RESUMO
The tuberous sclerosis complex (TSC) tumor suppressors form the TSC1-TSC2 complex, which limits cell growth in response to poor growth conditions. Through its GTPase-activating protein (GAP) activity toward Rheb, this complex inhibits the mechanistic target of rapamycin (mTOR) complex 1 (mTORC1), a key promoter of cell growth. Here, we identify and biochemically characterize TBC1D7 as a stably associated and ubiquitous third core subunit of the TSC1-TSC2 complex. We demonstrate that the TSC1-TSC2-TBC1D7 (TSC-TBC) complex is the functional complex that senses specific cellular growth conditions and possesses Rheb-GAP activity. Sequencing analyses of samples from TSC patients suggest that TBC1D7 is unlikely to represent TSC3. TBC1D7 knockdown decreases the association of TSC1 and TSC2 leading to decreased Rheb-GAP activity, without effects on the localization of TSC2 to the lysosome. Like the other TSC-TBC components, TBC1D7 knockdown results in increased mTORC1 signaling, delayed induction of autophagy, and enhanced cell growth under poor growth conditions.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Sequência de Aminoácidos , Proteínas de Transporte/genética , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Lisossomos/genética , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina , Dados de Sequência Molecular , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Complexos Multiproteicos , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Ligação Proteica , Proteínas/genética , Proteína Enriquecida em Homólogo de Ras do Encéfalo , Transdução de Sinais , Serina-Treonina Quinases TOR , Proteína 1 do Complexo Esclerose Tuberosa , Proteína 2 do Complexo Esclerose Tuberosa , Células Tumorais Cultivadas , Proteínas Supressoras de Tumor/genéticaRESUMO
Members of the phosphoinositide 3-kinase (PI3K) family collectively control multiple cellular responses, including proliferation, growth, chemotaxis, and survival. These diverse effects can partly be attributed to the broad range of downstream effectors being regulated by the products of these lipid kinases, the 3'-phosphoinositides. However, an additional layer of complexity is introduced by the existence of multiple PI3K enzyme isoforms. Much has been learned over the last years on the roles of the classes I and III PI3K members in cellular signaling, but little is known about the isoform-specific tasks done by the class II PI3Ks (C2alpha, beta, and gamma). In this study, we used quantitative reverse transcription-PCR and RNA interference in mammalian cells to gain further insight into the function of these lesser studied PI3K enzymes. We find that PI3K-C2alpha, but not PI3K-C2beta, has an important role in controlling cell survival and by using a panel of RNA interference reagents, we were able to determine a critical threshold of PI3K-C2alpha mRNA levels, below which the apoptotic program is switched on, via the intrinsic cell death pathway. In addition, knockdown of PI3K-C2alpha to levels that by themselves do not induce apoptosis sensitize cells to the anticancer agent Taxol (paclitaxel). Lastly, we report that lowering the levels of PI3K-C2alpha in a number of cancer cell lines reduces their proliferation and cell viability, arguing that PI3K inhibitors targeting not only the class Ialpha isoform but also class IIalpha may contribute to an effective anticancer strategy.
Assuntos
Apoptose , Regulação para Baixo , Fosfatidilinositol 3-Quinases/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Classe II de Fosfatidilinositol 3-Quinases , Perfilação da Expressão Gênica , Células HeLa , Humanos , Isoenzimas/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Fatores de TempoRESUMO
As a critical signaling node, ERK1/2 are attractive drug targets, particularly in tumors driven by activation of the MAPK pathway. Utility of targeting the MAPK pathway has been demonstrated by clinical responses to inhibitors of MEK1/2 or RAF kinases in some mutant BRAF-activated malignancies. Unlike tumors with mutations in BRAF, those with mutations in KRAS (>30% of all cancers and >90% of certain cancer types) are generally not responsive to inhibitors of MEK1/2 or RAF. Here, a covalent ERK1/2 inhibitor, CC-90003, was characterized and shown to be active in preclinical models of KRAS-mutant tumors. A unique occupancy assay was used to understand the mechanism of resistance in a KRAS-mutant patient-derived xenograft (PDX) model of colorectal cancer. Finally, combination of CC-90003 with docetaxel achieved full tumor regression and prevented tumor regrowth after cessation of treatment in a PDX model of lung cancer. This effect corresponded to changes in a stemness gene network, revealing a potential effect on tumor stem cell reprograming. IMPLICATIONS: Here, a covalent ERK1/2 inhibitor (CC-90003) is demonstrated to have preclinical efficacy in models of KRAS-mutant tumors, which present a therapeutic challenge for currently available therapies.
Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Camundongos Nus , MutaçãoRESUMO
Regulation of mast cell degranulation is dependent on the subtle interplay of cellular signaling proteins. The Src homology 2 (SH2) domain-containing inositol-5'-phosphatase (SHIP), which acts as the gatekeeper of degranulation, binds via both its SH2 domain and its phosphorylated NPXY motifs to the adapter protein Shc via the latter's phosphorylated tyrosines and phosphotyrosine-binding domain, respectively. This theoretically leaves Shc's SH2 domain available to bind proteins, which might be part of the SHIP/Shc complex. In a search for such proteins, protein kinase C-delta (PKC-delta) was found to coprecipitate in mast cells with Shc and to interact with Shc's SH2 domain following antigen or pervanadate stimulation. Phosphorylation of PKC-delta's Y(332), most likely by Lyn, was found to be responsible for PKC-delta's binding to Shc's SH2 domain. Using PKC-delta(-/-) bone marrow-derived mast cells (BMMCs), we found that the antigen-induced tyrosine phosphorylation of Shc was similar to that in wild-type (WT) BMMCs while that of SHIP was significantly increased. Moreover, increased translocation of PKC-delta to the membrane, as well as phosphorylation at T505, was observed in SHIP(-/-) BMMCs, demonstrating that while PKC-delta regulates SHIP phosphorylation, SHIP regulates PKC-delta localization and activation. Interestingly, stimulation of PKC-delta(-/-) BMMCs with suboptimal doses of antigen yielded a more sustained calcium mobilization and a significantly higher level of degranulation than that of WT cells. Altogether, our data suggest that PKC-delta is a negative regulator of antigen-induced mast cell degranulation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular , Antígenos/farmacologia , Isoenzimas/metabolismo , Mastócitos/fisiologia , Proteína Quinase C/metabolismo , Proteínas/metabolismo , Animais , Sítios de Ligação , Células da Medula Óssea/metabolismo , Cálcio/metabolismo , Degranulação Celular/efeitos dos fármacos , Ativação Enzimática , Isoenzimas/genética , Mastócitos/citologia , Mastócitos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Proteína Quinase C/genética , Proteína Quinase C-delta , Proteínas Adaptadoras da Sinalização Shc , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src , Tirosina/metabolismo , Domínios de Homologia de srcRESUMO
The variable fragment (Fv) of the monoclonal B1-8 antibody recognizes 3-nitro-4-hydroxy-phenylacetate (NP) and 5-iodo-NP (NIP) allowing for the affinity purification of the respective B cell antigen receptor with NP-sepharose and its specific elution with NIP-capronic acid (NIPcap). We generated an intracellular single-chain B1-8 Fv (iscFv), fused it to the N-terminus of the regulatory subunit (p85alpha) of phosphatidylinositol-3-kinase (PI3K) (isc-p85alpha), and examined the potential of this iscFv to serve as an intracellular elutable protein purification tag. The isc-p85alpha fusion protein could be specifically affinity-purified from the lysates of transfected Drosophila S2 cells with NP-sepharose and eluted with NIPcap, indicating the functional folding of the iscFv in the reducing environment of the cytosol. Furthermore, co-purification of the catalytic subunit of PI3K (p110) was achieved from lysates of co-transfected S2 cells as well as RBL-2H3 mast cells stably expressing isc-p85alpha. This indicates that the iscFv part of isc-p85alpha does not negatively influence p85alpha folding and interaction with p110. Moreover, successful incorporation of the p85alpha-moiety of isc-p85alpha into endogenous protein complexes in mast cells suggests the use of isc-containing fusion proteins for the native purification, elution, and analysis of intracellular signaling complexes.
