RESUMO
Protein phosphatase 2A (PP2A) is a critical human tumor suppressor. Cancerous inhibitor of PP2A (CIP2A) supports the activity of several critical cancer drivers (Akt, MYC, E2F1) and promotes malignancy in most cancer types via PP2A inhibition. However, the 3D structure of CIP2A has not been solved, and it remains enigmatic how it interacts with PP2A. Here, we show by yeast two-hybrid assays, and subsequent validation experiments, that CIP2A forms homodimers. The homodimerization of CIP2A is confirmed by solving the crystal structure of an N-terminal CIP2A fragment (amino acids 1-560) at 3.0 Å resolution, and by subsequent structure-based mutational analyses of the dimerization interface. We further describe that the CIP2A dimer interacts with the PP2A subunits B56α and B56γ. CIP2A binds to the B56 proteins via a conserved N-terminal region, and dimerization promotes B56 binding. Intriguingly, inhibition of either CIP2A dimerization or B56α/γ expression destabilizes CIP2A, indicating opportunities for controlled degradation. These results provide the first structure-function analysis of the interaction of CIP2A with PP2A/B56 and have direct implications for its targeting in cancer therapy.
Assuntos
Autoantígenos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Oncogênicas/metabolismo , Proteína Fosfatase 2/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Autoantígenos/química , Sítios de Ligação , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana/química , Modelos Moleculares , Mutação , Proteínas Oncogênicas/química , Ligação Proteica , Conformação Proteica , Mapeamento de Interação de Proteínas , Multimerização Proteica , Proteína Fosfatase 2/química , Proteína Fosfatase 2/genética , Estabilidade Proteica , Subunidades Proteicas/metabolismo , Relação Estrutura-Atividade , Proteínas Supressoras de Tumor/químicaRESUMO
Enhancing the tumour suppressive activity of protein phosphatase 2A (PP2A) has been suggested to be an anti-leukaemic strategy. KIAA1524 (also termed CIP2A), an oncoprotein inhibiting PP2A, is associated with disease progression in chronic myeloid leukaemia and may be prognostic in cytogenetically normal acute myeloid leukaemia. Here we demonstrated that the selective proteasome inhibitor, carfilzomib, induced apoptosis in sensitive primary leukaemia cells and in sensitive leukaemia cell lines, associated with KIAA1524 protein downregulation, increased PP2A activity and decreased p-Akt, but not with the proteasome inhibition effect of carfilzomib. Ectopic expression of KIAA1524, or pretreatment with the PP2A inhibitor, okadaic acid, suppressed carfilzomib-induced apoptosis and KIAA1524 downregulation in sensitive cells, whereas co-treatment with the PP2A agonist, forskolin, enhanced carfilzomib-induced apoptosis in resistant cells. Mechanistically, carfilzomib affected KIAA1524 transcription through disturbing ELK1 (Elk-1) binding to the KIAA1524 promoter. Moreover, the drug sensitivity and mechanism of carfilzomib in xenograft mouse models correlated well with the effects of carfilzomib on KIAA1524 and p-Akt expression, as well as PP2A activity. Our data disclosed a novel drug mechanism of carfilzomib in leukaemia cells and suggests the potential therapeutic implication of KIAA1524 in leukaemia treatment.
Assuntos
Leucemia/tratamento farmacológico , Oligopeptídeos/farmacologia , Adulto , Idoso , Animais , Apoptose/efeitos dos fármacos , Autoantígenos/metabolismo , Linhagem Celular Tumoral , Cicloeximida/farmacologia , Regulação para Baixo/efeitos dos fármacos , Feminino , Células HL-60 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Células K562 , Leucemia/fisiopatologia , Masculino , Proteínas de Membrana/metabolismo , Camundongos Nus , Pessoa de Meia-Idade , Transplante de Neoplasias/métodos , Ácido Okadáico/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Cancerous Inhibitor of Protein Phosphatase 2A (CIP2A) is a human oncoprotein, which exerts its cancer-promoting function through interaction with other proteins, for example Protein Phosphatase 2A (PP2A) and MYC. The lack of structural information for CIP2A significantly prevents the design of anti-cancer therapeutics targeting this protein. In an attempt to counteract this fact, we modeled the three-dimensional structure of the N-terminal domain (CIP2A-ArmRP), analyzed key areas and amino acids, and coupled the results to the existing literature. The model reliably shows a stable armadillo repeat fold with a positively charged groove. The fact that this conserved groove highly likely binds peptides is corroborated by the presence of a conserved polar ladder, which is essential for the proper peptide-binding mode of armadillo repeat proteins and, according to our results, several known CIP2A interaction partners appropriately possess an ArmRP-binding consensus motif. Moreover, we show that Arg229Gln, which has been linked to the development of cancer, causes a significant change in charge and surface properties of CIP2A-ArmRP. In conclusion, our results reveal that CIP2A-ArmRP shares the typical fold, protein-protein interaction site and interaction patterns with other natural armadillo proteins and that, presumably, several interaction partners bind into the central groove of the modeled CIP2A-ArmRP. By providing essential structural characteristics of CIP2A, the present study significantly increases our knowledge on how CIP2A interacts with other proteins in cancer progression and how to develop new therapeutics targeting CIP2A.