, regulated by CD45.

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Section: Methodsmentioning

confidence: 99%

Mutational Analysis of Lck in CD45-Negative T Cells: Dominant Role of Tyrosine 394 Phosphorylation in Kinase Activity

D’Oro

Sakaguchi

Appella

et al. 1996

Molecular and Cellular Biology

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“…All cDNAs were fully sequenced to ensure that they contained no unwanted mutations (data not shown). The cDNAs coding for constitutively activated FynT (FynT Y528F), myristylated CD45 (wild type or phosphatase inactive [C3S]), and ⌬SH2 Y505F Lck were described elsewhere (6,13,25).…”

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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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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“…Restoration of the TCR signal pathway can be achieved by transfection with CD45 (18 -20). Recently, it has been shown using chimeric molecules that the cytoplasmic portion of CD45 with a catalytically active membrane proximal phosphatase domain is necessary and sufficient for restoration of TCR signal transduction in CD45-deficient cell lines (21)(22)(23)(24)(25).…”

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confidence: 99%

An Activated Epidermal Growth Factor Receptor/Lck Chimera Restores Early T Cell Receptor-mediated Calcium Response in a CD45-deficient T Cell Line

Duplay

Alcover

Fargeas

et al. 1996

Journal of Biological Chemistry

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In T cells, cell surface expression of CD45, a transmembrane tyrosine phosphatase, is required for T cell receptor (TCR) signal transduction. Indirect evidence suggests that CD45 function in TCR signaling involves the dephosphorylation of the C-terminal negative regulatory site of p56 Stimulation of the T cell receptor (TCR 1 ) induces a signal transduction cascade that leads to activation of the mature T cell (1). Tyrosine phosphorylation of a number of cellular proteins is thought to be the initial signaling event initiated via the TCR (2). Two protein tyrosine kinases (PTKs) of the src family (p56 lck and p59 fyn ) (3-7) and two of the syk family (ZAP-70 and Syk) (8 -14) have been shown to participate in this process.Regulation of protein phosphorylation also involves specific protein tyrosine phosphatases (15). In T cells, CD45, a transmembrane glycoprotein, is the best characterized protein tyrosine phosphatase (16). Direct evidence for the involvement of CD45 in TCR signal transduction was demonstrated by functional analysis of CD45-deficient T cell mutants (17). In the absence of CD45 expression, TCR stimulation fails to generate proximal signaling events (i.e. induction of tyrosine phosphorylation, phosphoinositide hydrolysis, and increases in the concentration of intracellular Ca 2ϩ ) and distal events (i.e. interleukin 2 production and proliferation). Restoration of the TCR signal pathway can be achieved by transfection with . Recently, it has been shown using chimeric molecules that the cytoplasmic portion of CD45 with a catalytically active membrane proximal phosphatase domain is necessary and sufficient for restoration of TCR signal transduction in CD45-deficient cell lines (21-25).In addition, CD45 seems to be required not only to initiate but also to maintain TCR signaling. In a CD45 Ϫ T cell line in which a chimeric EGFR/CD45 restores signaling, the addition of EGF during TCR stimulation inhibits CD45 function and results in a rapid inactivation of TCR-mediated signals (22).Although the phosphatase activity of CD45 is clearly critical for TCR signaling, the mechanisms by which CD45 contributes to this process are poorly understood. However, increasing evidence suggests that there is an interplay of CD45 and tyrosine kinases during T cell activation. In particular, the regulation of p56 lck and p59 fyn by CD45 is well documented. The mechanism of activation of src PTKs is thought to involve phosphorylation of Tyr-394 (Lck) or Tyr-420 (Fyn) and dephosphorylation of Tyr-505 (Lck) or Tyr-531 (Fyn). A number of studies have shown that CD45 alters the state of phosphorylation of the C-terminal negative regulatory tyrosine residue of src PTKs (26 -31). The mechanism of CD45 regulation of p56 lck tyrosine kinase can be rationalized by a model involving an intramolecular interaction between the phosphotyrosine Tyr-505 and the SH2 domain of p56 lck (32). SH2 binding to the site of phosphorylated Tyr-505 imposes a "closed," presumably inactive, conformation on the p56 lck protein. Consistent with this model...

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

Cited by 12 publications

References 42 publications

Mutational Analysis of Lck in CD45-Negative T Cells: Dominant Role of Tyrosine 394 Phosphorylation in Kinase Activity

Mutational Analysis of Lck in CD45-Negative T Cells: Dominant Role of Tyrosine 394 Phosphorylation in Kinase Activity

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

An Activated Epidermal Growth Factor Receptor/Lck Chimera Restores Early T Cell Receptor-mediated Calcium Response in a CD45-deficient T Cell Line

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