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FBXO44-Mediated Degradation of RGS2 Protein Uniquely Depends on a Cullin 4B/DDB1 Complex.

Sjögren B, Swaney S, Neubig RR - PLoS ONE (2015)

Bottom Line: While the more typical F-box partners CUL1 and Skp1 can bind FBXO44, that E3 ligase complex does not bind RGS2 and is not involved in RGS2 degradation.These observations define an unexpected DDB1/CUL4B-containing FBXO44 E3 ligase complex.Pharmacological targeting of this mechanism provides a novel therapeutic approach to hypertension, anxiety, and other diseases associated with RGS2 dysregulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology & Toxicology, Michigan State University, 1355 Bogue Street, East Lansing, MI 48824, United States of America.

ABSTRACT
The ubiquitin-proteasome system for protein degradation plays a major role in regulating cell function and many signaling proteins are tightly controlled by this mechanism. Among these, Regulator of G Protein Signaling 2 (RGS2) is a target for rapid proteasomal degradation, however, the specific enzymes involved are not known. Using a genomic siRNA screening approach, we identified a novel E3 ligase complex containing cullin 4B (CUL4B), DNA damage binding protein 1 (DDB1) and F-box protein 44 (FBXO44) that mediates RGS2 protein degradation. While the more typical F-box partners CUL1 and Skp1 can bind FBXO44, that E3 ligase complex does not bind RGS2 and is not involved in RGS2 degradation. These observations define an unexpected DDB1/CUL4B-containing FBXO44 E3 ligase complex. Pharmacological targeting of this mechanism provides a novel therapeutic approach to hypertension, anxiety, and other diseases associated with RGS2 dysregulation.

No MeSH data available.


Related in: MedlinePlus

A. Increased RGS2 protein levels (PathHunter ProLabel assay) result from siRNA-mediated knockdown of CUL4B or FBXO44, but not of CUL4A B. Overexpression of CUL4B and FBXO44 decreases RGS2 protein levels whereas CUL4A overexpression has no effect. C-G. FBXO44 and CUL4B regulate RGS2 protein stability. HEK-293 cells were transiently transfected with RGS2-HA and treated with cycloheximide (10μg/ml) to inhibit protein synthesis. Representative Western blots (C) and time-course plots showing prolonged RGS2 protein half-life after either FBXO44 (D) or CUL4B (F) but not CUL4A (E) siRNA. Values were normalized to the level at 0 min (no cycloheximide), using actin as a loading control. G. Quantification of RGS2 protein half-life after siRNA-mediated knockdown of FBXO44, CUL4A and CUL4B. Data are presented as mean ± S.D. of 3 independent experiments run in triplicate; *P<0.05; **P<0.01; ***P<0.001 using one-way ANOVA with Bonferroni’s post hoc test for pairwise comparisons.
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pone.0123581.g002: A. Increased RGS2 protein levels (PathHunter ProLabel assay) result from siRNA-mediated knockdown of CUL4B or FBXO44, but not of CUL4A B. Overexpression of CUL4B and FBXO44 decreases RGS2 protein levels whereas CUL4A overexpression has no effect. C-G. FBXO44 and CUL4B regulate RGS2 protein stability. HEK-293 cells were transiently transfected with RGS2-HA and treated with cycloheximide (10μg/ml) to inhibit protein synthesis. Representative Western blots (C) and time-course plots showing prolonged RGS2 protein half-life after either FBXO44 (D) or CUL4B (F) but not CUL4A (E) siRNA. Values were normalized to the level at 0 min (no cycloheximide), using actin as a loading control. G. Quantification of RGS2 protein half-life after siRNA-mediated knockdown of FBXO44, CUL4A and CUL4B. Data are presented as mean ± S.D. of 3 independent experiments run in triplicate; *P<0.05; **P<0.01; ***P<0.001 using one-way ANOVA with Bonferroni’s post hoc test for pairwise comparisons.

Mentions: SiRNA-mediated knock-down of FBXO44 or CUL4B, but not CUL4A, resulted in a significant increase in RGS2 protein, in the PathHunter ProLabel assay (Fig 2A). Little is known about the function of FBXO44 and only a few specific functions have been attributed to CUL4B [45,46]. These results raise the possibility of a novel E3 ligase complex containing these two proteins in RGS2 degradation. Consistent with this, overexpression of either CUL4B or FLAG-FBXO44 significantly decreased RGS2 protein levels. In contrast, overexpression of CUL4A had no significant effect on RGS2 protein levels (Fig 2B).


