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Structure and vascular function of MEKK3-cerebral cavernous malformations 2 complex.

Fisher OS, Deng H, Liu D, Zhang Y, Wei R, Deng Y, Zhang F, Louvi A, Turk BE, Boggon TJ, Su B - Nat Commun (2015)

Bottom Line: Here we report that Mekk3 plays an intrinsic role in embryonic vascular development.We find Mekk3 deficiency impairs neurovascular integrity, which is partially dependent on Rho-ROCK signalling, and that disruption of MEKK3:CCM2 interaction leads to similar neurovascular leakage.We conclude that CCM2:MEKK3-mediated regulation of Rho signalling is required for maintenance of neurovascular integrity, unravelling a mechanism by which CCM2 loss leads to disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520, USA.

ABSTRACT
Cerebral cavernous malformations 2 (CCM2) loss is associated with the familial form of CCM disease. The protein kinase MEKK3 (MAP3K3) is essential for embryonic angiogenesis in mice and interacts physically with CCM2, but how this interaction is mediated and its relevance to cerebral vasculature are unknown. Here we report that Mekk3 plays an intrinsic role in embryonic vascular development. Inducible endothelial Mekk3 knockout in neonatal mice is lethal due to multiple intracranial haemorrhages and brain blood vessels leakage. We discover direct interaction between CCM2 harmonin homology domain (HHD) and the N terminus of MEKK3, and determine a 2.35 Å cocrystal structure. We find Mekk3 deficiency impairs neurovascular integrity, which is partially dependent on Rho-ROCK signalling, and that disruption of MEKK3:CCM2 interaction leads to similar neurovascular leakage. We conclude that CCM2:MEKK3-mediated regulation of Rho signalling is required for maintenance of neurovascular integrity, unravelling a mechanism by which CCM2 loss leads to disease.

No MeSH data available.


