Uncoordinated 119 Protein Controls Trafficking of Lck via the Rab11 Endosome and Is Critical for Immunological Synapse Formation

Górska, M.; Qiang, Liang; Karim, Zunayet; Alam, Rafeul

doi:10.4049/jimmunol.0900792

Cited by 90 publications

(113 citation statements)

References 49 publications

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“…3A), suggesting that endosomal Lck may play such role. Consistent with previous reports (20,21), we found that tyrosine kinase Lck is localized both at the plasma membrane and Rab5 and Rab11-positive recycling endosomes (Fig. S4G).…”

Section: Intracellular Organization Of Cd3ζsupporting

confidence: 81%

Imaging T-cell receptor activation reveals accumulation of tyrosine-phosphorylated CD3ζ in the endosomal compartment

Yudushkin

Vale²

2010

Proc. Natl. Acad. Sci. U.S.A.

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Phosphorylation of the T-cell receptor complex (TcR/CD3) mediates the survival and antigen-induced activation of T cells. TcR/CD3 phosphorylation is usually monitored using phospho-specific antibodies, which precludes dynamic measurements. Here, we have developed genetically encoded, live-cell reporters that enable simultaneous monitoring of the phosphorylation state and intracellular trafficking of CD3ζ, the major signal-transducing subunit of the TcR/CD3. We show that these reporters provide accurate readouts of TcR/CD3 phosphorylation and are sensitive to the local balance of kinase and phosphatase activities acting upon TcR/CD3. Using these reporters, we demonstrate that, in addition to the expected activation-dependent phosphorylation at the plasma membrane, tyrosine-phosphorylated CD3ζ accumulates on endosomal vesicles distinct from lysosomes. These results suggest that an intracellular pool of phosphorylated CD3ζ may help to sustain TcR/ CD3 signaling after the receptor internalization. Recent studies using high-resolution imaging have demonstrated that signaling via TcR/CD3 displays complex spatial organization. TcR/CD3 molecules are preclustered at the plasma membrane and, upon ligation, nucleate downstream signaling components seconds after T-cell activation (4, 5). TcR/CD3 is constitutively internalized and recycled to the plasma membrane in naïve and activated T cells (reviewed in ref. 6), but the function of this turnover as well as the phosphorylation state of internalized receptor remain unknown.Currently, phosphorylation of TcR/CD3 in cells and in vitro is monitored using phospho-specific antibodies, and there are no methods that enable dynamic monitoring of the spatial organization of CD3 phosphorylation in live cells. Here, we have constructed genetically encoded fluorescent reporters compatible with imaging of live and fixed cells and demonstrate that they accurately monitor the dynamics and intracellular organization of CD3ζ phosphorylation in Jurkat T cells. Using the reporters, we observed that in addition to the expected activationdependent phosphorylation at the plasma membrane, tyrosinephosphorylated CD3ζ accumulated in the perinuclear endosomal vesicles. Our results demonstrate that endosomal CD3ζ remains signaling-competent and suggest the possibility that internalized CD3ζ pool may help to sustain long-term signaling in T cells. Results Design and Characterization of the CD3ζ Phosphorylation Reporters.To generate a Förster's resonant energy transfer (FRET)-based monomolecular reporter for phosphorylation of the key CD3 signal-transducing subunit ζ, we fused the C terminus of CD3ζ to a pair of green and red fluorescent proteins, eGFP and mCherry, linked by a flexible spacer and followed by the tandem SH2 domains of human ZAP-70 (residues 1-259; tSH2 ). We reasoned that intramolecular binding of the SH2 domains to tyrosine-phosphorylated ITAMs of CD3ζ (7) would result in conformational rearrangement of the adjacent fluorescent proteins and change in the FRET efficiency between the dono...

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Section: Intracellular Organization Of Cd3ζsupporting

confidence: 81%

Imaging T-cell receptor activation reveals accumulation of tyrosine-phosphorylated CD3ζ in the endosomal compartment

Yudushkin

Vale²

2010

Proc. Natl. Acad. Sci. U.S.A.