Assuntos
Fragmentos de Imunoglobulinas/química , Região Variável de Imunoglobulina/química , Dobramento de Proteína , Receptores de Antígenos de Linfócitos B/isolamento & purificação , Proteínas Recombinantes de Fusão/química , Animais , Bovinos , Drosophila melanogaster/imunologia , Fragmentos de Imunoglobulinas/genética , Fragmentos de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologiaRESUMO
Signal transduction by the B-cell antigen receptor (BCR) regulates development, survival, and clonal expansion of B cells. The BCR complex comprises the membrane-bound immunoglobulin molecule and the Ig-alpha/Ig-beta heterodimer, and was shown to form oligomeric structures. Antigen-mediated engagement of the BCR results in the tyrosine phosphorylation of multiple signaling proteins leading to calcium mobilization and the activation of downstream serine/threonine kinases as well as transcription factors. In pervanadate (PV)-treated B cells, comparable pathways are activated on expression of the BCR, indicating that the BCR can signal in an antigen-independent fashion as well. In this chapter, we describe the analysis of antigen-dependent and -independent tyrosine phosphorylation events as well as a method to study calcium mobilization from differentially stimulated B cells. Furthermore, we emphasize the use of phospho-specific antibodies (Abs) and low-molecular-weight enzyme inhibitors in the process of mapping BCR-activated signaling pathways as well as determining activation states of signaling proteins.
Assuntos
Linfócitos B/metabolismo , Sinalização do Cálcio/imunologia , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais/imunologia , Tirosina/metabolismo , Animais , Linfócitos B/imunologia , Sinalização do Cálcio/efeitos dos fármacos , Galinhas , Pré-Escolar , Inibidores Enzimáticos/farmacologia , Citometria de Fluxo , Herpesvirus Humano 4/genética , Humanos , Masculino , Camundongos , Fosfatidilinositol 3-Quinases/imunologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células Tumorais Cultivadas , Tirosina/imunologia , Vanadatos/farmacologiaRESUMO
HER2/HER3 dimerization resulting from overexpression of HER2 or neuregulin (NRG1) in cancer leads to HER3-mediated oncogenic activation of phosphoinositide 3-kinase (PI3K) signaling. Although ligand-blocking HER3 antibodies inhibit NRG1-driven tumor growth, they are ineffective against HER2-driven tumor growth because HER2 activates HER3 in a ligand-independent manner. In this study, we describe a novel HER3 monoclonal antibody (LJM716) that can neutralize multiple modes of HER3 activation, making it a superior candidate for clinical translation as a therapeutic candidate. LJM716 was a potent inhibitor of HER3/AKT phosphorylation and proliferation in HER2-amplified and NRG1-expressing cancer cells, and it displayed single-agent efficacy in tumor xenograft models. Combining LJM716 with agents that target HER2 or EGFR produced synergistic antitumor activity in vitro and in vivo. In particular, combining LJM716 with trastuzumab produced a more potent inhibition of signaling and cell proliferation than trastuzumab/pertuzumab combinations with similar activity in vivo. To elucidate its mechanism of action, we solved the structure of LJM716 bound to HER3, finding that LJM716 bound to an epitope, within domains 2 and 4, that traps HER3 in an inactive conformation. Taken together, our findings establish that LJM716 possesses a novel mechanism of action that, in combination with HER2- or EGFR-targeted agents, may leverage their clinical efficacy in ErbB-driven cancers.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Neoplasias da Mama/patologia , Neuregulina-1/metabolismo , Conformação Proteica/efeitos dos fármacos , Receptor ErbB-3/antagonistas & inibidores , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/mortalidade , Proliferação de Células/efeitos dos fármacos , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Feminino , Humanos , Immunoblotting , Imunoprecipitação , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , Fosforilação/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/imunologia , Receptor ErbB-2/metabolismo , Receptor ErbB-3/química , Receptor ErbB-3/imunologia , Receptor ErbB-3/metabolismo , Transdução de Sinais , Taxa de Sobrevida , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Class IA phosphoinositide 3-kinases (PI3Ks) represent a group of heterodimeric lipid kinases with important functions in cellular signal transduction. The regulatory p85 subunit constitutively binds to the catalytic p110 subunit and mediates the recruitment of the heterodimer to various membrane-localized proteins upon activation by a vast array of stimuli. The functional characterization of protein domains that mediate p85 function has been hampered by a lack of structural data. Therefore, we investigated a 35-aa region in the inter-SH2 domain of p85, reported to be necessary for binding of p110, by site-directed mutagenesis and evaluated the importance of individual amino acids for PI3K heterodimer formation. This approach led to the identification of an 11-aa region required for p110 binding in vitro and mesoderm induction during early Xenopus development in vivo. Further analyses revealed two pairs of hydrophobic amino acids within this region, which are particularly important for high-affinity intersubunit interaction. Thus, our data provide further insight into the molecular mechanisms of PI3K intersubunit interaction and led to the identification of new p85 mutant proteins with varying degrees of dominant-negative effects that will be helpful for future PI3K-related research.