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Ndfip-mediated degradation of Jak1 tunes cytokine signalling to limit expansion of CD4+ effector T cells.

O'Leary CE, Riling CR, Spruce LA, Ding H, Kumar S, Deng G, Liu Y, Seeholzer SH, Oliver PM - Nat Commun (2016)

Bottom Line: Nedd4 family E3 ubiquitin ligases have been shown to restrict T-cell function and impact T-cell differentiation.We show here that Ndfip1 and Ndfip2, activators of Nedd4 family ligases, together limit accumulation and function of effector CD4+ T cells.In wild-type cells, Jak1 degradation lessens CD4+ cell sensitivity to cytokines during TCR stimulation, while in Ndfip-deficient cells cytokine responsiveness persists, promoting increased expansion and survival of pathogenic effector T cells.

View Article: PubMed Central - PubMed

Affiliation: Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

ABSTRACT
Nedd4 family E3 ubiquitin ligases have been shown to restrict T-cell function and impact T-cell differentiation. We show here that Ndfip1 and Ndfip2, activators of Nedd4 family ligases, together limit accumulation and function of effector CD4+ T cells. Using a three-part proteomics approach in primary T cells, we identify stabilization of Jak1 in Ndfip1/2-deficient T cells stimulated through the TCR. Jak1 degradation is aborted in activated T cells that lack Ndfips. In wild-type cells, Jak1 degradation lessens CD4+ cell sensitivity to cytokines during TCR stimulation, while in Ndfip-deficient cells cytokine responsiveness persists, promoting increased expansion and survival of pathogenic effector T cells. Thus, Ndfip1/Ndfip2 regulate the cross talk between the T-cell receptor and cytokine signalling pathways to limit inappropriate T-cell responses.

No MeSH data available.


Related in: MedlinePlus

Identification of differential ubiquitylation by proteomics.(a) Schematic of the three proteomics methods used. (b) Area proportional Venn diagram illustrating the reproducibility of proteins identified as having SILAC ratios after TUBE enrichment in three out of four biological replicates. (c) SILAC–TUBE ratios (WT/DKO) for each protein were corrected using ‘input' ratio (DKO/WT) as calculated by label-free quantification of whole proteome DKO and WT data sets. The average corrected ratio is plotted against the average number of unique peptides observed per protein across four whole proteome LC-MS/MS experiments. Plot limited to 40 average unique peptides for clarity. Itch and Nedd4-2 (indicated) have log2 transformed corrected ratio >1 at the protein level. Itch=1.57±0.17 and Nedd4-2=3.18±0.226. (d) Overlap of proteins with corrected SILAC–TUBE ratios (observed in at least three experiments) and proteins identified with at least one K-ɛ-GG peptide (in at least one of the three K-ɛ-GG immunoprecipitation experiments). (e) Heat map illustrating reproducibility of corrected SILAC–TUBE ratios (WT/DKO, log2 transformed, observed in at least three of four experiments) for proteins identified with K-ɛ-GG peptides. Reverse=SILAC labeling swapped.
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f5: Identification of differential ubiquitylation by proteomics.(a) Schematic of the three proteomics methods used. (b) Area proportional Venn diagram illustrating the reproducibility of proteins identified as having SILAC ratios after TUBE enrichment in three out of four biological replicates. (c) SILAC–TUBE ratios (WT/DKO) for each protein were corrected using ‘input' ratio (DKO/WT) as calculated by label-free quantification of whole proteome DKO and WT data sets. The average corrected ratio is plotted against the average number of unique peptides observed per protein across four whole proteome LC-MS/MS experiments. Plot limited to 40 average unique peptides for clarity. Itch and Nedd4-2 (indicated) have log2 transformed corrected ratio >1 at the protein level. Itch=1.57±0.17 and Nedd4-2=3.18±0.226. (d) Overlap of proteins with corrected SILAC–TUBE ratios (observed in at least three experiments) and proteins identified with at least one K-ɛ-GG peptide (in at least one of the three K-ɛ-GG immunoprecipitation experiments). (e) Heat map illustrating reproducibility of corrected SILAC–TUBE ratios (WT/DKO, log2 transformed, observed in at least three of four experiments) for proteins identified with K-ɛ-GG peptides. Reverse=SILAC labeling swapped.

Mentions: Having determined that Ndfip1 and Ndfip2 both negatively regulate activated effector CD4+ T cells, we wanted to understand the mechanism underlying the increased proliferation and survival of Ndfip-deficient CD4+ T cells. Both Ndfip1 and Ndfip2 have been shown in vitro to bind and activate several members of the Nedd4 family of E3 ubiquitin ligases. While putative substrates of Nedd4 family E3 ligases have been described in targeted studies, in primary lymphocytes unbiased screens to identify substrates of these and other ubiquitin ligases are lacking. To address this need, we developed a three-part proteomic workflow to identify Ndfip-dependent ubiquitylation in activated CD4+ T cells (Fig. 5a).


