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Impaired plasma membrane targeting of Grb2-murine son of sevenless (mSOS) complex and differential activation of the Fyn-T cell receptor (TCR)-zeta-Cbl pathway mediate T cell hyporesponsiveness in autoimmune nonobese diabetic mice.

Salojin K, Zhang J, Cameron M, Gill B, Arreaza G, Ochi A, Delovitch TL - J. Exp. Med. (1997)

Bottom Line: We previously showed that TCR-induced NOD T cell hyporesponsiveness is associated with a block in Ras activation and defective signaling along the PKC/Ras/MAPK pathway.We demonstrate that NOD T cell hyporesponsiveness is associated with the (a) enhanced TCR-beta-associated Fyn kinase activity and the differential activation of the Fyn-TCR-zeta-Cbl pathway, which may account for the impaired recruitment of ZAP70 to membrane-bound TCR-zeta; (b) relative inability of the murine son of sevenless (mSOS) Ras GDP releasing factor activity to translocate from the cytoplasm to the plasma membrane; and (c) exclusion of mSOS and PLC-gamma1 from the TCR-zeta-associated Grb2/pp36-38/ZAP70 signaling complex.These findings implicate mSOS as an important mediator of downregulation of Ras signaling in hyporesponsive NOD T cells.

View Article: PubMed Central - PubMed

Affiliation: Autoimmunity/Diabetes Group, The John P. Robarts Research Institute, London, Ontario, Canada N6G 2V4.

ABSTRACT
Nonobese diabetic (NOD) mouse thymocytes are hyporesponsive to T cell antigen receptor (TCR)-mediated stimulation of proliferation, and this T cell hyporesponsiveness may be causal to the onset of autoimmune diabetes in NOD mice. We previously showed that TCR-induced NOD T cell hyporesponsiveness is associated with a block in Ras activation and defective signaling along the PKC/Ras/MAPK pathway. Here, we report that several sequential changes in TCR-proximal signaling events may mediate this block in Ras activation. We demonstrate that NOD T cell hyporesponsiveness is associated with the (a) enhanced TCR-beta-associated Fyn kinase activity and the differential activation of the Fyn-TCR-zeta-Cbl pathway, which may account for the impaired recruitment of ZAP70 to membrane-bound TCR-zeta; (b) relative inability of the murine son of sevenless (mSOS) Ras GDP releasing factor activity to translocate from the cytoplasm to the plasma membrane; and (c) exclusion of mSOS and PLC-gamma1 from the TCR-zeta-associated Grb2/pp36-38/ZAP70 signaling complex. Our data suggest that altered tyrosine phosphorylation and targeting of the Grb2/pp36-38/ZAP70 complex to the plasma membrane and cytoskeleton and the deficient association of mSOS with this Grb2-containing complex may block the downstream activation of Ras and Ras-mediated amplification of TCR/CD3-mediated signals in hyporesponsive NOD T cells. These findings implicate mSOS as an important mediator of downregulation of Ras signaling in hyporesponsive NOD T cells.

