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Molecular basis and regulation of OTULIN-LUBAC interaction.

Elliott PR, Nielsen SV, Marco-Casanova P, Fiil BK, Keusekotten K, Mailand N, Freund SM, Gyrd-Hansen M, Komander D - Mol. Cell (2014)

Bottom Line: Moreover, LUBAC-OTULIN complex formation is regulated by OTULIN phosphorylation in the PIM.Phosphorylation of OTULIN prevents HOIP binding, whereas unphosphorylated OTULIN is part of the endogenous LUBAC complex.Our work exemplifies how coordination of ubiquitin assembly and disassembly activities in protein complexes regulates individual Ub linkage types.

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Affiliation: Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH, UK.

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Regulation of OTULIN-LUBAC Complex Formation by Phosphorylation(A) Gel filtration analysis of purified bacterial p97 hexamers and full-length OTULIN visualized by Coomassie staining and HEK293ET cell lysates probed with indicated antibodies.(B) Schematic of the OTULIN PIM indicating phosphorylation at Tyr56 as identified in 22 independent mass spectrometry experiments in http://phosphosite.org/proteinAction.do?id=2470471.(C) Fluorescence polarization assays of HOIP PUB domains with wild-type and Tyr56-phosphorylated FITC-Ahx-labeled OTULIN (49–67). Experiments were performed in triplicate, and errors represent SD from the mean.(D) HEK293ET lysates were prepared in absence of phosphatase inhibitors and probed for the same components as in (A). Only the OTULIN blot is shown.(E) Schematic model of the LUBAC-OTULIN complex indicating its regulation by protein phosphorylation.
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fig7: Regulation of OTULIN-LUBAC Complex Formation by Phosphorylation(A) Gel filtration analysis of purified bacterial p97 hexamers and full-length OTULIN visualized by Coomassie staining and HEK293ET cell lysates probed with indicated antibodies.(B) Schematic of the OTULIN PIM indicating phosphorylation at Tyr56 as identified in 22 independent mass spectrometry experiments in http://phosphosite.org/proteinAction.do?id=2470471.(C) Fluorescence polarization assays of HOIP PUB domains with wild-type and Tyr56-phosphorylated FITC-Ahx-labeled OTULIN (49–67). Experiments were performed in triplicate, and errors represent SD from the mean.(D) HEK293ET lysates were prepared in absence of phosphatase inhibitors and probed for the same components as in (A). Only the OTULIN blot is shown.(E) Schematic model of the LUBAC-OTULIN complex indicating its regulation by protein phosphorylation.

Mentions: Next, we wondered whether OTULIN was indeed part of LUBAC at the endogenous level. For this, we purified the endogenous LUBAC complex from human embryonic kidney 293ET (HEK293ET) cell lysates by gel filtration (Figure 7A). As reported previously (Kirisako et al., 2006), HOIP and HOIL-1 formed an approximately 600 kDa complex, and SHARPIN eluted quantitatively in this size range. The LUBAC complex is of similar size to recombinant p97 hexamers or to cellular p97 complexes. Bacterially purified OTULIN is monomeric and elutes according to its mass at ∼40 kDa. To our surprise, the majority of endogenous OTULIN in HEK293ET cells (>95%) eluted in a size range of ∼100–150 kDa, and only a small fraction seemed to coelute with the endogenous LUBAC complex (Figure 7A). Similar data were obtained in U2OS and RPE1 cells (Figure S8). This was in contrast to our findings that the HOIP PUB-OTULIN interaction was stable on gel filtration (Figure 1D). Although there were many potential reasons for why the interaction was unstable in cells, one intriguing possibility was that binding of OTULIN to HOIP was dynamically regulated. Indeed, OTULIN is phosphorylated in cells, and the prime site for phosphorylation is the PIM residue Tyr56 (http://phosphosite.org/proteinAction.do?id=2470471; Figure 7B). A Tyr56-phosphorylated PIM peptide was unable to bind HOIP, which is consistent with our structural data (Figure 7C). Importantly, the distribution of OTULIN changed significantly when HEK293ET lysates were prepared in the absence of phosphatase inhibitors. Although OTULIN eluted in a single peak when phosphatases are inhibited (Figure 7A), phosphatase activity resulted in two peaks at 600 and 40 kDa. This suggested that OTULIN is indeed phosphorylated in HEK293ET cell lysates and that dephosphorylation leads to quantitative association with LUBAC. OTULIN may be more abundant than LUBAC and HOIP, given that a significant fraction of dephosphorylated OTULIN is not bound to HOIP and elutes as a monomer. Altogether, this suggests that the abundance of OTULIN on LUBAC is regulated by phosphorylation of the OTULIN PIM.