Related in: MedlinePlus

Role of MEKK3:CCM2 interaction in signaling and localization.(a) Flag-tagged full-length CCM2 (Flag–CCM2) was cotransfected with HA–MEKK3FL, HA–MEKK3ΔNPB1 or HA–MEKK3ΔN into 293T cells as indicated. Transfected cells were subjected to immunoprecipitation (IP) and the immunoprecipitates and the whole-cell lysates were further analysed by IB with the indicated antibodies. (b) HA-tagged wild-type MEKK3 (WT) or its derived mutants A6D, L7D, A6D/L7D, I10D, L14D, I10D/L14D and A6D/I10D/L14D were cotransfected and analysed for interaction with Flag–CCM2 by co-IP. (c) HA–MEKK3 was cotransfected with Flag–CCM2, or its derived mutants L299E, M303E, L299E/M303E, A319D, L322D and A319D/L322D. The MEKK3–CCM2 interaction was then analysed by co-IP. (d) MBP–MKK3 was phosphorylated by GST–MEKK3 in the absence or presence of purified WT full-length CCM2 (CCM2FL) or its derived mutant (CCM2FL-A319D/A320D) as indicated. Loading of GST–MEKK3, HisMBP–MKK3, and CCM2 shown in the top and bottom panels, respectively. Phosphorylated HisMBP-MKK3 shown in the middle panel. Quantification was performed using a LiCor Odyssey Imager and shown below. Error bars indicate s.d. N=3. (e) 293T cells were transfected with HA–MEKK3FL or HA–MEKK3ΔNPB1. Cell extracts were prepared and analysed for p-JNK, p-ERK1/2 and p-p38 levels by IB with the indicated antibodies. Relative HA–MEKK3 expression levels shown by IB with an anti-HA antibody. (f) 293T cells were transfected with HA–MEKK3FL, HA–MEKK3ΔN, HA–MEKK3FL-A6D, HA–MEKK3FL-L7D or HA–MEKK3FL-A6D/L7D. Cell extracts were analysed for p-JNK and p-ERK1/2 levels by IB. Relative expression levels of MEKK3 and its mutants were determined by IB with an anti-HA antibody. (g) Representative confocal images showing that CCM2 colocalizes with WT MEKK3 (MEKK3FL), but not its mutants MEKK3ΔNPB1, MEKK3ΔN or MEKK3FL-A6D/L7D. Visualized with a LM8 Leica confocal microscopy. White scale bar, 10 μm; cyan scale bar, 1 μm. Power of objective lens: 63 × . (h) MEKK3N-peptide disrupts CCM2–MEKK3 interaction. Flag–CCM2 and HA–MEKK3 were cotransfected into 293T cells. After 24 h, the transfected cells were administrated with cell-permeable peptides MEKK3N-peptide or MEKK3mutant-N-peptide. Flag–CCM2 and HA–MEKK3 interaction was determined by co-IP. (i) MEKK3N-peptide increases pMLC2 S19 phosphorylation. Mouse brain endothelial cells from WT pups were isolated, cultured for 3 days and treated with either MEKK3N-peptide or MEKK3mutant-N-peptide.
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f3: Role of MEKK3:CCM2 interaction in signaling and localization.(a) Flag-tagged full-length CCM2 (Flag–CCM2) was cotransfected with HA–MEKK3FL, HA–MEKK3ΔNPB1 or HA–MEKK3ΔN into 293T cells as indicated. Transfected cells were subjected to immunoprecipitation (IP) and the immunoprecipitates and the whole-cell lysates were further analysed by IB with the indicated antibodies. (b) HA-tagged wild-type MEKK3 (WT) or its derived mutants A6D, L7D, A6D/L7D, I10D, L14D, I10D/L14D and A6D/I10D/L14D were cotransfected and analysed for interaction with Flag–CCM2 by co-IP. (c) HA–MEKK3 was cotransfected with Flag–CCM2, or its derived mutants L299E, M303E, L299E/M303E, A319D, L322D and A319D/L322D. The MEKK3–CCM2 interaction was then analysed by co-IP. (d) MBP–MKK3 was phosphorylated by GST–MEKK3 in the absence or presence of purified WT full-length CCM2 (CCM2FL) or its derived mutant (CCM2FL-A319D/A320D) as indicated. Loading of GST–MEKK3, HisMBP–MKK3, and CCM2 shown in the top and bottom panels, respectively. Phosphorylated HisMBP-MKK3 shown in the middle panel. Quantification was performed using a LiCor Odyssey Imager and shown below. Error bars indicate s.d. N=3. (e) 293T cells were transfected with HA–MEKK3FL or HA–MEKK3ΔNPB1. Cell extracts were prepared and analysed for p-JNK, p-ERK1/2 and p-p38 levels by IB with the indicated antibodies. Relative HA–MEKK3 expression levels shown by IB with an anti-HA antibody. (f) 293T cells were transfected with HA–MEKK3FL, HA–MEKK3ΔN, HA–MEKK3FL-A6D, HA–MEKK3FL-L7D or HA–MEKK3FL-A6D/L7D. Cell extracts were analysed for p-JNK and p-ERK1/2 levels by IB. Relative expression levels of MEKK3 and its mutants were determined by IB with an anti-HA antibody. (g) Representative confocal images showing that CCM2 colocalizes with WT MEKK3 (MEKK3FL), but not its mutants MEKK3ΔNPB1, MEKK3ΔN or MEKK3FL-A6D/L7D. Visualized with a LM8 Leica confocal microscopy. White scale bar, 10 μm; cyan scale bar, 1 μm. Power of objective lens: 63 × . (h) MEKK3N-peptide disrupts CCM2–MEKK3 interaction. Flag–CCM2 and HA–MEKK3 were cotransfected into 293T cells. After 24 h, the transfected cells were administrated with cell-permeable peptides MEKK3N-peptide or MEKK3mutant-N-peptide. Flag–CCM2 and HA–MEKK3 interaction was determined by co-IP. (i) MEKK3N-peptide increases pMLC2 S19 phosphorylation. Mouse brain endothelial cells from WT pups were isolated, cultured for 3 days and treated with either MEKK3N-peptide or MEKK3mutant-N-peptide.

Mentions: We found that in accordance with the in vitro experiments, introduction of the structure-designed mutations into the full-length proteins disrupted their interaction in cultured cells as measured by coimmunoprecipitation (Fig. 3a–c). As MEKK3 is a protein kinase we next wondered whether the interaction with CCM2 would impact kinase activity. We found that CCM2 had no effect on MEKK3 activity toward a purified substrate (MKK3) in vitro (Fig. 3d). Likewise, the interaction with CCM2 did not alter MEKK3 signalling to the ERK1/2, p38 or JNK MAPK cascades in cells (Fig. 3e,f) suggesting an alternate role for CCM2 interaction with MEKK3 in vascular function. Fluorescently tagged full-length MEKK3 and CCM2 colocalized in the para-plasma membrane region in HeLa cells (Fig. 3g). In contrast, coexpression of MEKK3 lacking either the NPB1 region or the N-terminal helix alone (MEKK3ΔNPB1, MEKK3ΔN) did not colocalize with CCM2. MEKK3 remained predominantly at the membrane, but CCM2 appeared to be diffused throughout the cytosol rather than close to the membrane (Fig. 3g). Likewise, coexpression of CCM2 with MEKK3 that included a double mutation (A6D/L7D), which prevents interaction with CCM2 (MEKK3FL-A6D/L7D) failed to show colocalization. We therefore conclude that the interaction between CCM2 and MEKK3 is important for correct subcellular localization of CCM2. It is also formally possible that CCM2 is required for activation of a spatially restricted pool of MEKK3.


