Endosomes: A legitimate platform for the signaling train

Murphy, Jane E.; Padilla, Benjamin E.; Hasdemir, Burcu; Cottrell, Graeme S.; Bunnett, Nigel W.

doi:10.1073/pnas.0906541106

Cited by 338 publications

(358 citation statements)

References 99 publications

(120 reference statements)

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“…Although active signaling from within endosomes has been reported for a selected group of membrane receptors (reviewed in refs. [23][24][25], here we directly demonstrate that internalized CD3ζ is signaling competent. The important, albeit nontrivial, questions that remain to be answered are whether distinct TcR/CD3-dependent signaling pathways can be traced specifically to the plasma-membrane and intracellular pools of CD3ζ and how these pathways depend on TcR/CD3 trafficking within the cell.…”

Section: Discussionmentioning

confidence: 84%

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: Discussionmentioning

confidence: 84%

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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“…Unlike other G‐protein‐coupled receptors that rapidly internalize following stimulation to terminate signaling,69 CXCR4 exhibits a prolonged stimulatory capacity attributed to endosomal signaling. Recruitment of signaling complexes, which can include both inhibitors or activators of signaling, to endosomes determines the signal type 70. Given that the presence of sCD74 did not inhibit MIF‐induced internalization of either CXCR2 or CXCR4, sCD74 might diminish MIF‐mediated endosomal signaling 69.…”

Section: Discussionmentioning

confidence: 99%

Soluble CD74 Reroutes MIF/CXCR4/AKT‐Mediated Survival of Cardiac Myofibroblasts to Necroptosis

Soppert

Kraemer

Beckers

et al. 2018

JAHA

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BackgroundAlthough macrophage migration inhibitory factor (MIF) has been demonstrated to mediate cardioprotection in ischemia/reperfusion injury and antagonize fibrotic effects through its receptor, CD74, the function of the soluble CD74 receptor ectodomain (sCD74) and its interaction with circulating MIF have not been explored in cardiac disease.Methods and ResultsCardiac fibroblasts were isolated from hearts of neonatal mice and differentiated into myofibroblasts. Co‐treatment with recombinant MIF and sCD74 induced cell death (P<0.001), which was mediated by receptor‐interacting serine/threonine‐protein kinase (RIP)1/RIP3‐dependent necroptosis (P=0.0376). This effect was specific for cardiac fibroblasts and did not affect cardiomyocytes. Gene expression analyses using microarray and RT‐qPCR technology revealed a 4‐fold upregulation of several interferon‐induced genes upon co‐treatment of myofibroblasts with sCD74 and MIF (Ifi44: P=0.011; Irg1: P=0.022; Clec4e: P=0.011). Furthermore, Western blot analysis confirmed the role of sCD74 as a modulator of MIF signaling by diminishing MIF‐mediated protein kinase B (AKT) activation (P=0.0197) and triggering p38 activation (P=0.0641). We obtained evidence that sCD74 inhibits MIF‐mediated survival pathway through the C‐X‐C chemokine receptor 4/AKT axis, enabling the induction of CD74‐dependent necroptotic processes in cardiac myofibroblasts. Preliminary clinical data revealed a lowered sCD74/MIF ratio in heart failure patients (17.47±10.09 versus 1.413±0.6244).ConclusionsThese findings suggest that treatment of cardiac myofibroblasts with sCD74 and MIF induces necroptosis, offering new insights into the mechanism of myofibroblast depletion during scar maturation. Preliminary clinical data provided first evidence about a clinical relevance of the sCD74/MIF axis in heart failure, suggesting that these proteins may be a promising target to modulate cardiac remodeling and disease progression in heart failure.

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“…Subsequent studies demonstrated that EGFR and other RTKs, are also capable of recruiting signaling molecules and transmitting signals from endosomes (7,9,10). Moreover, it was proposed that following EGFR activation clathrin mediated endocytosis plays an important regulatory role in control of sustained activation of the MAPK/ERK signaling pathway and in Akt stimulation (4,11).…”

mentioning

confidence: 99%

“…Moreover, it was proposed that following EGFR activation clathrin mediated endocytosis plays an important regulatory role in control of sustained activation of the MAPK/ERK signaling pathway and in Akt stimulation (4,11). The current prevailing view is that signals induced by activated EGFR and other RTKs can be transmitted from the plasma membrane as well as from endosomes and that the spatial localizations of RTKs play an important role in the control of signal specificity, duration, and robustness (4,(9)(10)(11)(12).…”

mentioning

confidence: 99%

Suppression of EGFR endocytosis by dynamin depletion reveals that EGFR signaling occurs primarily at the plasma membrane

Sousa¹,

Lax²,

Shen

et al. 2012

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

142

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The role of endocytosis in the control of EGF receptor (EGFR) activation and cell signaling was explored by using mouse fibroblasts in which dynamin was conditionally depleted. Dynamin is a GTPase shown to play an important role in the control clathrin mediated endocytosis of EGFR and other cell surface receptors. In this report, we demonstrate that EGF binding activity and the display of high and low affinity EGFRs on the cell surface are not affected by dynamin depletion. By contrast, dynamin depletion leads to a strong inhibition of EGFR endocytosis, robust enhancement of EGFR autophosphorylation and ubiquitination, and slower kinetics of EGFR degradation. Surprisingly, MAPK stimulation induced by either low or high EGF concentrations is not affected by dynamin depletion. While a similar initial Akt response is detected in control or dynamin depleted fibroblasts, a somewhat more sustained Akt stimulation is detected in the dynamin depleted cells. These experiments demonstrate that dynamin-mediated endocytosis leads to attenuation of EGFR activation and degradation and that stimulation of the MAPK response and Akt activation are primarily mediated by activated EGFR located in the plasma membrane.membrane receptors | tyrosine kinases

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Endosomes: A legitimate platform for the signaling train

Cited by 338 publications

References 99 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

Soluble CD74 Reroutes MIF/CXCR4/AKT‐Mediated Survival of Cardiac Myofibroblasts to Necroptosis

Suppression of EGFR endocytosis by dynamin depletion reveals that EGFR signaling occurs primarily at the plasma membrane

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