RESUMO
Mutations in CCM1, CCM2, or CCM3 lead to cerebral cavernous malformations, one of the most common hereditary vascular diseases of the brain. Endothelial cells within these lesions are the main disease compartments. Here, we show that adenoviral CCM3 expression inhibits endothelial cell migration, proliferation, and tube formation while downregulation of endogenous CCM3 results in increased formation of tube-like structures. Adenoviral CCM3 expression does not induce apoptosis under normal endothelial cell culture conditions but protects endothelial cells from staurosporine-induced cell death. Tyrosine kinase activity profiling suggests that CCM3 supports PDPK-1/Akt-mediated endothelial cell quiescence and survival.
Assuntos
Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/fisiologia , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/fisiologia , Neoplasias Encefálicas/irrigação sanguínea , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Movimento Celular/fisiologia , Proliferação de Células , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Células Endoteliais/fisiologia , Hemangioma Cavernoso do Sistema Nervoso Central/irrigação sanguínea , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Hemangioma Cavernoso do Sistema Nervoso Central/patologia , Humanos , Mutação , Neovascularização Fisiológica/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Estaurosporina/farmacologia , TransfecçãoRESUMO
Cerebral cavernous malformations (CCMs) may cause recurrent headaches, seizures, and hemorrhagic stroke and have been associated with loss-of-function mutations in CCM1/KRIT1, CCM2, and CCM3/programmed cell death 10 (PDCD10). The CCM3/PDCD10 amino acid sequence does not reveal significant homologies to protein domains with known structure. With the help of the only published human in-frame deletion of the CCM3 gene (c.97-?_150+?del), CCM3:p.L33_K50del, we have identified the interaction domain of CCM3 with the oxidant stress response serine/threonine kinase 25 (STK25, YSK1, SOK1) and with the mammalian Ste20-like kinase 4 (MST4, MASK). Consistently, nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) analyses revealed two STK25 phosphorylation sites at serine 39 and threonine 43. The corresponding in-frame deletion of zebrafish ccm3a, dccm3:p.L31_K48del, also resulted in impaired interaction with STK25 and MST4. In agreement with the observed redundant biochemical functionality of zebrafish ccm3a and its duplicate ccm3b, simultaneous inactivation of both genes resulted in a progressive cardiovascular phenotype in zebrafish indistinguishable from ccm1 and ccm2 mutants. The pronounced cardiovascular dilatations could be recapitulated by morpholino-induced in-frame skipping of the exon encoding the STK25 and MST4 binding site of zebrafish Ccm3a if Ccm3b was repressed in parallel. Using a novel zebrafish model of CCM, we could thus demonstrate that the newly mapped STK25 and MST4 interaction domain within the CCM3 protein plays a crucial role for vascular development in zebrafish.
Assuntos
Aminoácidos/genética , Proteínas Reguladoras de Apoptose/genética , Mutação da Fase de Leitura/genética , Proteínas de Membrana/genética , Mapeamento de Interação de Proteínas , Proteínas Proto-Oncogênicas/genética , Deleção de Sequência , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/metabolismo , Anormalidades Cardiovasculares/embriologia , Anormalidades Cardiovasculares/metabolismo , Proteínas de Transporte/metabolismo , Linhagem Celular , Éxons/genética , Técnicas de Silenciamento de Genes , Coração/embriologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína KRIT1 , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Fenótipo , Fosforilação , Fosfosserina/metabolismo , Fosfotreonina/metabolismo , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/metabolismo , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/químicaRESUMO
Cerebral cavernous malformations (CCM) are prevalent cerebrovascular lesions predisposing to chronic headaches, epilepsy, and hemorrhagic stroke. Using a combination of direct sequencing and MLPA analyses, we identified 15 novel and eight previously published CCM1 (KRIT1), CCM2, and CCM3 (PDCD10) mutations. The mutation detection rate was >90% for familial cases and >60% for isolated cases with multiple malformations. Splice site mutations constituted almost 20% of all CCM mutations identified. One of these proved to be a de novo mutation of the most 3' acceptor splice site of the CCM1 gene resulting in retention of intron 19. A further mutation affected the 3' splice site of CCM2 intron 2 leading to cryptic splice site utilization in both CCM2 and its transcript variant lacking exon 2. With the exception of one in-frame deletion of CCM2 exon 2, which corresponds to the naturally occurring splice variant of CCM2 on the RNA level and is predicted to result in the omission of 58 amino acids (CCM2:p.P11_K68del), all mutations lead to the introduction of premature stop codons. To gain insight into the likely mechanisms underlying the only known CCM2 in-frame deletion, we analyzed the functional consequences of loss of CCM2 exon 2. The CCM2:p.P11_K68del protein could be expressed in cell culture and complexed with CCM3. However, its ability to interact with CCM1 and to form a CCM1/CCM2/CCM3 complex was lost. These data are in agreement with a loss-of-function mechanism for CCM mutations, uncover an N-terminal CCM2 domain required for CCM1 binding, and demonstrate full-length CCM2 as the essential core protein in the CCM1/CCM2/CCM3 complex.
Assuntos
Proteínas Reguladoras de Apoptose/genética , Proteínas de Transporte/genética , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Proteínas de Membrana/genética , Proteínas Associadas aos Microtúbulos/genética , Mutação , Proteínas Proto-Oncogênicas/genética , Deleção de Sequência , Proteínas Reguladoras de Apoptose/metabolismo , Sequência de Bases , Proteínas de Transporte/metabolismo , Linhagem Celular , Primers do DNA , Feminino , Humanos , Proteína KRIT1 , Masculino , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Linhagem , Ligação Proteica , Proteínas Proto-Oncogênicas/metabolismoRESUMO
Individuals carrying a mutation in one of the three cerebral cavernous malformation genes (CCM1/KRIT1, CCM2, CCM3) cannot be clinically distinguished, raising the possibility that they act within common molecular pathways. In this study, we demonstrate that CCM3 (PDCD10) coprecipitates and colocalizes with CCM2. We also show that CCM3 directly binds to serine/threonine kinase 25 (STK25, YSK1, SOK1) and the phosphatase domain of Fas-associated phosphatase-1 (FAP-1, PTPN13, PTP-Bas, PTP-BL). CCM3 is phosphorylated by STK25 but not by its other Yeast-Two hybrid interactor STK24, whereas the C-terminal catalytic domain of FAP-1 dephosphorylates CCM3. Finally, our experiments reveal that STK25 forms a protein complex with CCM2. Thus, our data link two proteins of unknown function, CCM3 and STK25, with CCM2, which is part of signaling pathways essential for vascular development and CCM pathogenesis.