Structure and vascular function of MEKK3-cerebral cavernous malformations 2 complex.

Fisher OS, Deng H, Liu D, Zhang Y, Wei R, Deng Y, Zhang F, Louvi A, Turk BE, Boggon TJ, Su B - Nat Commun (2015)

Role of MEKK3:CCM2 interaction in signaling and localization.(a) Flag-tagged full-length CCM2 (Flag–CCM2) was cotransfected with HA–MEKK3FL, HA–MEKK3ΔNPB1 or HA–MEKK3ΔN into 293T cells as indicated. Transfected cells were subjected to immunoprecipitation (IP) and the immunoprecipitates and the whole-cell lysates were further analysed by IB with the indicated antibodies. (b) HA-tagged wild-type MEKK3 (WT) or its derived mutants A6D, L7D, A6D/L7D, I10D, L14D, I10D/L14D and A6D/I10D/L14D were cotransfected and analysed for interaction with Flag–CCM2 by co-IP. (c) HA–MEKK3 was cotransfected with Flag–CCM2, or its derived mutants L299E, M303E, L299E/M303E, A319D, L322D and A319D/L322D. The MEKK3–CCM2 interaction was then analysed by co-IP. (d) MBP–MKK3 was phosphorylated by GST–MEKK3 in the absence or presence of purified WT full-length CCM2 (CCM2FL) or its derived mutant (CCM2FL-A319D/A320D) as indicated. Loading of GST–MEKK3, HisMBP–MKK3, and CCM2 shown in the top and bottom panels, respectively. Phosphorylated HisMBP-MKK3 shown in the middle panel. Quantification was performed using a LiCor Odyssey Imager and shown below. Error bars indicate s.d. N=3. (e) 293T cells were transfected with HA–MEKK3FL or HA–MEKK3ΔNPB1. Cell extracts were prepared and analysed for p-JNK, p-ERK1/2 and p-p38 levels by IB with the indicated antibodies. Relative HA–MEKK3 expression levels shown by IB with an anti-HA antibody. (f) 293T cells were transfected with HA–MEKK3FL, HA–MEKK3ΔN, HA–MEKK3FL-A6D, HA–MEKK3FL-L7D or HA–MEKK3FL-A6D/L7D. Cell extracts were analysed for p-JNK and p-ERK1/2 levels by IB. Relative expression levels of MEKK3 and its mutants were determined by IB with an anti-HA antibody. (g) Representative confocal images showing that CCM2 colocalizes with WT MEKK3 (MEKK3FL), but not its mutants MEKK3ΔNPB1, MEKK3ΔN or MEKK3FL-A6D/L7D. Visualized with a LM8 Leica confocal microscopy. White scale bar, 10 μm; cyan scale bar, 1 μm. Power of objective lens: 63 × . (h) MEKK3N-peptide disrupts CCM2–MEKK3 interaction. Flag–CCM2 and HA–MEKK3 were cotransfected into 293T cells. After 24 h, the transfected cells were administrated with cell-permeable peptides MEKK3N-peptide or MEKK3mutant-N-peptide. Flag–CCM2 and HA–MEKK3 interaction was determined by co-IP. (i) MEKK3N-peptide increases pMLC2 S19 phosphorylation. Mouse brain endothelial cells from WT pups were isolated, cultured for 3 days and treated with either MEKK3N-peptide or MEKK3mutant-N-peptide.
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f3: Role of MEKK3:CCM2 interaction in signaling and localization.(a) Flag-tagged full-length CCM2 (Flag–CCM2) was cotransfected with HA–MEKK3FL, HA–MEKK3ΔNPB1 or HA–MEKK3ΔN into 293T cells as indicated. Transfected cells were subjected to immunoprecipitation (IP) and the immunoprecipitates and the whole-cell lysates were further analysed by IB with the indicated antibodies. (b) HA-tagged wild-type MEKK3 (WT) or its derived mutants A6D, L7D, A6D/L7D, I10D, L14D, I10D/L14D and A6D/I10D/L14D were cotransfected and analysed for interaction with Flag–CCM2 by co-IP. (c) HA–MEKK3 was cotransfected with Flag–CCM2, or its derived mutants L299E, M303E, L299E/M303E, A319D, L322D and A319D/L322D. The MEKK3–CCM2 interaction was then analysed by co-IP. (d) MBP–MKK3 was phosphorylated by GST–MEKK3 in the absence or presence of purified WT full-length CCM2 (CCM2FL) or its derived mutant (CCM2FL-A319D/A320D) as indicated. Loading of GST–MEKK3, HisMBP–MKK3, and CCM2 shown in the top and bottom panels, respectively. Phosphorylated HisMBP-MKK3 shown in the middle panel. Quantification was performed using a LiCor Odyssey Imager and shown below. Error bars indicate s.d. N=3. (e) 293T cells were transfected with HA–MEKK3FL or HA–MEKK3ΔNPB1. Cell extracts were prepared and analysed for p-JNK, p-ERK1/2 and p-p38 levels by IB with the indicated antibodies. Relative HA–MEKK3 expression levels shown by IB with an anti-HA antibody. (f) 293T cells were transfected with HA–MEKK3FL, HA–MEKK3ΔN, HA–MEKK3FL-A6D, HA–MEKK3FL-L7D or HA–MEKK3FL-A6D/L7D. Cell extracts were analysed for p-JNK and p-ERK1/2 levels by IB. Relative expression levels of MEKK3 and its mutants were determined by IB with an anti-HA antibody. (g) Representative confocal images showing that CCM2 colocalizes with WT MEKK3 (MEKK3FL), but not its mutants MEKK3ΔNPB1, MEKK3ΔN or MEKK3FL-A6D/L7D. Visualized with a LM8 Leica confocal microscopy. White scale bar, 10 μm; cyan scale bar, 1 μm. Power of objective lens: 63 × . (h) MEKK3N-peptide disrupts CCM2–MEKK3 interaction. Flag–CCM2 and HA–MEKK3 were cotransfected into 293T cells. After 24 h, the transfected cells were administrated with cell-permeable peptides MEKK3N-peptide or MEKK3mutant-N-peptide. Flag–CCM2 and HA–MEKK3 interaction was determined by co-IP. (i) MEKK3N-peptide increases pMLC2 S19 phosphorylation. Mouse brain endothelial cells from WT pups were isolated, cultured for 3 days and treated with either MEKK3N-peptide or MEKK3mutant-N-peptide.
Mentions: We found that in accordance with the in vitro experiments, introduction of the structure-designed mutations into the full-length proteins disrupted their interaction in cultured cells as measured by coimmunoprecipitation (Fig. 3a–c). As MEKK3 is a protein kinase we next wondered whether the interaction with CCM2 would impact kinase activity. We found that CCM2 had no effect on MEKK3 activity toward a purified substrate (MKK3) in vitro (Fig. 3d). Likewise, the interaction with CCM2 did not alter MEKK3 signalling to the ERK1/2, p38 or JNK MAPK cascades in cells (Fig. 3e,f) suggesting an alternate role for CCM2 interaction with MEKK3 in vascular function. Fluorescently tagged full-length MEKK3 and CCM2 colocalized in the para-plasma membrane region in HeLa cells (Fig. 3g). In contrast, coexpression of MEKK3 lacking either the NPB1 region or the N-terminal helix alone (MEKK3ΔNPB1, MEKK3ΔN) did not colocalize with CCM2. MEKK3 remained predominantly at the membrane, but CCM2 appeared to be diffused throughout the cytosol rather than close to the membrane (Fig. 3g). Likewise, coexpression of CCM2 with MEKK3 that included a double mutation (A6D/L7D), which prevents interaction with CCM2 (MEKK3FL-A6D/L7D) failed to show colocalization. We therefore conclude that the interaction between CCM2 and MEKK3 is important for correct subcellular localization of CCM2. It is also formally possible that CCM2 is required for activation of a spatially restricted pool of MEKK3.

