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On the Control of TCR Phosphorylation.

Fernandes RA, Huo J, Lui Y, Felce JH, Davis SJ - Front Immunol (2012)

View Article: PubMed Central - PubMed

Affiliation: MRC Human Immunology Unit, Nuffield Department of Clinical Medicine, John Radcliffe Hospital, University of Oxford Headington, Oxford, UK.

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This results in a net increase in the number and/or half-life of phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs) present in the CD3 subunits of the TCR (Smith-Garvin et al., )... Signaling via tyrosine-containing motifs phosphorylated by Src tyrosine kinases is not unique to the TCR complex; in fact, a large number of receptors present in lymphocytes likely signal in the same manner (Davis and van der Merwe, )... The possibility that positively charged residues present in the CD3ε and CD3ζ cytoplasmic domains interact with the cell membrane offered a novel explanation: that membrane association prevents kinase access to the TCR (Xu et al., )... However, mutation of these positively charged residues does not lead to receptor phosphorylation and has little or no effect on T-cell development (Deford-Watts et al., ; Fernandes et al., )... One possible explanation for this is that, if they occur at all, transient interactions of the relatively long and flexible CD3 cytoplasmic domains with the cell membrane could increase the frequency of productive encounters between ITAMs and the active sites of tyrosine kinases, whose positions are largely fixed by N-terminal membrane attachment... For now, whether or not receptors and kinases are pre-segregated within the membrane prior to triggering remains an open question... Further supporting this idea, receptor-type PTPs such as CD45 account for ∼10% of the cell surface protein content in a T-cell (Williams and Barclay, ), and have broad specificity (Barr et al., ) and very high catalytic activity (Fischer et al., )... Such effects might not be restricted to antigen receptors, or lymphocytes... Such a system would be highly sensitive to changes in the levels of expression of each individual component, and should respond with compensatory changes that return the status quo... We found that shRNAs that acutely down-regulate CD45 in Jurkat T-cells rapidly induce strong, Src kinase-dependent TCR phosphorylation and T-cell activation... Small-angle X-ray solution scattering and molecular dynamic simulations have revealed that unphosphorylated forms of Src kinases mainly adopt two distinct states in solution, with around 84% of Src or Hck assuming a closed/inactive conformation and 16% an open/active state; phosphorylated Tyr Src forms a single, closed conformation (Bernadó et al., ; Yang et al., )... Nika et al. have used the levels of phosphorylation of Tyr and Tyr to make inferences about the distribution of Lck states in vivo, and concluded that up to 40% of Lck is constitutively active... Classical “feed-forward” regulation of this type is observed for c-Src, Hck, and the closely related Fes and Abl kinases (see e.g., Alexandropoulos and Baltimore, ), but does it apply to Lck? We have found that phosphorylated CD3 ITAM-derived peptides strongly activate Lck measured with enolase as substrate (R... A final point is that Src kinase SH2 domains seem unique among the SH2 domains expressed by T-cells insofar as the purified SH2 domains bind the tyrosine-phosphorylated motifs of most receptors we have tested in surface plasmon resonance-based assays (J.

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Related in: MedlinePlus

Analysis of protein interactions using bioluminescence resonance energy transfer (BRET). (A) Schematic showing a monomeric protein expressed at the cell surface as chimeras with luciferase (Luc) and a green fluorescent protein (GFP) acting as donors and acceptors, respectively. The blue circle represents the 100 Å sphere within which random or stable co-association of Luc and GFP allows energy transfer. (B) Graphical representation of the relationship of energy transfer efficiency (BRETeff) to the acceptor/donor ratio for dimeric (green) and monomeric (blue) protein pairs expressed at the membrane, versus that measured for a pair of proteins one of which is expressed at the membrane (mem) and the other in the cytoplasm (cyt; James et al., 2006, 2011). In practice, the transfer efficiency is normalized against that measured for a soluble GFP/Luc fusion protein (sGFP–Luc) expressed in the cytoplasm. (C) The efficiency of energy transfer arising via random interactions is explained by the high density of membrane surface proteins and by their high mobility. Protein density is illustrated to scale, assuming that there are 20,000 molecules/μm2, and that each protein is 4 nm in diameter (Grasberger et al., 1986). It takes 0.2–0.3 s for a protein to move from position A to position B, based on measurements of the TCR diffusion rate (James et al., 2007). Since many of the proteins are likely diffusing at comparable rates, numerous random interactions seem unavoidable. Some estimates for the expression levels of cell surface proteins are substantially higher (Quinn et al., 1984).
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Figure 1: Analysis of protein interactions using bioluminescence resonance energy transfer (BRET). (A) Schematic showing a monomeric protein expressed at the cell surface as chimeras with luciferase (Luc) and a green fluorescent protein (GFP) acting as donors and acceptors, respectively. The blue circle represents the 100 Å sphere within which random or stable co-association of Luc and GFP allows energy transfer. (B) Graphical representation of the relationship of energy transfer efficiency (BRETeff) to the acceptor/donor ratio for dimeric (green) and monomeric (blue) protein pairs expressed at the membrane, versus that measured for a pair of proteins one of which is expressed at the membrane (mem) and the other in the cytoplasm (cyt; James et al., 2006, 2011). In practice, the transfer efficiency is normalized against that measured for a soluble GFP/Luc fusion protein (sGFP–Luc) expressed in the cytoplasm. (C) The efficiency of energy transfer arising via random interactions is explained by the high density of membrane surface proteins and by their high mobility. Protein density is illustrated to scale, assuming that there are 20,000 molecules/μm2, and that each protein is 4 nm in diameter (Grasberger et al., 1986). It takes 0.2–0.3 s for a protein to move from position A to position B, based on measurements of the TCR diffusion rate (James et al., 2007). Since many of the proteins are likely diffusing at comparable rates, numerous random interactions seem unavoidable. Some estimates for the expression levels of cell surface proteins are substantially higher (Quinn et al., 1984).

