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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

MEKK3 uses its NPB1 region to interact with the HHD domain of CCM2.(a) Immunoprecipitation (IP) of Flag–CCM2 after cotransfection with HA–MEKK3FL or HA–MEKK3ΔNPB1 into 293T cells. Whole-cell lysates and immunoprecipitates were analysed by immunoblotting (IB) with the indicated antibodies. (b) Domain schematic of CCM2 (top) and MEKK3 (bottom). Black lines indicate constructs used in this study. Domains are indicated as PTB (Phosphotyrosine Binding), HHD (Harmonin homology domain), PB1 (Phox/Bem1p) and kinase. N and C termini are indicated. (c) Pull-down assay. GST–MEKK3NPB1 was immobilized on glutathione Sepharose beads and tested for ability to pull-down purified regions of CCM2. Coomassie stained SDS–PAGE analysis of washed beads. (d) Pull-down assay. Immobilized GST–MEKK3NPB1, GST–MEKK3N or GST–MEKK3PB1 were incubated with purified CCM2HHD. Coomassie-stained SDS–PAGE of washed beads. (e) Cocrystal structure of MEKK3NPB1 in complex with CCM2HHD. CCM2HHD is shown in green and MEKK3NPB1 in purple. Secondary structure elements are labelled. Disordered residues indicated by a dotted line. N and C termini are indicated. (f) Close-up views of the main interaction sites between MEKK3NPB1 and CCM2HHD. Left, CCM2 helix H1 with MEKK3 helix αN. Centre, CCM2 helix H2 with MEKK3 helix αN and PB1 domain. Right, CCM2 helices H2 and H3 with MEKK3 PB1 domain. Hydrogen-bonds shown as dotted lines. Residues mutated in this study are boxed in red. Structural figures generated using CCP4mg36. (g) Map of MEKK3NPB1:CCM2HHD interactions. MEKK3 residues are shown in purple and CCM2 residues in green. Residues mutated in this study are shown in red. Dotted lines indicate hydrophobic interactions and solid lines ionic interactions. Interactions defined by PDBSum37. (h) Pull-down assay. Crystallographically determined mutations to GST–MEKK3NPB1 interrupts pull-down of wild-type CCM2HHD. MEKK3 mutants, A6D/L7D, D13R, I10D/L14D and L119E/L121E are indicated. Coomassie stained SDS–PAGE of washed beads. (i) Pull-down assay. Introduction of crystallographically determined mutations to CCM2HHD interrupts pull-down with wild-type GST–MEKK3NPB1. CCM2 mutants A319D/L322D and A319D/A320D are indicated. Coomassie stained SDS–PAGE of washed beads. (j) Isothermal titration calorimetry. CCM2HHD was titrated into MEKK3NPB1. Crystallographically defined mutants tested for MEKK3 were D13R and A6D/L7D and for CCM2 was A319D/A320D38.
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f2: MEKK3 uses its NPB1 region to interact with the HHD domain of CCM2.(a) Immunoprecipitation (IP) of Flag–CCM2 after cotransfection with HA–MEKK3FL or HA–MEKK3ΔNPB1 into 293T cells. Whole-cell lysates and immunoprecipitates were analysed by immunoblotting (IB) with the indicated antibodies. (b) Domain schematic of CCM2 (top) and MEKK3 (bottom). Black lines indicate constructs used in this study. Domains are indicated as PTB (Phosphotyrosine Binding), HHD (Harmonin homology domain), PB1 (Phox/Bem1p) and kinase. N and C termini are indicated. (c) Pull-down assay. GST–MEKK3NPB1 was immobilized on glutathione Sepharose beads and tested for ability to pull-down purified regions of CCM2. Coomassie stained SDS–PAGE analysis of washed beads. (d) Pull-down assay. Immobilized GST–MEKK3NPB1, GST–MEKK3N or GST–MEKK3PB1 were incubated with purified CCM2HHD. Coomassie-stained SDS–PAGE of washed beads. (e) Cocrystal structure of MEKK3NPB1 in complex with CCM2HHD. CCM2HHD is shown in green and MEKK3NPB1 in purple. Secondary structure elements are labelled. Disordered residues indicated by a dotted line. N and C termini are indicated. (f) Close-up views of the main interaction sites between MEKK3NPB1 and CCM2HHD. Left, CCM2 helix H1 with MEKK3 helix αN. Centre, CCM2 helix H2 with MEKK3 helix αN and PB1 domain. Right, CCM2 helices H2 and H3 with MEKK3 PB1 domain. Hydrogen-bonds shown as dotted lines. Residues mutated in this study are boxed in red. Structural figures generated using CCP4mg36. (g) Map of MEKK3NPB1:CCM2HHD interactions. MEKK3 residues are shown in purple and CCM2 residues in green. Residues mutated in this study are shown in red. Dotted lines indicate hydrophobic interactions and solid lines ionic interactions. Interactions defined by PDBSum37. (h) Pull-down assay. Crystallographically determined mutations to GST–MEKK3NPB1 interrupts pull-down of wild-type CCM2HHD. MEKK3 mutants, A6D/L7D, D13R, I10D/L14D and L119E/L121E are indicated. Coomassie stained SDS–PAGE of washed beads. (i) Pull-down assay. Introduction of crystallographically determined mutations to CCM2HHD interrupts pull-down with wild-type GST–MEKK3NPB1. CCM2 mutants A319D/L322D and A319D/A320D are indicated. Coomassie stained SDS–PAGE of washed beads. (j) Isothermal titration calorimetry. CCM2HHD was titrated into MEKK3NPB1. Crystallographically defined mutants tested for MEKK3 were D13R and A6D/L7D and for CCM2 was A319D/A320D38.