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“…Second, the sensitivity of the block in MC formation by overexpression of Unc119 indicated that specific anterograde transport processes of LAT are inhibited in the presence of Nef. Unc119 acts as adaptor protein in the transport of Lck to the plasma membrane (59) and may facilitate intracellular trafficking of LAT via similar mechanisms. Nef might interfere with these processes by, for example, prevention of the association of transport cargo with the Unc119 transport machinery or by modulating the activity of the associated Rab11 GTPase (40,59,60).…”

Section: Discussionmentioning

confidence: 99%

“…Unc119 acts as adaptor protein in the transport of Lck to the plasma membrane (59) and may facilitate intracellular trafficking of LAT via similar mechanisms. Nef might interfere with these processes by, for example, prevention of the association of transport cargo with the Unc119 transport machinery or by modulating the activity of the associated Rab11 GTPase (40,59,60). In this scenario, the differential destination of Lck (TGN) and LAT (undefined) upon disruption of their anterograde transport in the presence of Nef might reflect their specific topology as peripheral and integral membrane protein, respectively.…”

Section: Discussionmentioning

confidence: 99%

HIV-1 Nef Limits Communication between Linker of Activated T Cells and SLP-76 To Reduce Formation of SLP-76–Signaling Microclusters following TCR Stimulation

Abraham

Bankhead

Pan

et al. 2012

The Journal of Immunology

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Signal initiation by engagement of the TCR triggers actin rearrangements, receptor clustering, and dynamic organization of signaling complexes to elicit and sustain downstream signaling. Nef, a pathogenicity factor of HIV, disrupts early TCR signaling in target T cells. To define the mechanism underlying this Nef-mediated signal disruption, we employed quantitative single-cell microscopy following surface-mediated TCR stimulation that allows for dynamic visualization of distinct signaling complexes as microclusters (MCs). Despite marked inhibition of actin remodeling and cell spreading, the induction of MCs containing TCR-CD3 or ZAP70 was not affected significantly by Nef. However, Nef potently inhibited the subsequent formation of MCs positive for the signaling adaptor Src homology-2 domain-containing leukocyte protein of 76 kDa (SLP-76) to reduce MC density in Nef-expressing and HIV-1–infected T cells. Further analyses suggested that Nef prevents formation of SLP-76 MCs at the level of the upstream adaptor protein, linker of activated T cells (LAT), that couples ZAP70 to SLP-76. Nef did not disrupt pre-existing MCs positive for LAT. However, the presence of the viral protein prevented de novo recruitment of active LAT into MCs due to retargeting of LAT to an intracellular compartment. These modulations in MC formation and composition depended on Nef’s ability to simultaneously disrupt both actin remodeling and subcellular localization of TCR-proximal machinery. Nef thus employs a dual mechanism to disturb early TCR signaling by limiting the communication between LAT and SLP-76 and preventing the dynamic formation of SLP-76–signaling MCs.

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“…We subsequently showed that Unc119 controls the constitutive transport of Lck to the plasma membrane via endosomes. Unc119 is also critical for stimulation of the Lck enzymatic activity upon TCR triggering (14,15). The effect of Unc119 on Fyn is short-lasting and of significantly lower magnitude (14,16).…”

mentioning

confidence: 99%

Uncoordinated 119 Preferentially Induces Th2 Differentiation and Promotes the Development of Asthma

Górska

Goplen²,

Qiang³

et al. 2010

The Journal of Immunology

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The Th2 bias is a hallmark of allergic diseases. In this study, we show that the Th1 versus Th2 balance and the development of allergic asthma are strongly affected by the signaling protein uncoordinated 119 (Unc119). The expression of this adaptor protein is significantly increased in Th2 cells. Unc119 activates the Src family and inhibits the Abl family of tyrosine kinases. The activated Src family kinase Lck stimulates the activity of Itk and the expression of the transcription factor JunB. As a result, Unc119 promotes IL-4 production. Through inhibition of Abl kinases, Unc119 dampens IFN-γ production. Using adoptive transfer of Unc119-knockdown CD4 T cells, we show a critical role for Unc119 in the development of eosinophilic inflammation of airways, mucus production, and bronchial hyperreactivity in a mouse model. Intriguingly, the expression of the Unc119 protein is enhanced in CD4 T cells from patients with asthma. We speculate that the heightened expression of Unc119 promotes Th2, inhibits Th1 differentiation, and contributes to the pathogenesis of asthma in humans.

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Uncoordinated 119 Protein Controls Trafficking of Lck via the Rab11 Endosome and Is Critical for Immunological Synapse Formation

Cited by 90 publications

References 49 publications

Imaging T-cell receptor activation reveals accumulation of tyrosine-phosphorylated CD3ζ in the endosomal compartment

Imaging T-cell receptor activation reveals accumulation of tyrosine-phosphorylated CD3ζ in the endosomal compartment

HIV-1 Nef Limits Communication between Linker of Activated T Cells and SLP-76 To Reduce Formation of SLP-76–Signaling Microclusters following TCR Stimulation

Uncoordinated 119 Preferentially Induces Th2 Differentiation and Promotes the Development of Asthma

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