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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

Ndfip1 and Ndfip2 promote Itch and Nedd4-2 activity.(a) Immunoblot of Itch and Nedd4-2 isolated from activated WT or Ndfip1/Ndfip2 DKO CD4+ T-cell lysates by GST pulldown of Ndfip1 and Ndfip2 cytosolic domain GST fusion proteins. Coomassie stain for the GST fusion proteins revealed full-length products for each construct as well as a GST cleavage product. (b) Itch activity, in the absence or presence of Ndfip1 or Ndfip2, was analysed via TR-FRET polyubiquitylation assay. (c) E2/E3 transthiolation assay was used to test whether Ndfip2 promotes ubiquitin ‘charging' of Itch. Biotinylated ubiquitin non-covalently bound to Itch was analysed by western blot using fluorescent streptavidin (top); total protein was visualized by Coomassie stain (bottom). (d,e) As in b, human Nedd4L activity was assessed alone or in the presence of Ndfip1 or Ndfip2 by TR-FRET. Data for each panel is representative of a minimum of three independent experiments.
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f6: Ndfip1 and Ndfip2 promote Itch and Nedd4-2 activity.(a) Immunoblot of Itch and Nedd4-2 isolated from activated WT or Ndfip1/Ndfip2 DKO CD4+ T-cell lysates by GST pulldown of Ndfip1 and Ndfip2 cytosolic domain GST fusion proteins. Coomassie stain for the GST fusion proteins revealed full-length products for each construct as well as a GST cleavage product. (b) Itch activity, in the absence or presence of Ndfip1 or Ndfip2, was analysed via TR-FRET polyubiquitylation assay. (c) E2/E3 transthiolation assay was used to test whether Ndfip2 promotes ubiquitin ‘charging' of Itch. Biotinylated ubiquitin non-covalently bound to Itch was analysed by western blot using fluorescent streptavidin (top); total protein was visualized by Coomassie stain (bottom). (d,e) As in b, human Nedd4L activity was assessed alone or in the presence of Ndfip1 or Ndfip2 by TR-FRET. Data for each panel is representative of a minimum of three independent experiments.

Mentions: Itch is known to interact with, and be activated by, Ndfip1 in T cells, while Nedd4-2 has been shown to work with either Ndfip1 or Ndfip2 (refs 8, 13). In Ndfip doubly deficient T cells, decreased activity of Itch/Nedd4-2 could indicate E3 ligase dependence on both Ndfip1 and Ndfip2, or either Ndfip1 or Ndfip2 alone. We first assessed binding of Ndfips to Nedd4 family E3 ligases using the cytoplasmic domains of either Ndfip1 or Ndfip2 to isolate the endogenous E3 ligases from T-cell lysates. Both Ndfip1 and Ndfip2 were capable of isolating Itch and Nedd4-2 from WT CD4+ T cells in this pulldown assay (Fig. 6a). Analysis of Ndfip1/2-deficient cells yielded similar results (Fig. 6a).


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)

Ndfip1 and Ndfip2 promote Itch and Nedd4-2 activity.(a) Immunoblot of Itch and Nedd4-2 isolated from activated WT or Ndfip1/Ndfip2 DKO CD4+ T-cell lysates by GST pulldown of Ndfip1 and Ndfip2 cytosolic domain GST fusion proteins. Coomassie stain for the GST fusion proteins revealed full-length products for each construct as well as a GST cleavage product. (b) Itch activity, in the absence or presence of Ndfip1 or Ndfip2, was analysed via TR-FRET polyubiquitylation assay. (c) E2/E3 transthiolation assay was used to test whether Ndfip2 promotes ubiquitin ‘charging' of Itch. Biotinylated ubiquitin non-covalently bound to Itch was analysed by western blot using fluorescent streptavidin (top); total protein was visualized by Coomassie stain (bottom). (d,e) As in b, human Nedd4L activity was assessed alone or in the presence of Ndfip1 or Ndfip2 by TR-FRET. Data for each panel is representative of a minimum of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6: Ndfip1 and Ndfip2 promote Itch and Nedd4-2 activity.(a) Immunoblot of Itch and Nedd4-2 isolated from activated WT or Ndfip1/Ndfip2 DKO CD4+ T-cell lysates by GST pulldown of Ndfip1 and Ndfip2 cytosolic domain GST fusion proteins. Coomassie stain for the GST fusion proteins revealed full-length products for each construct as well as a GST cleavage product. (b) Itch activity, in the absence or presence of Ndfip1 or Ndfip2, was analysed via TR-FRET polyubiquitylation assay. (c) E2/E3 transthiolation assay was used to test whether Ndfip2 promotes ubiquitin ‘charging' of Itch. Biotinylated ubiquitin non-covalently bound to Itch was analysed by western blot using fluorescent streptavidin (top); total protein was visualized by Coomassie stain (bottom). (d,e) As in b, human Nedd4L activity was assessed alone or in the presence of Ndfip1 or Ndfip2 by TR-FRET. Data for each panel is representative of a minimum of three independent experiments.
Mentions: Itch is known to interact with, and be activated by, Ndfip1 in T cells, while Nedd4-2 has been shown to work with either Ndfip1 or Ndfip2 (refs 8, 13). In Ndfip doubly deficient T cells, decreased activity of Itch/Nedd4-2 could indicate E3 ligase dependence on both Ndfip1 and Ndfip2, or either Ndfip1 or Ndfip2 alone. We first assessed binding of Ndfips to Nedd4 family E3 ligases using the cytoplasmic domains of either Ndfip1 or Ndfip2 to isolate the endogenous E3 ligases from T-cell lysates. Both Ndfip1 and Ndfip2 were capable of isolating Itch and Nedd4-2 from WT CD4+ T cells in this pulldown assay (Fig. 6a). Analysis of Ndfip1/2-deficient cells yielded similar results (Fig. 6a).

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