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A Toll-like receptor-responsive kinase, protein kinase R, is inactivated in endotoxin tolerance through differential K63/K48 ubiquitination.

Perkins DJ, Qureshi N, Vogel SN - MBio (2010)

Bottom Line: As a safeguard, the innate immune system has evolved to counter excessive inflammation through the induction of "tolerance." In endotoxin tolerance, TLR signaling is inhibited and/or attenuated by multiple mechanisms that mitigate the ability of lipopolysaccharide (LPS) to activate critical kinases through TLR4.In tolerance, the kinetics of this differential Ub is altered, resulting in a predominance of K48-linked chains, concomitant with a loss of PKR activation.These findings provide a novel mechanism by which a TLR-responsive kinase may be rendered inactive in tolerance.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Maryland, Baltimore (UMB), School of Medicine, Baltimore, Maryland, USA.

ABSTRACT
Overwhelming inflammation triggered by systemic infection in bacterial sepsis contributes to the pathology of this condition. Toll-like receptors (TLRs) are important in early septic inflammation. As a safeguard, the innate immune system has evolved to counter excessive inflammation through the induction of "tolerance." In endotoxin tolerance, TLR signaling is inhibited and/or attenuated by multiple mechanisms that mitigate the ability of lipopolysaccharide (LPS) to activate critical kinases through TLR4. Here, we describe a novel mechanism. Protein kinase R (PKR), a kinase normally activated by a subset of TLRs, is rendered unresponsive to LPS in endotoxin-tolerized cells. In its naive state, PKR is subject to K63-linked ubiquitination (Ub), followed by K48-linked Ub, in response to LPS. In tolerance, the kinetics of this differential Ub is altered, resulting in a predominance of K48-linked chains, concomitant with a loss of PKR activation. These findings provide a novel mechanism by which a TLR-responsive kinase may be rendered inactive in tolerance.

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SOCS-1 physically interacts with and negatively regulates PKR. (A) Primary peritoneal macrophages were stimulated for 18 h with medium (M), LPS (10 ng/ml), P3C (100 ng/ml), or p(I ⋅ C) (10 µg/ml). Cells were harvested, and the levels of SOCS-1 protein were examined by Western analysis. (B and C) HEK293T cells were transfected with 200 ng V5-tagged WT PKR and either empty vector or an increasing amount of cDNA expressing FLAG-tagged SOCS-1 (B) or SOCS-2 (C). Twenty-four hours following transfection, whole-cell lysates were subjected to Western analysis with antibodies against the indicated species. (D) HEK293T cells transfected with V5-PKR alone or in conjunction with FLAG-tagged SOCS-1. Cell lysates were immunoprecipitated with anti-FLAG antibody and separated by SDS-PAGE, followed by Western blotting with the indicated antibodies. These data are representative of 3 independent experiments. (E and F) WT and SOCS-1−/− MEFs were cultured overnight in medium alone (E) or in medium supplemented with LPS (100 ng/ml) (F). Following 18 h of treatment, cells were washed and restimulated with LPS (250 ng/ml) for the indicated times. Whole-cell lysates were resolved by SDS-PAGE and probed with antibodies against phosphorylated or total PKR. These data are representative of 3 independent experiments.
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f5: SOCS-1 physically interacts with and negatively regulates PKR. (A) Primary peritoneal macrophages were stimulated for 18 h with medium (M), LPS (10 ng/ml), P3C (100 ng/ml), or p(I ⋅ C) (10 µg/ml). Cells were harvested, and the levels of SOCS-1 protein were examined by Western analysis. (B and C) HEK293T cells were transfected with 200 ng V5-tagged WT PKR and either empty vector or an increasing amount of cDNA expressing FLAG-tagged SOCS-1 (B) or SOCS-2 (C). Twenty-four hours following transfection, whole-cell lysates were subjected to Western analysis with antibodies against the indicated species. (D) HEK293T cells transfected with V5-PKR alone or in conjunction with FLAG-tagged SOCS-1. Cell lysates were immunoprecipitated with anti-FLAG antibody and separated by SDS-PAGE, followed by Western blotting with the indicated antibodies. These data are representative of 3 independent experiments. (E and F) WT and SOCS-1−/− MEFs were cultured overnight in medium alone (E) or in medium supplemented with LPS (100 ng/ml) (F). Following 18 h of treatment, cells were washed and restimulated with LPS (250 ng/ml) for the indicated times. Whole-cell lysates were resolved by SDS-PAGE and probed with antibodies against phosphorylated or total PKR. These data are representative of 3 independent experiments.

