RESUMO
Cushing's disease is caused by corticotroph adenomas of the pituitary. To explore the molecular mechanisms of endocrine autonomy in these tumors, we performed exome sequencing of 10 corticotroph adenomas. We found somatic mutations in the USP8 deubiquitinase gene in 4 of 10 adenomas. The mutations clustered in the 14-3-3 protein binding motif and enhanced the proteolytic cleavage and catalytic activity of USP8. Cleavage of USP8 led to increased deubiqutination of the EGF receptor, impairing its downregulation and sustaining EGF signaling. USP8 mutants enhanced promoter activity of the gene encoding proopiomelanocortin. In summary, our data show that dominant mutations in USP8 cause Cushing's disease via activation of EGF receptor signaling.
Assuntos
Adenoma Hipofisário Secretor de ACT/genética , Endopeptidases/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Mutação , Proteínas de Neoplasias/genética , Hipersecreção Hipofisária de ACTH/genética , Neoplasias Hipofisárias/genética , Ubiquitina Tiolesterase/genética , Proteínas 14-3-3/metabolismo , Adenoma Hipofisário Secretor de ACT/complicações , Adenoma Hipofisário Secretor de ACT/metabolismo , Adenoma/genética , Hormônio Adrenocorticotrópico/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Endopeptidases/fisiologia , Complexos Endossomais de Distribuição Requeridos para Transporte/fisiologia , Receptores ErbB/metabolismo , Exoma/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Dados de Sequência Molecular , Proteínas de Neoplasias/fisiologia , Neoplasias Hipofisárias/complicações , Neoplasias Hipofisárias/metabolismo , Pró-Opiomelanocortina/biossíntese , Pró-Opiomelanocortina/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Ubiquitina Tiolesterase/fisiologiaRESUMO
Sec2p is a guanine nucleotide exchange factor that promotes exocytosis by activating the Rab GTPase Sec4p. Sec2p is highly phosphorylated, and we have explored the role of phosphorylation in the regulation of its function. We have identified three phosphosites and demonstrate that phosphorylation regulates the interaction of Sec2p with its binding partners Ypt32p, Sec15p, and phosphatidyl-inositol-4-phosphate. In its nonphosphorylated form, Sec2p binds preferentially to the upstream Rab, Ypt32p-GTP, thus forming a Rab guanine nucleotide exchange factor cascade that leads to the activation of the downstream Rab, Sec4p. The nonphosphorylated form of Sec2p also binds to the Golgi-associated phosphatidyl-inositol-4-phosphate, which works in concert with Ypt32p-GTP to recruit Sec2p to Golgi-derived secretory vesicles. In contrast, the phosphorylated form of Sec2p binds preferentially to Sec15p, a downstream effector of Sec4p and a component of the exocyst tethering complex, thus forming a positive-feedback loop that prepares the secretory vesicle for fusion with the plasma membrane. Our results suggest that the phosphorylation state of Sec2p can direct a switch in its regulatory binding partners that facilitates maturation of the secretory vesicle and helps to promote the directionality of vesicular transport.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Vesículas Transportadoras/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Transporte Biológico/fisiologia , Eletroforese em Gel de Poliacrilamida , Fatores de Troca do Nucleotídeo Guanina/genética , Imunoprecipitação , Microscopia Eletrônica , Microscopia de Fluorescência , Mutagênese Sítio-Dirigida , Fosfatos de Fosfatidilinositol/metabolismo , Fosforilação , Ligação Proteica , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Transporte Vesicular/metabolismoRESUMO
Sec2p is the guanine nucleotide exchange factor (GEF) that activates the Rab GTPase Sec4p on secretory vesicles. Sec2p also binds a Rab acting earlier in the secretory pathway, Ypt32-GTP, forming a Rab GEF cascade. Ypt32p and the Sec4p effector Sec15p (a component of the exocyst complex) compete for binding to Sec2p. Indeed Ypt32p initially recruits Sec2p, but subsequently allows a handoff of active Sec2p/Sec4p to Sec15p. Intriguingly, Golgi-associated phosphatidylinositol 4-phosphate (PI4P) works together with Ypt32-GTP in this context. PI4P inhibits Sec2p-Sec15p interactions, promoting recruitment of Sec2p by Ypt32p as secretory vesicles form. However, PI4P levels appear to decline as vesicles reach secretory sites, allowing Sec15p to replace Ypt32p as vesicles mature. In this way, the regulation of PI4P levels may switch Sec2p/Sec4p function during vesicle maturation, from a Rab GEF recruitment cascade involving Ypt32p to an effector positive feedback loop involving Sec15p.