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(A) Time course of  activation of TCR-β–associated  kinase activity in TCR-β–stimulated NOD and B6 thymocytes.  NOD and control B6 thymocytes (2 × 107 cells/lane)  were incubated for 3 min in the  presence of biotinylated anti– TCR-β. Cell-bound mAbs were  either not cross-linked (0 min)  or were cross-linked for the indicated times in the presence of  protein G. Cells were washed to  remove unbound mAbs, and  TCR-β–immune complexes  were immunoprecipitated from  precleared postnuclear fractions  of thymocyte lysates using  streptavidin immobilized on 4%  beaded agarose and then assayed  for their associated in vitro kinase activity. Membranes were  then immunoblotted serially  with different mAbs, and overlay  of autoradiograms and immunoblots demonstrated equal loading in each lane (data not shown)  and confirmed the nature of the  proteins phosphorylated in  vitro. The positions of molecular  mass markers are shown on the  left. The results shown are representative of one of three separate  reproducible experiments. (B) Tyrosine phosphorylation of Fyn is markedly increased in NOD thymocytes. NOD and control B6 thymocytes (2 × 107  cells/lane) were either unstimulated (None) or stimulated for 1.5 min with anti–TCR-β or anti–TCR-β plus anti-CD4. Fyn was immunoprecipitated  from precleared postnuclear fractions of thymocyte lysates, and immunoprecipitates were immunoblotted with an anti–p-Tyr mAb. (C) Stimulation of  Fyn-associated kinase activity and diminished association of Fyn-independent TCR-ζ with ZAP70 in response to TCR-β or TCR-β–CD4 treatment of  NOD and B6 thymocytes. NOD and B6 thymocyte lysates were immunoprecipitated with anti-Fyn and assayed for Fyn-associated kinase activity in in  vitro kinase assays. Overlay of autoradiograms and immunoblots demonstrated equal loading in each lane and confirmed the nature of the detected  phosphoproteins (anti-Fyn immunoblotting, data not shown; anti–TCR-ζ immunoblotting, middle). Supernatants precleared of Fyn were immunoprecipitated with anti-ZAP70, and the amounts of residual (Fyn-independent) TCR-ζ in these precipitates were analyzed by immunoblotting (bottom). (D)  The level of Tyr528 phosphorylation of Fyn is decreased in quiescent NOD thymocytes. [32P]Phosphate labeling and peptide mapping of Fyn immobilized to membrane. [32P]Phosphate-labeled Fyn fragments were resolved by SDS-24% PAGE in a Tricine cathode buffer, and the signal intensities from  autoradiographs were quantified. Results are expressed as the ratio of the signal intensity of the potential NH2-terminal sites of serine, threonine, and tyrosine phosphorylation (NH2) relative to the Tyr528-containing regulatory COOH-terminal site (COOH). The results shown are representative of one of  two separate reproducible experiments.
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Figure 4: (A) Time course of activation of TCR-β–associated kinase activity in TCR-β–stimulated NOD and B6 thymocytes. NOD and control B6 thymocytes (2 × 107 cells/lane) were incubated for 3 min in the presence of biotinylated anti– TCR-β. Cell-bound mAbs were either not cross-linked (0 min) or were cross-linked for the indicated times in the presence of protein G. Cells were washed to remove unbound mAbs, and TCR-β–immune complexes were immunoprecipitated from precleared postnuclear fractions of thymocyte lysates using streptavidin immobilized on 4% beaded agarose and then assayed for their associated in vitro kinase activity. Membranes were then immunoblotted serially with different mAbs, and overlay of autoradiograms and immunoblots demonstrated equal loading in each lane (data not shown) and confirmed the nature of the proteins phosphorylated in vitro. The positions of molecular mass markers are shown on the left. The results shown are representative of one of three separate reproducible experiments. (B) Tyrosine phosphorylation of Fyn is markedly increased in NOD thymocytes. NOD and control B6 thymocytes (2 × 107 cells/lane) were either unstimulated (None) or stimulated for 1.5 min with anti–TCR-β or anti–TCR-β plus anti-CD4. Fyn was immunoprecipitated from precleared postnuclear fractions of thymocyte lysates, and immunoprecipitates were immunoblotted with an anti–p-Tyr mAb. (C) Stimulation of Fyn-associated kinase activity and diminished association of Fyn-independent TCR-ζ with ZAP70 in response to TCR-β or TCR-β–CD4 treatment of NOD and B6 thymocytes. NOD and B6 thymocyte lysates were immunoprecipitated with anti-Fyn and assayed for Fyn-associated kinase activity in in vitro kinase assays. Overlay of autoradiograms and immunoblots demonstrated equal loading in each lane and confirmed the nature of the detected phosphoproteins (anti-Fyn immunoblotting, data not shown; anti–TCR-ζ immunoblotting, middle). Supernatants precleared of Fyn were immunoprecipitated with anti-ZAP70, and the amounts of residual (Fyn-independent) TCR-ζ in these precipitates were analyzed by immunoblotting (bottom). (D) The level of Tyr528 phosphorylation of Fyn is decreased in quiescent NOD thymocytes. [32P]Phosphate labeling and peptide mapping of Fyn immobilized to membrane. [32P]Phosphate-labeled Fyn fragments were resolved by SDS-24% PAGE in a Tricine cathode buffer, and the signal intensities from autoradiographs were quantified. Results are expressed as the ratio of the signal intensity of the potential NH2-terminal sites of serine, threonine, and tyrosine phosphorylation (NH2) relative to the Tyr528-containing regulatory COOH-terminal site (COOH). The results shown are representative of one of two separate reproducible experiments.

