RESUMO
Obinutuzumab is thought to exert its effects through its high antibody-dependent cellular cytotoxicity (ADCC) via glyco-engineering of the Fc region. In addition, obinutuzumab causes direct binding-induced cell death (DCD) only by specifically binding to its target CD20, a Ca2+ channel. However, the specific features of CD20 related to obinutuzumab binding-induction of cell death are not clearly understood. In this study, we evaluated the relationship between the Ca2+ channel features of CD20 as a store-operated Ca2+ channel (SOC) and obinutuzumab binding-induced cell death. Ca2+ channel function and biochemical analysis revealed that CD20 is an Orai1- and stromal interaction molecule (STIM1)-dependent Ca2+ pore. However, binding of obinutuzumab on CD20 did not have any effect on Ca2+ influx activity of CD20; the direct cell death rate mediated by obinutuzumab binding was almost equivalent with or without the extracellular Ca2+ condition. Given the apparent interaction between STIM1 and CD20, we observed Triton-X solubilized obinutuzumab-bound CD20 accompanied by STIM1. Subsequently, obinutuzumab binding and cell death were decreased by STIM1 knock-down in Ramos B cells. Thus, STIM1 directly contributes to cell death by increasing the affinity of cells for obinutuzumab by transferring CD20 to the Triton-soluble membrane region.
Assuntos
Anticorpos Monoclonais Humanizados/imunologia , Antígenos CD20/imunologia , Membrana Celular/imunologia , Técnicas de Silenciamento de Genes , Proteínas de Neoplasias/imunologia , Molécula 1 de Interação Estromal/imunologia , Animais , Antígenos CD20/genética , Células CHO , Membrana Celular/genética , Cricetulus , Humanos , Proteínas de Neoplasias/genética , Octoxinol/química , Solubilidade , Molécula 1 de Interação Estromal/genéticaRESUMO
Prostate cancer is initially androgen-dependent but, over time, usually develops hormone- and chemo-resistance. The present study investigated a role for p21-activated kinase 4 (PAK4) in prostate cancer progression. PAK4 activation was markedly inhibited by H89, a specific protein kinase A (PKA) inhibitor, and PAK4 was activated by the elevation of cAMP. The catalytic subunit of PKA interacted with the regulatory domain of PAK4, and directly phosphorylated PAK4 at serine 474 (S474). Catalytically active PAK4 enhanced the transcriptional activity of CREB independent of S133 phosphorylation. Stable knockdown of PAK4 in PC-3 and DU145 prostate cancer cells inhibited tumor formation in nude mice. Decreased tumorigenicity correlated with decreased expression of CREB and its targets, including Bcl-2 and cyclin A1. Additionally, in androgen-dependent LNCap-FGC cells, PAK4 regulated cAMP-induced neuroendocrine differentiation, which is known to promote tumor progression. Finally, PAK4 enhanced survival and decreased apoptosis following chemotherapy. These results suggested that PAK4 regulates progression toward hormone- and chemo-resistance in prostate cancer, and this study identified both a novel activation mechanism and potential downstream effector pathways. Therefore, PAK4 may be a promising therapeutic target in prostate cancer.
Assuntos
Proteína de Ligação a CREB/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Quinases Ativadas por p21/metabolismo , Animais , Proteína de Ligação a CREB/genética , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Progressão da Doença , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Fosforilação , Neoplasias da Próstata/enzimologia , Transplante Heterólogo , Quinases Ativadas por p21/genéticaRESUMO
Ca(2)+-ATPases are key regulators of Ca(2+) ion efflux in all eukaryotes. Animal cells have two distinct families of Ca(2+) pumps, with calmodulin-stimulated pumps (type IIB pumps) found exclusively at the plasma membrane. In plants, no equivalent type IIB pump located at the plasma membrane has been identified at the molecular level, although related isoforms have been identified in non-plasma membrane locations. Here, we identify a plant cDNA, designated SCA1 (for soybean Ca(2+)-ATPase 1), that encodes Ca(2+)-ATPase and is located at the plasma membrane. The plasma membrane localization was determined by sucrose gradient and aqueous two-phase membrane fractionations and was confirmed by the localization of SCA1p tagged with a green fluorescent protein. The Ca(2+)-ATPase activity of the SCA1p was increased approximately sixfold by calmodulin (K(1/2) approximately 10 nM). Two calmodulin binding sequences were identified in the N-terminal domain. An N-terminal truncation mutant that deletes sequence through the two calmodulin binding sites was able to complement a yeast mutant (K616) that was deficient in two endogenous Ca(2+) pumps. Our results indicate that SCA1p is structurally distinct from the plasma membrane-localized Ca(2+) pump in animal cells, belonging instead to a novel family of plant type IIB pumps found in multiple subcellular locations. In plant cells from soybean, expression of this plasma membrane pump was highly and rapidly induced by salt (NaCl) stress and a fungal elicitor but not by osmotic stress.
Assuntos
ATPases Transportadoras de Cálcio/metabolismo , Calmodulina/farmacologia , Membrana Celular/enzimologia , Glycine max/enzimologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação , Cálcio/farmacologia , ATPases Transportadoras de Cálcio/química , ATPases Transportadoras de Cálcio/genética , Calmodulina/metabolismo , Fracionamento Celular , Membrana Celular/efeitos dos fármacos , Clonagem Molecular , Ativação Enzimática/efeitos dos fármacos , Teste de Complementação Genética , Dados de Sequência Molecular , Especificidade de Órgãos , Concentração Osmolar , Estrutura Terciária de Proteína , RNA Mensageiro/análise , RNA Mensageiro/genética , RNA de Plantas/análise , RNA de Plantas/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Sais/farmacologia , Alinhamento de Sequência , Deleção de Sequência/genética , Glycine max/citologia , Glycine max/efeitos dos fármacos , Leveduras/citologia , Leveduras/genética , Leveduras/metabolismoRESUMO
The Ca2+ signal is essential for the activation of plant defense responses, but downstream components of the signaling pathway are still poorly defined. Here we demonstrate that specific calmodulin (CaM) isoforms are activated by infection or pathogen-derived elicitors and participate in Ca2+-mediated induction of plant disease resistance responses. Soybean CaM (SCaM)-4 and SCaM-5 genes, which encode for divergent CaM isoforms, were induced within 30 min by a fungal elicitor or pathogen, whereas other SCaM genes encoding highly conserved CaM isoforms did not show such response. This pathogen-triggered induction of these genes specifically depended on the increase of intracellular Ca2+ level. Constitutive expression of SCaM-4 and SCaM-5 in transgenic tobacco plants triggered spontaneous induction of lesions and induces an array of systemic acquired resistance (SAR)-associated genes. Surprisingly, these transgenic plants have normal levels of endogenous salicylic acid (SA). Furthermore, coexpression of nahG gene did not block the induction of SAR-associated genes in these transgenic plants, indicating that SA is not involved in the SAR gene induction mediated by SCaM-4 or SCaM-5. The transgenic plants exhibit enhanced resistance to a wide spectrum of virulent and avirulent pathogens, including bacteria, fungi, and virus. These results suggest that specific CaM isoforms are components of a SA-independent signal transduction chain leading to disease resistance.