Mentions: Because our data strongly suggest that ubiquitin-mediated proteolysis plays a role in the inactivation of PKR in LPS-tolerized cells, we next sought to ascertain the identity of the K48 ligase that is responsible. Among the LPS-inducible K48 ligases, the SOCS family of ubiquitin ligases has been linked extensively to the negative regulation of cytokine and inflammatory processes (37). In particular, SOCS-1 is upregulated by LPS and has been reported to catalyze the destruction of proximal elements of TLR signal complexes (31). In addition, SOCS-1-deficient mice have been reported to be impaired in their capacity to induce endotoxin tolerance (20, 38), although others have reached different conclusions in regard to a role for SOCS-1 as a mediator of tolerance (39). We examined the steady-state levels of SOCS-1 by Western analysis of both naive macrophages and macrophages that had been rendered tolerant by preincubation with medium only or with LPS, P3C, or p(I ⋅ C). In the naive state, SOCS-1 protein levels were comparatively low (Fig. 5A). However, in tolerant macrophages, SOCS-1 levels were significantly elevated regardless of stimulus used (Fig. 5A). We therefore investigated the possibility that PKR and SOCS-1 interact functionally in vitro, resulting in K48 Ub of PKR. To test this hypothesis, PKR was overexpressed in HEK293T cells, along with increasing concentrations of a SOCS-1 expression vector. We observed a striking dose-dependent reduction in basal PKR protein expression levels in response to an increase in SOCS-1 following 24 h of incubation (Fig. 5B, top). This was not the result of widespread nonspecific degradation of TLR4-responsive kinases because endogenous levels of the TLR-responsive MAPK p38 were not reduced, even at the highest levels of transfected SOCS-1 (Fig. 5A, bottom). Since overexpression of K48 ligases can, in some instances, lead to a loss of target specificity, we repeated this experiment using a closely related family member, SOCS-2, in lieu of SOCS-1. Importantly, SOCS-2 overexpression had a negligible effect on the basal level of PKR, even at the highest dose (Fig. 5C). Since the effects of SOCS-1 on PKR protein levels may be the result of regulation of an intermediate element, we evaluated the potential for SOCS-1 and PKR to interact physically in our overexpression system. V5-tagged WT PKR was transfected into HEK293T cells without or with concomitant transfection of FLAG-tagged SOCS-1, and immunoprecipitations were carried out with anti-FLAG monoclonal antibody. PKR was immunoprecipitated only in the presence of cotransfected SOCS-1 (Fig. 5D, top). Western analysis of whole-cell lysates revealed the expected reduction in PKR expression levels when coexpressed with SOCS-1 (Fig. 5D, bottom). The capacity of SOCS-1 to interact physically with and catalyze degradation of PKR in HEK293T cells supports the possibility that SOCS-1 regulates PKR protein levels during the TLR4-mediated response to LPS. To assay for the potential in vivo significance of SOCS-1 in regulating PKR activity, we obtained SOCS-1 knockout mouse embryonic fibroblasts (MEFs) and ascertained LPS-responsive PKR activation in naive and LPS-tolerized cells. Stimulation of wild-type, naive MEFs produced a modest and transient activation of PKR (Fig. 5E). Remarkably, PKR activity was dramatically enhanced and maximal in unstimulated SOCS-1−/− MEFs and could not be further enhanced by LPS treatment at any dose used, demonstrating a role for SOCS-1 in regulating PKR activity (Fig. 5E). In LPS-tolerized MEFs, however, PKR activity could not be stimulated by LPS in either the SOCS-1+/+ or SOCS-1−/− genotype, suggesting that a combination of mechanisms play a role in enforcing PKR tolerance (Fig. 5F).