Mentions: In anergic CD4+ peripheral T cells, Fyn activity is elevated (33, 34) and Fyn, but neither Lck nor ZAP70, associates with TCR-ζ (29). To test whether similar events occur in hyporesponsive NOD thymocytes, the levels and kinetics of TCR-associated Fyn activity and the relative capacities of Fyn to bind to TCR-ζ induced upon TCR ligation were determined. A rapid increase in tyrosine phosphorylation of membrane-localized Fyn, CD3, and TCR-ζ was noted (Fig. 4 A). The most striking difference between NOD and control C57BL/6J thymocytes was the elevated autophosphorylation of membrane-bound Fyn both in quiescent (no cross-linking) and TCR-β–stimulated NOD thymocytes. This was accompanied by the transient tyrosine hyperphosphorylation of CD3 and TCR-ζ. At each time of analysis (0–20 min), slightly increased amounts of Fyn coprecipitated with TCR-β before and after cross-linking, indicating that Fyn is constitutively associated with TCR in the membrane. Anti-Lck and anti-ZAP70 mAb immunoblotting confirmed that increased Fyn activity was not due to the presence of Lck or ZAP70 in TCR-β immunoprecipitates (data not shown). A significant increase in basal (no cross-linking) and TCR-β cross-linking–induced tyrosine phosphorylation of total cellular Fyn was also observed in NOD thymocytes (Fig. 4 B), and a similar result was obtained using an in vitro kinase assay (Fig. 4 C).


Impaired plasma membrane targeting of Grb2-murine son of sevenless (mSOS) complex and differential activation of the Fyn-T cell receptor (TCR)-zeta-Cbl pathway mediate T cell hyporesponsiveness in autoimmune nonobese diabetic mice.

Salojin K, Zhang J, Cameron M, Gill B, Arreaza G, Ochi A, Delovitch TL - J. Exp. Med. (1997)

(A) Time course of  activation of TCR-β–associated  kinase activity in TCR-β–stimulated NOD and B6 thymocytes.  NOD and control B6 thymocytes (2 × 107 cells/lane)  were incubated for 3 min in the  presence of biotinylated anti– TCR-β. Cell-bound mAbs were  either not cross-linked (0 min)  or were cross-linked for the indicated times in the presence of  protein G. Cells were washed to  remove unbound mAbs, and  TCR-β–immune complexes  were immunoprecipitated from  precleared postnuclear fractions  of thymocyte lysates using  streptavidin immobilized on 4%  beaded agarose and then assayed  for their associated in vitro kinase activity. Membranes were  then immunoblotted serially  with different mAbs, and overlay  of autoradiograms and immunoblots demonstrated equal loading in each lane (data not shown)  and confirmed the nature of the  proteins phosphorylated in  vitro. The positions of molecular  mass markers are shown on the  left. The results shown are representative of one of three separate  reproducible experiments. (B) Tyrosine phosphorylation of Fyn is markedly increased in NOD thymocytes. NOD and control B6 thymocytes (2 × 107  cells/lane) were either unstimulated (None) or stimulated for 1.5 min with anti–TCR-β or anti–TCR-β plus anti-CD4. Fyn was immunoprecipitated  from precleared postnuclear fractions of thymocyte lysates, and immunoprecipitates were immunoblotted with an anti–p-Tyr mAb. (C) Stimulation of  Fyn-associated kinase activity and diminished association of Fyn-independent TCR-ζ with ZAP70 in response to TCR-β or TCR-β–CD4 treatment of  NOD and B6 thymocytes. NOD and B6 thymocyte lysates were immunoprecipitated with anti-Fyn and assayed for Fyn-associated kinase activity in in  vitro kinase assays. Overlay of autoradiograms and immunoblots demonstrated equal loading in each lane and confirmed the nature of the detected  phosphoproteins (anti-Fyn immunoblotting, data not shown; anti–TCR-ζ immunoblotting, middle). Supernatants precleared of Fyn were immunoprecipitated with anti-ZAP70, and the amounts of residual (Fyn-independent) TCR-ζ in these precipitates were analyzed by immunoblotting (bottom). (D)  The level of Tyr528 phosphorylation of Fyn is decreased in quiescent NOD thymocytes. [32P]Phosphate labeling and peptide mapping of Fyn immobilized to membrane. [32P]Phosphate-labeled Fyn fragments were resolved by SDS-24% PAGE in a Tricine cathode buffer, and the signal intensities from  autoradiographs were quantified. Results are expressed as the ratio of the signal intensity of the potential NH2-terminal sites of serine, threonine, and tyrosine phosphorylation (NH2) relative to the Tyr528-containing regulatory COOH-terminal site (COOH). The results shown are representative of one of  two separate reproducible experiments.
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Related In: Results  -  Collection

