RESUMO
Serum- and glucocorticoid-regulated kinase 3 (Sgk3) is a serine/threonine protein kinase activated by the phospholipid phosphatidylinositol 3-phosphate (PI3P) downstream of growth factor signaling via class I phosphatidylinositol 3-kinase (PI3K) signaling and by class III PI3K/Vps34-mediated PI3P production on endosomes. Upregulation of Sgk3 activity has recently been linked to a number of human cancers; however, the precise mechanism of activation of Sgk3 is unknown. Here, we use a wide range of cell biological, biochemical, and biophysical techniques, including hydrogen-deuterium exchange mass spectrometry, to investigate the mechanism of activation of Sgk3 by PI3P. We show that Sgk3 is regulated by a combination of phosphorylation and allosteric activation. We demonstrate that binding of Sgk3 to PI3P via its regulatory phox homology (PX) domain induces large conformational changes in Sgk3 associated with its activation and that the PI3P-binding pocket of the PX domain of Sgk3 is sequestered in its inactive conformation. Finally, we reconstitute Sgk3 activation via Vps34-mediated PI3P synthesis on phosphatidylinositol liposomes in vitro. In addition to identifying the mechanism of Sgk3 activation by PI3P, our findings open up potential therapeutic avenues in allosteric inhibitor development to target Sgk3 in cancer.
Assuntos
Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Endossomos/metabolismo , Lipossomos/química , Neoplasias/patologia , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Humanos , Técnicas In Vitro , Lipossomos/metabolismo , Espectrometria de Massas/métodos , Neoplasias/enzimologia , Fosfatos de Fosfatidilinositol/química , Proteínas Serina-Treonina Quinases/química , Elementos Estruturais de Proteínas , Transdução de SinaisRESUMO
The GTPase Rab5 and phosphatidylinositol-3 phosphate [PI(3)P] coordinately regulate endosome trafficking. Rab5 recruits Vps34, the class III phosphoinositide 3-kinase (PI3K), to generate PI(3)P and recruit PI(3)P-binding proteins. Loss of Rab5 and loss of Vps34 have opposite effects on endosome size, suggesting that our understanding of how Rab5 and PI(3)P cooperate is incomplete. Here, we report a novel regulatory loop whereby Caenorhabditis elegans VPS-34 inactivates RAB-5 via recruitment of the TBC-2 Rab GTPase-activating protein. We found that loss of VPS-34 caused a phenotype with large late endosomes, as with loss of TBC-2, and that Rab5 activity (mice have two Rab5 isoforms, Rab5a and Rab5b) is increased in Vps34-knockout mouse embryonic fibroblasts (Vps34 is also known as PIK3C3 in mammals). We found that VPS-34 is required for TBC-2 endosome localization and that the pleckstrin homology (PH) domain of TBC-2 bound PI(3)P. Deletion of the PH domain enhanced TBC-2 localization to endosomes in a VPS-34-dependent manner. Thus, PI(3)P binding of the PH domain might be permissive for another PI(3)P-regulated interaction that recruits TBC-2 to endosomes. Therefore, VPS-34 recruits TBC-2 to endosomes to inactivate RAB-5 to ensure the directionality of endosome maturation.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Endossomos/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans , Membrana Celular/metabolismo , Fibroblastos/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Lipossomos/química , Camundongos , Camundongos Knockout , Mutação , Fenótipo , Plasmídeos/metabolismo , Domínios Proteicos , Interferência de RNARESUMO
Autophagy is an intracellular pathway that can contribute to innate antiviral immunity by delivering viruses to lysosomes for degradation or can be beneficial for viruses by providing specialized membranes for virus replication. Here, we show that the picornavirus foot-and-mouth disease virus (FMDV) induces the formation of autophagosomes. Induction was dependent on Atg5, involved processing of LC3 to LC3II, and led to a redistribution of LC3 from the cytosol to punctate vesicles indicative of authentic autophagosomes. Furthermore, FMDV yields were reduced in cells lacking Atg5, suggesting that autophagy may facilitate FMDV infection. However, induction of autophagosomes by FMDV appeared to differ from starvation, as the generation of LC3 punctae was not inhibited by wortmannin, implying that FMDV-induced autophagosome formation does not require the class III phosphatidylinositol 3-kinase (PI3-kinase) activity of vps34. Unlike other picornaviruses, for which there is strong evidence that autophagosome formation is linked to expression of viral nonstructural proteins, FMDV induced autophagosomes very early during infection. Furthermore, autophagosomes could be triggered by either UV-inactivated virus or empty FMDV capsids, suggesting that autophagosome formation was activated during cell entry. Unlike other picornaviruses, FMDV-induced autophagosomes did not colocalize with the viral 3A or 3D protein. In contrast, â¼50% of the autophagosomes induced by FMDV colocalized with VP1. LC3 and VP1 also colocalized with the cellular adaptor protein p62, which normally targets ubiquitinated proteins to autophagosomes. These results suggest that FMDV induces autophagosomes during cell entry to facilitate infection, but not to provide membranes for replication.
Assuntos
Autofagia/fisiologia , Vírus da Febre Aftosa/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Fagossomos/virologia , Internalização do Vírus , Androstadienos , Animais , Proteína 5 Relacionada à Autofagia , Western Blotting , Células CHO , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Cricetinae , Cricetulus , Proteínas de Fluorescência Verde , Camundongos , Microscopia de Fluorescência , Proteínas Associadas aos Microtúbulos/deficiência , WortmaninaRESUMO
Charcot-Marie-Tooth Disorder Type 4B (CMT4B) is a demyelinating peripheral neuropathy caused by mutations in myotubularin-related (MTMR) proteins 2, 13, or 5 (CMT4B1/2/3), which regulate phosphoinositide turnover and endosomal trafficking. Although mouse models of CMT4B2 exist, an in vitro model would make possible pharmacological and reverse genetic experiments needed to clarify the role of MTMR13 in myelination. We have generated such a model using Schwann cell-dorsal root ganglion (SC-DRG) explants from Mtmr13-/- mice. Myelin sheaths in mutant cultures contain outfoldings highly reminiscent of those observed in the nerves of Mtmr13-/- mice and CMT4B2 patients. Mtmr13-/- SC-DRG explants also contain reduced Mtmr2, further supporting a role of Mtmr13 in stabilizing Mtmr2. Elevated PI(3,5)P2 has been implicated as a cause of myelin outfoldings in Mtmr2-/- models. In contrast, the role of elevated PI3P or PI(3,5)P2 in promoting outfoldings in Mtmr13-/- models is unclear. We found that over-expression of MTMR2 in Mtmr13-/- SC-DRGs moderately reduced the prevalence of myelin outfoldings. Thus, a manipulation predicted to lower PI3P and PI(3,5)P2 partially suppressed the phenotype caused by Mtmr13 deficiency. We also explored the relationship between CMT4B2-like myelin outfoldings and kinases that produce PI3P and PI(3,5)P2 by analyzing nerve pathology in mice lacking both Mtmr13 and one of two specific PI 3-kinases. Intriguingly, the loss of vacuolar protein sorting 34 or PI3K-C2ß in Mtmr13-/- mice had no impact on the prevalence of myelin outfoldings. In aggregate, our findings suggest that the MTMR13 scaffold protein likely has critical functions other than stabilizing MTMR2 to achieve an adequate level of PI 3-phosphatase activity.