Mentions: Another proposed mechanism protecting the TCR from random phosphorylation is based on the physical segregation of the complex from Src kinases, predicated on the existence of “lipid rafts” (He and Marguet, 2008). Recently, in situ ultra high-resolution approaches, i.e., stimulated emission depletion far-field fluorescence microscopy and fluctuation correlation spectroscopy, were used to study the “nano-scale” organization of membrane lipids in situ (Eggeling et al., 2009). This showed that domains containing sphingolipids and glycosylphosphatidylinositol-anchored proteins, i.e., lipid rafts, might be as small as <20 nm diameter and very short-lived (∼10–20 ms; Eggeling et al., 2009). Whether such structures nevertheless prevent the interaction in resting cells of, e.g., receptors and Src kinases, could be probed using Förster resonance energy transfer (RET; Figure 1A), which is highly sensitive to random and non-random interactions of proteins within membranes, at roughly this length-scale (<10 nm; James et al., 2006). For now, whether or not receptors and kinases are pre-segregated within the membrane prior to triggering remains an open question.


On the Control of TCR Phosphorylation.

Fernandes RA, Huo J, Lui Y, Felce JH, Davis SJ - Front Immunol (2012)

Analysis of protein interactions using bioluminescence resonance energy transfer (BRET). (A) Schematic showing a monomeric protein expressed at the cell surface as chimeras with luciferase (Luc) and a green fluorescent protein (GFP) acting as donors and acceptors, respectively. The blue circle represents the 100 Å sphere within which random or stable co-association of Luc and GFP allows energy transfer. (B) Graphical representation of the relationship of energy transfer efficiency (BRETeff) to the acceptor/donor ratio for dimeric (green) and monomeric (blue) protein pairs expressed at the membrane, versus that measured for a pair of proteins one of which is expressed at the membrane (mem) and the other in the cytoplasm (cyt; James et al., 2006, 2011). In practice, the transfer efficiency is normalized against that measured for a soluble GFP/Luc fusion protein (sGFP–Luc) expressed in the cytoplasm. (C) The efficiency of energy transfer arising via random interactions is explained by the high density of membrane surface proteins and by their high mobility. Protein density is illustrated to scale, assuming that there are 20,000 molecules/μm2, and that each protein is 4 nm in diameter (Grasberger et al., 1986). It takes 0.2–0.3 s for a protein to move from position A to position B, based on measurements of the TCR diffusion rate (James et al., 2007). Since many of the proteins are likely diffusing at comparable rates, numerous random interactions seem unavoidable. Some estimates for the expression levels of cell surface proteins are substantially higher (Quinn et al., 1984).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3345363&req=5