A Toll-like receptor-responsive kinase, protein kinase R, is inactivated in endotoxin tolerance through differential K63/K48 ubiquitination.

Perkins DJ, Qureshi N, Vogel SN - MBio (2010)

SOCS-1 physically interacts with and negatively regulates PKR. (A) Primary peritoneal macrophages were stimulated for 18 h with medium (M), LPS (10 ng/ml), P3C (100 ng/ml), or p(I ⋅ C) (10 µg/ml). Cells were harvested, and the levels of SOCS-1 protein were examined by Western analysis. (B and C) HEK293T cells were transfected with 200 ng V5-tagged WT PKR and either empty vector or an increasing amount of cDNA expressing FLAG-tagged SOCS-1 (B) or SOCS-2 (C). Twenty-four hours following transfection, whole-cell lysates were subjected to Western analysis with antibodies against the indicated species. (D) HEK293T cells transfected with V5-PKR alone or in conjunction with FLAG-tagged SOCS-1. Cell lysates were immunoprecipitated with anti-FLAG antibody and separated by SDS-PAGE, followed by Western blotting with the indicated antibodies. These data are representative of 3 independent experiments. (E and F) WT and SOCS-1−/− MEFs were cultured overnight in medium alone (E) or in medium supplemented with LPS (100 ng/ml) (F). Following 18 h of treatment, cells were washed and restimulated with LPS (250 ng/ml) for the indicated times. Whole-cell lysates were resolved by SDS-PAGE and probed with antibodies against phosphorylated or total PKR. These data are representative of 3 independent experiments.
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Related In: Results  -  Collection

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f5: SOCS-1 physically interacts with and negatively regulates PKR. (A) Primary peritoneal macrophages were stimulated for 18 h with medium (M), LPS (10 ng/ml), P3C (100 ng/ml), or p(I ⋅ C) (10 µg/ml). Cells were harvested, and the levels of SOCS-1 protein were examined by Western analysis. (B and C) HEK293T cells were transfected with 200 ng V5-tagged WT PKR and either empty vector or an increasing amount of cDNA expressing FLAG-tagged SOCS-1 (B) or SOCS-2 (C). Twenty-four hours following transfection, whole-cell lysates were subjected to Western analysis with antibodies against the indicated species. (D) HEK293T cells transfected with V5-PKR alone or in conjunction with FLAG-tagged SOCS-1. Cell lysates were immunoprecipitated with anti-FLAG antibody and separated by SDS-PAGE, followed by Western blotting with the indicated antibodies. These data are representative of 3 independent experiments. (E and F) WT and SOCS-1−/− MEFs were cultured overnight in medium alone (E) or in medium supplemented with LPS (100 ng/ml) (F). Following 18 h of treatment, cells were washed and restimulated with LPS (250 ng/ml) for the indicated times. Whole-cell lysates were resolved by SDS-PAGE and probed with antibodies against phosphorylated or total PKR. These data are representative of 3 independent experiments.
Mentions: Because our data strongly suggest that ubiquitin-mediated proteolysis plays a role in the inactivation of PKR in LPS-tolerized cells, we next sought to ascertain the identity of the K48 ligase that is responsible. Among the LPS-inducible K48 ligases, the SOCS family of ubiquitin ligases has been linked extensively to the negative regulation of cytokine and inflammatory processes (37). In particular, SOCS-1 is upregulated by LPS and has been reported to catalyze the destruction of proximal elements of TLR signal complexes (31). In addition, SOCS-1-deficient mice have been reported to be impaired in their capacity to induce endotoxin tolerance (20, 38), although others have reached different conclusions in regard to a role for SOCS-1 as a mediator of tolerance (39). We examined the steady-state levels of SOCS-1 by Western analysis of both naive macrophages and macrophages that had been rendered tolerant by preincubation with medium only or with LPS, P3C, or p(I ⋅ C). In the naive state, SOCS-1 protein levels were comparatively low (Fig. 5A). However, in tolerant macrophages, SOCS-1 levels were significantly elevated regardless of stimulus used (Fig. 5A). We therefore investigated the possibility that PKR and SOCS-1 interact functionally in vitro, resulting in K48 Ub of PKR. To test this hypothesis, PKR was overexpressed in HEK293T cells, along with increasing concentrations of a SOCS-1 expression vector. We observed a striking dose-dependent reduction in basal PKR protein expression levels in response to an increase in SOCS-1 following 24 h of incubation (Fig. 5B, top). This was not the result of widespread nonspecific degradation of TLR4-responsive kinases because endogenous levels of the TLR-responsive MAPK p38 were not reduced, even at the highest levels of transfected SOCS-1 (Fig. 5A, bottom). Since overexpression of K48 ligases can, in some instances, lead to a loss of target specificity, we repeated this experiment using a closely related family member, SOCS-2, in lieu of SOCS-1. Importantly, SOCS-2 overexpression had a negligible effect on the basal level of PKR, even at the highest dose (Fig. 5C). Since the effects of SOCS-1 on PKR protein levels may be the result of regulation of an intermediate element, we evaluated the potential for SOCS-1 and PKR to interact physically in our overexpression system. V5-tagged WT PKR was transfected into HEK293T cells without or with concomitant transfection of FLAG-tagged SOCS-1, and immunoprecipitations were carried out with anti-FLAG monoclonal antibody. PKR was immunoprecipitated only in the presence of cotransfected SOCS-1 (Fig. 5D, top). Western analysis of whole-cell lysates revealed the expected reduction in PKR expression levels when coexpressed with SOCS-1 (Fig. 5D, bottom). The capacity of SOCS-1 to interact physically with and catalyze degradation of PKR in HEK293T cells supports the possibility that SOCS-1 regulates PKR protein levels during the TLR4-mediated response to LPS. To assay for the potential in vivo significance of SOCS-1 in regulating PKR activity, we obtained SOCS-1 knockout mouse embryonic fibroblasts (MEFs) and ascertained LPS-responsive PKR activation in naive and LPS-tolerized cells. Stimulation of wild-type, naive MEFs produced a modest and transient activation of PKR (Fig. 5E). Remarkably, PKR activity was dramatically enhanced and maximal in unstimulated SOCS-1−/− MEFs and could not be further enhanced by LPS treatment at any dose used, demonstrating a role for SOCS-1 in regulating PKR activity (Fig. 5E). In LPS-tolerized MEFs, however, PKR activity could not be stimulated by LPS in either the SOCS-1+/+ or SOCS-1−/− genotype, suggesting that a combination of mechanisms play a role in enforcing PKR tolerance (Fig. 5F).