Mentions: As the Mekk3 iEC−/− data above support a genetic link between Mekk3 and Ccm2, we next asked whether these neurovascular phenotypes directly relate to an interaction between MEKK3 and CCM2. As previously reported5, we found that full-length MEKK3 and full-length CCM2 co-immunoprecipitate (Fig. 2a). MEKK3 contains only two defined domains: an N-terminal Phox/Bem1p (PB1) domain and a C-terminal catalytic domain. Because MEKK3's PB1 domain is a known protein interaction domain we tested whether MEKK3 lacking this region (MEKK3ΔNPB1) could bind CCM2 and found that the interaction was lost, indicating that the PB1 domain was important for MEKK3–CCM2 interaction (Fig. 2a). To probe this interaction more deeply we generated a number of constructs of CCM2 and MEKK3 and examined direct binding in vitro (Fig. 2b). We found a direct interaction between the N terminus of MEKK3 (MEKK3NPB1) and full-length CCM2 (CCM2FL) using purified components (Fig. 2c). We further tested constructs of CCM2 encoding its phosphotyrosine binding (PTB) domain12 (CCM2PTB), its C terminus (CCM2CT), and its HHD (CCM2HHD)13 (Fig. 2b) and found that CCM2HHD directly interacts with GST–MEKK3NPB1, but that CCM2PTB does not (Fig. 2c). This represents the first binding partner identified for the CCM2 HHD. Further pull-down analysis showed that neither the PB1 domain (GST–MEKK3PB1) nor the predicted α-helical N terminus of MEKK3 (GST–MEKK3N) could appreciably pull-down CCM2HHD on their own, but that a construct encoding both of these regions of MEKK3 (GST–MEKK3NPB1) could (Fig. 2d). This suggested that both the canonical protein interaction PB1 domain of MEKK3 and a short region N-terminal of this domain play roles in its interaction with CCM2.


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)