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Figure 4: (A) Time course of activation of TCR-β–associated kinase activity in TCR-β–stimulated NOD and B6 thymocytes. NOD and control B6 thymocytes (2 × 107 cells/lane) were incubated for 3 min in the presence of biotinylated anti– TCR-β. Cell-bound mAbs were either not cross-linked (0 min) or were cross-linked for the indicated times in the presence of protein G. Cells were washed to remove unbound mAbs, and TCR-β–immune complexes were immunoprecipitated from precleared postnuclear fractions of thymocyte lysates using streptavidin immobilized on 4% beaded agarose and then assayed for their associated in vitro kinase activity. Membranes were then immunoblotted serially with different mAbs, and overlay of autoradiograms and immunoblots demonstrated equal loading in each lane (data not shown) and confirmed the nature of the proteins phosphorylated in vitro. The positions of molecular mass markers are shown on the left. The results shown are representative of one of three separate reproducible experiments. (B) Tyrosine phosphorylation of Fyn is markedly increased in NOD thymocytes. NOD and control B6 thymocytes (2 × 107 cells/lane) were either unstimulated (None) or stimulated for 1.5 min with anti–TCR-β or anti–TCR-β plus anti-CD4. Fyn was immunoprecipitated from precleared postnuclear fractions of thymocyte lysates, and immunoprecipitates were immunoblotted with an anti–p-Tyr mAb. (C) Stimulation of Fyn-associated kinase activity and diminished association of Fyn-independent TCR-ζ with ZAP70 in response to TCR-β or TCR-β–CD4 treatment of NOD and B6 thymocytes. NOD and B6 thymocyte lysates were immunoprecipitated with anti-Fyn and assayed for Fyn-associated kinase activity in in vitro kinase assays. Overlay of autoradiograms and immunoblots demonstrated equal loading in each lane and confirmed the nature of the detected phosphoproteins (anti-Fyn immunoblotting, data not shown; anti–TCR-ζ immunoblotting, middle). Supernatants precleared of Fyn were immunoprecipitated with anti-ZAP70, and the amounts of residual (Fyn-independent) TCR-ζ in these precipitates were analyzed by immunoblotting (bottom). (D) The level of Tyr528 phosphorylation of Fyn is decreased in quiescent NOD thymocytes. [32P]Phosphate labeling and peptide mapping of Fyn immobilized to membrane. [32P]Phosphate-labeled Fyn fragments were resolved by SDS-24% PAGE in a Tricine cathode buffer, and the signal intensities from autoradiographs were quantified. Results are expressed as the ratio of the signal intensity of the potential NH2-terminal sites of serine, threonine, and tyrosine phosphorylation (NH2) relative to the Tyr528-containing regulatory COOH-terminal site (COOH). The results shown are representative of one of two separate reproducible experiments.
Mentions: In anergic CD4+ peripheral T cells, Fyn activity is elevated (33, 34) and Fyn, but neither Lck nor ZAP70, associates with TCR-ζ (29). To test whether similar events occur in hyporesponsive NOD thymocytes, the levels and kinetics of TCR-associated Fyn activity and the relative capacities of Fyn to bind to TCR-ζ induced upon TCR ligation were determined. A rapid increase in tyrosine phosphorylation of membrane-localized Fyn, CD3, and TCR-ζ was noted (Fig. 4 A). The most striking difference between NOD and control C57BL/6J thymocytes was the elevated autophosphorylation of membrane-bound Fyn both in quiescent (no cross-linking) and TCR-β–stimulated NOD thymocytes. This was accompanied by the transient tyrosine hyperphosphorylation of CD3 and TCR-ζ. At each time of analysis (0–20 min), slightly increased amounts of Fyn coprecipitated with TCR-β before and after cross-linking, indicating that Fyn is constitutively associated with TCR in the membrane. Anti-Lck and anti-ZAP70 mAb immunoblotting confirmed that increased Fyn activity was not due to the presence of Lck or ZAP70 in TCR-β immunoprecipitates (data not shown). A significant increase in basal (no cross-linking) and TCR-β cross-linking–induced tyrosine phosphorylation of total cellular Fyn was also observed in NOD thymocytes (Fig. 4 B), and a similar result was obtained using an in vitro kinase assay (Fig. 4 C).