Bottom Line: Here we report that Mekk3 plays an intrinsic role in embryonic vascular development.We find Mekk3 deficiency impairs neurovascular integrity, which is partially dependent on Rho-ROCK signalling, and that disruption of MEKK3:CCM2 interaction leads to similar neurovascular leakage.We conclude that CCM2:MEKK3-mediated regulation of Rho signalling is required for maintenance of neurovascular integrity, unravelling a mechanism by which CCM2 loss leads to disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520, USA.

ABSTRACT
Cerebral cavernous malformations 2 (CCM2) loss is associated with the familial form of CCM disease. The protein kinase MEKK3 (MAP3K3) is essential for embryonic angiogenesis in mice and interacts physically with CCM2, but how this interaction is mediated and its relevance to cerebral vasculature are unknown. Here we report that Mekk3 plays an intrinsic role in embryonic vascular development. Inducible endothelial Mekk3 knockout in neonatal mice is lethal due to multiple intracranial haemorrhages and brain blood vessels leakage. We discover direct interaction between CCM2 harmonin homology domain (HHD) and the N terminus of MEKK3, and determine a 2.35 Å cocrystal structure. We find Mekk3 deficiency impairs neurovascular integrity, which is partially dependent on Rho-ROCK signalling, and that disruption of MEKK3:CCM2 interaction leads to similar neurovascular leakage. We conclude that CCM2:MEKK3-mediated regulation of Rho signalling is required for maintenance of neurovascular integrity, unravelling a mechanism by which CCM2 loss leads to disease.

No MeSH data available.


Related in: MedlinePlus