MEKK3 uses its NPB1 region to interact with the HHD domain of CCM2.(a) Immunoprecipitation (IP) of Flag–CCM2 after cotransfection with HA–MEKK3FL or HA–MEKK3ΔNPB1 into 293T cells. Whole-cell lysates and immunoprecipitates were analysed by immunoblotting (IB) with the indicated antibodies. (b) Domain schematic of CCM2 (top) and MEKK3 (bottom). Black lines indicate constructs used in this study. Domains are indicated as PTB (Phosphotyrosine Binding), HHD (Harmonin homology domain), PB1 (Phox/Bem1p) and kinase. N and C termini are indicated. (c) Pull-down assay. GST–MEKK3NPB1 was immobilized on glutathione Sepharose beads and tested for ability to pull-down purified regions of CCM2. Coomassie stained SDS–PAGE analysis of washed beads. (d) Pull-down assay. Immobilized GST–MEKK3NPB1, GST–MEKK3N or GST–MEKK3PB1 were incubated with purified CCM2HHD. Coomassie-stained SDS–PAGE of washed beads. (e) Cocrystal structure of MEKK3NPB1 in complex with CCM2HHD. CCM2HHD is shown in green and MEKK3NPB1 in purple. Secondary structure elements are labelled. Disordered residues indicated by a dotted line. N and C termini are indicated. (f) Close-up views of the main interaction sites between MEKK3NPB1 and CCM2HHD. Left, CCM2 helix H1 with MEKK3 helix αN. Centre, CCM2 helix H2 with MEKK3 helix αN and PB1 domain. Right, CCM2 helices H2 and H3 with MEKK3 PB1 domain. Hydrogen-bonds shown as dotted lines. Residues mutated in this study are boxed in red. Structural figures generated using CCP4mg36. (g) Map of MEKK3NPB1:CCM2HHD interactions. MEKK3 residues are shown in purple and CCM2 residues in green. Residues mutated in this study are shown in red. Dotted lines indicate hydrophobic interactions and solid lines ionic interactions. Interactions defined by PDBSum37. (h) Pull-down assay. Crystallographically determined mutations to GST–MEKK3NPB1 interrupts pull-down of wild-type CCM2HHD. MEKK3 mutants, A6D/L7D, D13R, I10D/L14D and L119E/L121E are indicated. Coomassie stained SDS–PAGE of washed beads. (i) Pull-down assay. Introduction of crystallographically determined mutations to CCM2HHD interrupts pull-down with wild-type GST–MEKK3NPB1. CCM2 mutants A319D/L322D and A319D/A320D are indicated. Coomassie stained SDS–PAGE of washed beads. (j) Isothermal titration calorimetry. CCM2HHD was titrated into MEKK3NPB1. Crystallographically defined mutants tested for MEKK3 were D13R and A6D/L7D and for CCM2 was A319D/A320D38.
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f2: MEKK3 uses its NPB1 region to interact with the HHD domain of CCM2.(a) Immunoprecipitation (IP) of Flag–CCM2 after cotransfection with HA–MEKK3FL or HA–MEKK3ΔNPB1 into 293T cells. Whole-cell lysates and immunoprecipitates were analysed by immunoblotting (IB) with the indicated antibodies. (b) Domain schematic of CCM2 (top) and MEKK3 (bottom). Black lines indicate constructs used in this study. Domains are indicated as PTB (Phosphotyrosine Binding), HHD (Harmonin homology domain), PB1 (Phox/Bem1p) and kinase. N and C termini are indicated. (c) Pull-down assay. GST–MEKK3NPB1 was immobilized on glutathione Sepharose beads and tested for ability to pull-down purified regions of CCM2. Coomassie stained SDS–PAGE analysis of washed beads. (d) Pull-down assay. Immobilized GST–MEKK3NPB1, GST–MEKK3N or GST–MEKK3PB1 were incubated with purified CCM2HHD. Coomassie-stained SDS–PAGE of washed beads. (e) Cocrystal structure of MEKK3NPB1 in complex with CCM2HHD. CCM2HHD is shown in green and MEKK3NPB1 in purple. Secondary structure elements are labelled. Disordered residues indicated by a dotted line. N and C termini are indicated. (f) Close-up views of the main interaction sites between MEKK3NPB1 and CCM2HHD. Left, CCM2 helix H1 with MEKK3 helix αN. Centre, CCM2 helix H2 with MEKK3 helix αN and PB1 domain. Right, CCM2 helices H2 and H3 with MEKK3 PB1 domain. Hydrogen-bonds shown as dotted lines. Residues mutated in this study are boxed in red. Structural figures generated using CCP4mg36. (g) Map of MEKK3NPB1:CCM2HHD interactions. MEKK3 residues are shown in purple and CCM2 residues in green. Residues mutated in this study are shown in red. Dotted lines indicate hydrophobic interactions and solid lines ionic interactions. Interactions defined by PDBSum37. (h) Pull-down assay. Crystallographically determined mutations to GST–MEKK3NPB1 interrupts pull-down of wild-type CCM2HHD. MEKK3 mutants, A6D/L7D, D13R, I10D/L14D and L119E/L121E are indicated. Coomassie stained SDS–PAGE of washed beads. (i) Pull-down assay. Introduction of crystallographically determined mutations to CCM2HHD interrupts pull-down with wild-type GST–MEKK3NPB1. CCM2 mutants A319D/L322D and A319D/A320D are indicated. Coomassie stained SDS–PAGE of washed beads. (j) Isothermal titration calorimetry. CCM2HHD was titrated into MEKK3NPB1. Crystallographically defined mutants tested for MEKK3 were D13R and A6D/L7D and for CCM2 was A319D/A320D38.
Mentions: As the Mekk3 iEC−/− data above support a genetic link between Mekk3 and Ccm2, we next asked whether these neurovascular phenotypes directly relate to an interaction between MEKK3 and CCM2. As previously reported5, we found that full-length MEKK3 and full-length CCM2 co-immunoprecipitate (Fig. 2a). MEKK3 contains only two defined domains: an N-terminal Phox/Bem1p (PB1) domain and a C-terminal catalytic domain. Because MEKK3's PB1 domain is a known protein interaction domain we tested whether MEKK3 lacking this region (MEKK3ΔNPB1) could bind CCM2 and found that the interaction was lost, indicating that the PB1 domain was important for MEKK3–CCM2 interaction (Fig. 2a). To probe this interaction more deeply we generated a number of constructs of CCM2 and MEKK3 and examined direct binding in vitro (Fig. 2b). We found a direct interaction between the N terminus of MEKK3 (MEKK3NPB1) and full-length CCM2 (CCM2FL) using purified components (Fig. 2c). We further tested constructs of CCM2 encoding its phosphotyrosine binding (PTB) domain12 (CCM2PTB), its C terminus (CCM2CT), and its HHD (CCM2HHD)13 (Fig. 2b) and found that CCM2HHD directly interacts with GST–MEKK3NPB1, but that CCM2PTB does not (Fig. 2c). This represents the first binding partner identified for the CCM2 HHD. Further pull-down analysis showed that neither the PB1 domain (GST–MEKK3PB1) nor the predicted α-helical N terminus of MEKK3 (GST–MEKK3N) could appreciably pull-down CCM2HHD on their own, but that a construct encoding both of these regions of MEKK3 (GST–MEKK3NPB1) could (Fig. 2d). This suggested that both the canonical protein interaction PB1 domain of MEKK3 and a short region N-terminal of this domain play roles in its interaction with CCM2.

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