FBXO44-Mediated Degradation of RGS2 Protein Uniquely Depends on a Cullin 4B/DDB1 Complex.

Sjögren B, Swaney S, Neubig RR - PLoS ONE (2015)

A. Increased RGS2 protein levels (PathHunter ProLabel assay) result from siRNA-mediated knockdown of CUL4B or FBXO44, but not of CUL4A B. Overexpression of CUL4B and FBXO44 decreases RGS2 protein levels whereas CUL4A overexpression has no effect. C-G. FBXO44 and CUL4B regulate RGS2 protein stability. HEK-293 cells were transiently transfected with RGS2-HA and treated with cycloheximide (10μg/ml) to inhibit protein synthesis. Representative Western blots (C) and time-course plots showing prolonged RGS2 protein half-life after either FBXO44 (D) or CUL4B (F) but not CUL4A (E) siRNA. Values were normalized to the level at 0 min (no cycloheximide), using actin as a loading control. G. Quantification of RGS2 protein half-life after siRNA-mediated knockdown of FBXO44, CUL4A and CUL4B. Data are presented as mean ± S.D. of 3 independent experiments run in triplicate; *P<0.05; **P<0.01; ***P<0.001 using one-way ANOVA with Bonferroni’s post hoc test for pairwise comparisons.
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pone.0123581.g002: A. Increased RGS2 protein levels (PathHunter ProLabel assay) result from siRNA-mediated knockdown of CUL4B or FBXO44, but not of CUL4A B. Overexpression of CUL4B and FBXO44 decreases RGS2 protein levels whereas CUL4A overexpression has no effect. C-G. FBXO44 and CUL4B regulate RGS2 protein stability. HEK-293 cells were transiently transfected with RGS2-HA and treated with cycloheximide (10μg/ml) to inhibit protein synthesis. Representative Western blots (C) and time-course plots showing prolonged RGS2 protein half-life after either FBXO44 (D) or CUL4B (F) but not CUL4A (E) siRNA. Values were normalized to the level at 0 min (no cycloheximide), using actin as a loading control. G. Quantification of RGS2 protein half-life after siRNA-mediated knockdown of FBXO44, CUL4A and CUL4B. Data are presented as mean ± S.D. of 3 independent experiments run in triplicate; *P<0.05; **P<0.01; ***P<0.001 using one-way ANOVA with Bonferroni’s post hoc test for pairwise comparisons.
Mentions: SiRNA-mediated knock-down of FBXO44 or CUL4B, but not CUL4A, resulted in a significant increase in RGS2 protein, in the PathHunter ProLabel assay (Fig 2A). Little is known about the function of FBXO44 and only a few specific functions have been attributed to CUL4B [45,46]. These results raise the possibility of a novel E3 ligase complex containing these two proteins in RGS2 degradation. Consistent with this, overexpression of either CUL4B or FLAG-FBXO44 significantly decreased RGS2 protein levels. In contrast, overexpression of CUL4A had no significant effect on RGS2 protein levels (Fig 2B).

Bottom Line: While the more typical F-box partners CUL1 and Skp1 can bind FBXO44, that E3 ligase complex does not bind RGS2 and is not involved in RGS2 degradation.These observations define an unexpected DDB1/CUL4B-containing FBXO44 E3 ligase complex.Pharmacological targeting of this mechanism provides a novel therapeutic approach to hypertension, anxiety, and other diseases associated with RGS2 dysregulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology & Toxicology, Michigan State University, 1355 Bogue Street, East Lansing, MI 48824, United States of America.

ABSTRACT
The ubiquitin-proteasome system for protein degradation plays a major role in regulating cell function and many signaling proteins are tightly controlled by this mechanism. Among these, Regulator of G Protein Signaling 2 (RGS2) is a target for rapid proteasomal degradation, however, the specific enzymes involved are not known. Using a genomic siRNA screening approach, we identified a novel E3 ligase complex containing cullin 4B (CUL4B), DNA damage binding protein 1 (DDB1) and F-box protein 44 (FBXO44) that mediates RGS2 protein degradation. While the more typical F-box partners CUL1 and Skp1 can bind FBXO44, that E3 ligase complex does not bind RGS2 and is not involved in RGS2 degradation. These observations define an unexpected DDB1/CUL4B-containing FBXO44 E3 ligase complex. Pharmacological targeting of this mechanism provides a novel therapeutic approach to hypertension, anxiety, and other diseases associated with RGS2 dysregulation.

No MeSH data available.


Related in: MedlinePlus