Figure 1: Analysis of protein interactions using bioluminescence resonance energy transfer (BRET). (A) Schematic showing a monomeric protein expressed at the cell surface as chimeras with luciferase (Luc) and a green fluorescent protein (GFP) acting as donors and acceptors, respectively. The blue circle represents the 100 Å sphere within which random or stable co-association of Luc and GFP allows energy transfer. (B) Graphical representation of the relationship of energy transfer efficiency (BRETeff) to the acceptor/donor ratio for dimeric (green) and monomeric (blue) protein pairs expressed at the membrane, versus that measured for a pair of proteins one of which is expressed at the membrane (mem) and the other in the cytoplasm (cyt; James et al., 2006, 2011). In practice, the transfer efficiency is normalized against that measured for a soluble GFP/Luc fusion protein (sGFP–Luc) expressed in the cytoplasm. (C) The efficiency of energy transfer arising via random interactions is explained by the high density of membrane surface proteins and by their high mobility. Protein density is illustrated to scale, assuming that there are 20,000 molecules/μm2, and that each protein is 4 nm in diameter (Grasberger et al., 1986). It takes 0.2–0.3 s for a protein to move from position A to position B, based on measurements of the TCR diffusion rate (James et al., 2007). Since many of the proteins are likely diffusing at comparable rates, numerous random interactions seem unavoidable. Some estimates for the expression levels of cell surface proteins are substantially higher (Quinn et al., 1984).
Mentions: Another proposed mechanism protecting the TCR from random phosphorylation is based on the physical segregation of the complex from Src kinases, predicated on the existence of “lipid rafts” (He and Marguet, 2008). Recently, in situ ultra high-resolution approaches, i.e., stimulated emission depletion far-field fluorescence microscopy and fluctuation correlation spectroscopy, were used to study the “nano-scale” organization of membrane lipids in situ (Eggeling et al., 2009). This showed that domains containing sphingolipids and glycosylphosphatidylinositol-anchored proteins, i.e., lipid rafts, might be as small as <20 nm diameter and very short-lived (∼10–20 ms; Eggeling et al., 2009). Whether such structures nevertheless prevent the interaction in resting cells of, e.g., receptors and Src kinases, could be probed using Förster resonance energy transfer (RET; Figure 1A), which is highly sensitive to random and non-random interactions of proteins within membranes, at roughly this length-scale (<10 nm; James et al., 2006). For now, whether or not receptors and kinases are pre-segregated within the membrane prior to triggering remains an open question.

View Article: PubMed Central - PubMed

Affiliation: MRC Human Immunology Unit, Nuffield Department of Clinical Medicine, John Radcliffe Hospital, University of Oxford Headington, Oxford, UK.

AUTOMATICALLY GENERATED EXCERPT
Please rate it.

This results in a net increase in the number and/or half-life of phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs) present in the CD3 subunits of the TCR (Smith-Garvin et al., )... Signaling via tyrosine-containing motifs phosphorylated by Src tyrosine kinases is not unique to the TCR complex; in fact, a large number of receptors present in lymphocytes likely signal in the same manner (Davis and van der Merwe, )... The possibility that positively charged residues present in the CD3ε and CD3ζ cytoplasmic domains interact with the cell membrane offered a novel explanation: that membrane association prevents kinase access to the TCR (Xu et al., )... However, mutation of these positively charged residues does not lead to receptor phosphorylation and has little or no effect on T-cell development (Deford-Watts et al., ; Fernandes et al., )... One possible explanation for this is that, if they occur at all, transient interactions of the relatively long and flexible CD3 cytoplasmic domains with the cell membrane could increase the frequency of productive encounters between ITAMs and the active sites of tyrosine kinases, whose positions are largely fixed by N-terminal membrane attachment... For now, whether or not receptors and kinases are pre-segregated within the membrane prior to triggering remains an open question... Further supporting this idea, receptor-type PTPs such as CD45 account for ∼10% of the cell surface protein content in a T-cell (Williams and Barclay, ), and have broad specificity (Barr et al., ) and very high catalytic activity (Fischer et al., )... Such effects might not be restricted to antigen receptors, or lymphocytes... Such a system would be highly sensitive to changes in the levels of expression of each individual component, and should respond with compensatory changes that return the status quo... We found that shRNAs that acutely down-regulate CD45 in Jurkat T-cells rapidly induce strong, Src kinase-dependent TCR phosphorylation and T-cell activation... Small-angle X-ray solution scattering and molecular dynamic simulations have revealed that unphosphorylated forms of Src kinases mainly adopt two distinct states in solution, with around 84% of Src or Hck assuming a closed/inactive conformation and 16% an open/active state; phosphorylated Tyr Src forms a single, closed conformation (Bernadó et al., ; Yang et al., )... Nika et al. have used the levels of phosphorylation of Tyr and Tyr to make inferences about the distribution of Lck states in vivo, and concluded that up to 40% of Lck is constitutively active... Classical “feed-forward” regulation of this type is observed for c-Src, Hck, and the closely related Fes and Abl kinases (see e.g., Alexandropoulos and Baltimore, ), but does it apply to Lck? We have found that phosphorylated CD3 ITAM-derived peptides strongly activate Lck measured with enolase as substrate (R... A final point is that Src kinase SH2 domains seem unique among the SH2 domains expressed by T-cells insofar as the purified SH2 domains bind the tyrosine-phosphorylated motifs of most receptors we have tested in surface plasmon resonance-based assays (J.

No MeSH data available.


Related in: MedlinePlus