Bottom Line: We previously showed that TCR-induced NOD T cell hyporesponsiveness is associated with a block in Ras activation and defective signaling along the PKC/Ras/MAPK pathway.We demonstrate that NOD T cell hyporesponsiveness is associated with the (a) enhanced TCR-beta-associated Fyn kinase activity and the differential activation of the Fyn-TCR-zeta-Cbl pathway, which may account for the impaired recruitment of ZAP70 to membrane-bound TCR-zeta; (b) relative inability of the murine son of sevenless (mSOS) Ras GDP releasing factor activity to translocate from the cytoplasm to the plasma membrane; and (c) exclusion of mSOS and PLC-gamma1 from the TCR-zeta-associated Grb2/pp36-38/ZAP70 signaling complex.These findings implicate mSOS as an important mediator of downregulation of Ras signaling in hyporesponsive NOD T cells.

View Article: PubMed Central - PubMed

Affiliation: Autoimmunity/Diabetes Group, The John P. Robarts Research Institute, London, Ontario, Canada N6G 2V4.

ABSTRACT
Nonobese diabetic (NOD) mouse thymocytes are hyporesponsive to T cell antigen receptor (TCR)-mediated stimulation of proliferation, and this T cell hyporesponsiveness may be causal to the onset of autoimmune diabetes in NOD mice. We previously showed that TCR-induced NOD T cell hyporesponsiveness is associated with a block in Ras activation and defective signaling along the PKC/Ras/MAPK pathway. Here, we report that several sequential changes in TCR-proximal signaling events may mediate this block in Ras activation. We demonstrate that NOD T cell hyporesponsiveness is associated with the (a) enhanced TCR-beta-associated Fyn kinase activity and the differential activation of the Fyn-TCR-zeta-Cbl pathway, which may account for the impaired recruitment of ZAP70 to membrane-bound TCR-zeta; (b) relative inability of the murine son of sevenless (mSOS) Ras GDP releasing factor activity to translocate from the cytoplasm to the plasma membrane; and (c) exclusion of mSOS and PLC-gamma1 from the TCR-zeta-associated Grb2/pp36-38/ZAP70 signaling complex. Our data suggest that altered tyrosine phosphorylation and targeting of the Grb2/pp36-38/ZAP70 complex to the plasma membrane and cytoskeleton and the deficient association of mSOS with this Grb2-containing complex may block the downstream activation of Ras and Ras-mediated amplification of TCR/CD3-mediated signals in hyporesponsive NOD T cells. These findings implicate mSOS as an important mediator of downregulation of Ras signaling in hyporesponsive NOD T cells.

Show MeSH
Related in: MedlinePlus