Molecular basis and regulation of OTULIN-LUBAC interaction.

Elliott PR, Nielsen SV, Marco-Casanova P, Fiil BK, Keusekotten K, Mailand N, Freund SM, Gyrd-Hansen M, Komander D - Mol. Cell (2014)

Regulation of OTULIN-LUBAC Complex Formation by Phosphorylation(A) Gel filtration analysis of purified bacterial p97 hexamers and full-length OTULIN visualized by Coomassie staining and HEK293ET cell lysates probed with indicated antibodies.(B) Schematic of the OTULIN PIM indicating phosphorylation at Tyr56 as identified in 22 independent mass spectrometry experiments in http://phosphosite.org/proteinAction.do?id=2470471.(C) Fluorescence polarization assays of HOIP PUB domains with wild-type and Tyr56-phosphorylated FITC-Ahx-labeled OTULIN (49–67). Experiments were performed in triplicate, and errors represent SD from the mean.(D) HEK293ET lysates were prepared in absence of phosphatase inhibitors and probed for the same components as in (A). Only the OTULIN blot is shown.(E) Schematic model of the LUBAC-OTULIN complex indicating its regulation by protein phosphorylation.
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fig7: Regulation of OTULIN-LUBAC Complex Formation by Phosphorylation(A) Gel filtration analysis of purified bacterial p97 hexamers and full-length OTULIN visualized by Coomassie staining and HEK293ET cell lysates probed with indicated antibodies.(B) Schematic of the OTULIN PIM indicating phosphorylation at Tyr56 as identified in 22 independent mass spectrometry experiments in http://phosphosite.org/proteinAction.do?id=2470471.(C) Fluorescence polarization assays of HOIP PUB domains with wild-type and Tyr56-phosphorylated FITC-Ahx-labeled OTULIN (49–67). Experiments were performed in triplicate, and errors represent SD from the mean.(D) HEK293ET lysates were prepared in absence of phosphatase inhibitors and probed for the same components as in (A). Only the OTULIN blot is shown.(E) Schematic model of the LUBAC-OTULIN complex indicating its regulation by protein phosphorylation.
Mentions: Next, we wondered whether OTULIN was indeed part of LUBAC at the endogenous level. For this, we purified the endogenous LUBAC complex from human embryonic kidney 293ET (HEK293ET) cell lysates by gel filtration (Figure 7A). As reported previously (Kirisako et al., 2006), HOIP and HOIL-1 formed an approximately 600 kDa complex, and SHARPIN eluted quantitatively in this size range. The LUBAC complex is of similar size to recombinant p97 hexamers or to cellular p97 complexes. Bacterially purified OTULIN is monomeric and elutes according to its mass at ∼40 kDa. To our surprise, the majority of endogenous OTULIN in HEK293ET cells (>95%) eluted in a size range of ∼100–150 kDa, and only a small fraction seemed to coelute with the endogenous LUBAC complex (Figure 7A). Similar data were obtained in U2OS and RPE1 cells (Figure S8). This was in contrast to our findings that the HOIP PUB-OTULIN interaction was stable on gel filtration (Figure 1D). Although there were many potential reasons for why the interaction was unstable in cells, one intriguing possibility was that binding of OTULIN to HOIP was dynamically regulated. Indeed, OTULIN is phosphorylated in cells, and the prime site for phosphorylation is the PIM residue Tyr56 (http://phosphosite.org/proteinAction.do?id=2470471; Figure 7B). A Tyr56-phosphorylated PIM peptide was unable to bind HOIP, which is consistent with our structural data (Figure 7C). Importantly, the distribution of OTULIN changed significantly when HEK293ET lysates were prepared in the absence of phosphatase inhibitors. Although OTULIN eluted in a single peak when phosphatases are inhibited (Figure 7A), phosphatase activity resulted in two peaks at 600 and 40 kDa. This suggested that OTULIN is indeed phosphorylated in HEK293ET cell lysates and that dephosphorylation leads to quantitative association with LUBAC. OTULIN may be more abundant than LUBAC and HOIP, given that a significant fraction of dephosphorylated OTULIN is not bound to HOIP and elutes as a monomer. Altogether, this suggests that the abundance of OTULIN on LUBAC is regulated by phosphorylation of the OTULIN PIM.

Bottom Line: Moreover, LUBAC-OTULIN complex formation is regulated by OTULIN phosphorylation in the PIM.Phosphorylation of OTULIN prevents HOIP binding, whereas unphosphorylated OTULIN is part of the endogenous LUBAC complex.Our work exemplifies how coordination of ubiquitin assembly and disassembly activities in protein complexes regulates individual Ub linkage types.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH, UK.

Show MeSH
Related in: MedlinePlus