Bottom Line: As a safeguard, the innate immune system has evolved to counter excessive inflammation through the induction of "tolerance." In endotoxin tolerance, TLR signaling is inhibited and/or attenuated by multiple mechanisms that mitigate the ability of lipopolysaccharide (LPS) to activate critical kinases through TLR4.In tolerance, the kinetics of this differential Ub is altered, resulting in a predominance of K48-linked chains, concomitant with a loss of PKR activation.These findings provide a novel mechanism by which a TLR-responsive kinase may be rendered inactive in tolerance.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Maryland, Baltimore (UMB), School of Medicine, Baltimore, Maryland, USA.

ABSTRACT
Overwhelming inflammation triggered by systemic infection in bacterial sepsis contributes to the pathology of this condition. Toll-like receptors (TLRs) are important in early septic inflammation. As a safeguard, the innate immune system has evolved to counter excessive inflammation through the induction of "tolerance." In endotoxin tolerance, TLR signaling is inhibited and/or attenuated by multiple mechanisms that mitigate the ability of lipopolysaccharide (LPS) to activate critical kinases through TLR4. Here, we describe a novel mechanism. Protein kinase R (PKR), a kinase normally activated by a subset of TLRs, is rendered unresponsive to LPS in endotoxin-tolerized cells. In its naive state, PKR is subject to K63-linked ubiquitination (Ub), followed by K48-linked Ub, in response to LPS. In tolerance, the kinetics of this differential Ub is altered, resulting in a predominance of K48-linked chains, concomitant with a loss of PKR activation. These findings provide a novel mechanism by which a TLR-responsive kinase may be rendered inactive in tolerance.

Show MeSH
Related in: MedlinePlus