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1.
Hum Mol Genet ; 25(1): 109-22, 2016 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-26546614

RESUMO

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common known cause of inherited Parkinson's disease (PD), and LRRK2 is a risk factor for idiopathic PD. How LRRK2 function is regulated is not well understood. Recently, the highly conserved 14-3-3 proteins, which play a key role in many cellular functions including cell death, have been shown to interact with LRRK2. In this study, we investigated whether 14-3-3s can regulate mutant LRRK2-induced neurite shortening and kinase activity. In the presence of 14-3-3θ overexpression, neurite length of primary neurons from BAC transgenic G2019S-LRRK2 mice returned back to wild-type levels. Similarly, 14-3-3θ overexpression reversed neurite shortening in neuronal cultures from BAC transgenic R1441G-LRRK2 mice. Conversely, inhibition of 14-3-3s by the pan-14-3-3 inhibitor difopein or dominant-negative 14-3-3θ further reduced neurite length in G2019S-LRRK2 cultures. Since G2019S-LRRK2 toxicity is likely mediated through increased kinase activity, we examined 14-3-3θ's effects on LRRK2 kinase activity. 14-3-3θ overexpression reduced the kinase activity of G2019S-LRRK2, while difopein promoted the kinase activity of G2019S-LRRK2. The ability of 14-3-3θ to reduce LRRK2 kinase activity required direct binding of 14-3-3θ with LRRK2. The potentiation of neurite shortening by difopein in G2019S-LRRK2 neurons was reversed by LRRK2 kinase inhibitors. Taken together, we conclude that 14-3-3θ can regulate LRRK2 and reduce the toxicity of mutant LRRK2 through a reduction of kinase activity.


Assuntos
Proteínas 14-3-3/fisiologia , Neuritos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Crescimento Celular , Células Cultivadas , Células HEK293 , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Neuritos/enzimologia , Neurônios/citologia , Neurônios/metabolismo , Doença de Parkinson/metabolismo , Fosforilação , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas/genética , Serina/metabolismo
2.
Neuro Oncol ; 14(3): 266-77, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22259051

RESUMO

Malignant peripheral nerve sheath tumors (MPNSTs) are rapidly progressive Schwann cell neoplasms. The erbB family of membrane tyrosine kinases has been implicated in MPNST mitogenesis and invasion and, thus, is a potential therapeutic target. However, tyrosine kinase inhibitors (TKIs) used alone have limited tumoricidal activity. Manipulating the autophagy lysosomal pathway in cells treated with cytostatic agents can promote apoptotic cell death in some cases. The goal of this study was to establish a mechanistic basis for formulating drug combinations to effectively trigger death in MPNST cells. We assessed the effects of the pan erbB inhibitor PD168393 on MPNST cell survival, caspase activation, and autophagy. PD168393 induced a cytostatic but not a cytotoxic response in MPNST cells that was accompanied by suppression of Akt and mTOR activation and increased autophagic activity. The effects of autophagy modulation on MPNST survival were then assessed following the induction of chloroquine (CQ)-induced lysosomal stress. In CQ-treated cells, suppression of autophagy was accompanied by increased caspase activation. In contrast, increased autophagy induction by inhibition of mTOR did not trigger cytotoxicity, possibly because of Akt activation. We thus hypothesized that dual targeting of mTOR and Akt by PD168393 would significantly increase cytotoxicity in cells exposed to lysosomal stress. We found that PD168393 and CQ in combination significantly increased cytotoxicity. We conclude that combinatorial therapies with erbB inhibitors and agents inducing lysosomal dysfunction may be an effective means of treating MPNSTs.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose , Cloroquina/farmacologia , Receptores ErbB/antagonistas & inibidores , Lisossomos/efeitos dos fármacos , Neoplasias de Bainha Neural/tratamento farmacológico , Quinazolinas/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Autofagia/efeitos dos fármacos , Caspases/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cloroquina/uso terapêutico , Genes erbB/efeitos dos fármacos , Humanos , Terapia de Alvo Molecular , Neoplasias de Bainha Neural/enzimologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Quinazolinas/uso terapêutico , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR/antagonistas & inibidores
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