Ndfip-mediated degradation of Jak1 tunes cytokine signalling to limit expansion of CD4+ effector T cells.

O'Leary CE, Riling CR, Spruce LA, Ding H, Kumar S, Deng G, Liu Y, Seeholzer SH, Oliver PM - Nat Commun (2016)

Identification of differential ubiquitylation by proteomics.(a) Schematic of the three proteomics methods used. (b) Area proportional Venn diagram illustrating the reproducibility of proteins identified as having SILAC ratios after TUBE enrichment in three out of four biological replicates. (c) SILAC–TUBE ratios (WT/DKO) for each protein were corrected using ‘input' ratio (DKO/WT) as calculated by label-free quantification of whole proteome DKO and WT data sets. The average corrected ratio is plotted against the average number of unique peptides observed per protein across four whole proteome LC-MS/MS experiments. Plot limited to 40 average unique peptides for clarity. Itch and Nedd4-2 (indicated) have log2 transformed corrected ratio >1 at the protein level. Itch=1.57±0.17 and Nedd4-2=3.18±0.226. (d) Overlap of proteins with corrected SILAC–TUBE ratios (observed in at least three experiments) and proteins identified with at least one K-ɛ-GG peptide (in at least one of the three K-ɛ-GG immunoprecipitation experiments). (e) Heat map illustrating reproducibility of corrected SILAC–TUBE ratios (WT/DKO, log2 transformed, observed in at least three of four experiments) for proteins identified with K-ɛ-GG peptides. Reverse=SILAC labeling swapped.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4837450&req=5

f5: Identification of differential ubiquitylation by proteomics.(a) Schematic of the three proteomics methods used. (b) Area proportional Venn diagram illustrating the reproducibility of proteins identified as having SILAC ratios after TUBE enrichment in three out of four biological replicates. (c) SILAC–TUBE ratios (WT/DKO) for each protein were corrected using ‘input' ratio (DKO/WT) as calculated by label-free quantification of whole proteome DKO and WT data sets. The average corrected ratio is plotted against the average number of unique peptides observed per protein across four whole proteome LC-MS/MS experiments. Plot limited to 40 average unique peptides for clarity. Itch and Nedd4-2 (indicated) have log2 transformed corrected ratio >1 at the protein level. Itch=1.57±0.17 and Nedd4-2=3.18±0.226. (d) Overlap of proteins with corrected SILAC–TUBE ratios (observed in at least three experiments) and proteins identified with at least one K-ɛ-GG peptide (in at least one of the three K-ɛ-GG immunoprecipitation experiments). (e) Heat map illustrating reproducibility of corrected SILAC–TUBE ratios (WT/DKO, log2 transformed, observed in at least three of four experiments) for proteins identified with K-ɛ-GG peptides. Reverse=SILAC labeling swapped.
Mentions: Having determined that Ndfip1 and Ndfip2 both negatively regulate activated effector CD4+ T cells, we wanted to understand the mechanism underlying the increased proliferation and survival of Ndfip-deficient CD4+ T cells. Both Ndfip1 and Ndfip2 have been shown in vitro to bind and activate several members of the Nedd4 family of E3 ubiquitin ligases. While putative substrates of Nedd4 family E3 ligases have been described in targeted studies, in primary lymphocytes unbiased screens to identify substrates of these and other ubiquitin ligases are lacking. To address this need, we developed a three-part proteomic workflow to identify Ndfip-dependent ubiquitylation in activated CD4+ T cells (Fig. 5a).

Bottom Line: Nedd4 family E3 ubiquitin ligases have been shown to restrict T-cell function and impact T-cell differentiation.We show here that Ndfip1 and Ndfip2, activators of Nedd4 family ligases, together limit accumulation and function of effector CD4+ T cells.In wild-type cells, Jak1 degradation lessens CD4+ cell sensitivity to cytokines during TCR stimulation, while in Ndfip-deficient cells cytokine responsiveness persists, promoting increased expansion and survival of pathogenic effector T cells.

View Article: PubMed Central - PubMed

Affiliation: Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

ABSTRACT
Nedd4 family E3 ubiquitin ligases have been shown to restrict T-cell function and impact T-cell differentiation. We show here that Ndfip1 and Ndfip2, activators of Nedd4 family ligases, together limit accumulation and function of effector CD4+ T cells. Using a three-part proteomics approach in primary T cells, we identify stabilization of Jak1 in Ndfip1/2-deficient T cells stimulated through the TCR. Jak1 degradation is aborted in activated T cells that lack Ndfips. In wild-type cells, Jak1 degradation lessens CD4+ cell sensitivity to cytokines during TCR stimulation, while in Ndfip-deficient cells cytokine responsiveness persists, promoting increased expansion and survival of pathogenic effector T cells. Thus, Ndfip1/Ndfip2 regulate the cross talk between the T-cell receptor and cytokine signalling pathways to limit inappropriate T-cell responses.

No MeSH data available.


Related in: MedlinePlus