RESUMO
AF9/MLLT3 contributes to the regulation of the gene encoding the epithelial sodium channel alpha, ENaCalpha, in renal tubular cells. Specifically, increases in AF9 protein lead to a reduction in ENaCalpha expression and changes in AF9 activity appear to be an important component of aldosterone signaling in the kidney. Whereas AF9 is found in the nucleus where it interacts with the histone H3 lysine 79 methyltransferase, Dot1, AF9 is also present in the cytoplasm. Data presented in this report indicate that the heat shock protein Hsp90 directly and specifically interacts with AF9 as part of an Hsp90-Hsp70-p60/Hop chaperone complex. Experimental manipulation of Hsp90 function by the inhibitor novobiocin, but not 17-AAG, results in redistribution of AF9 from a primarily nuclear to cytoplasmic location. Knockdown of Hsp90 with siRNA mimics the effect elicited by novobiocin. As expected, a shift in AF9 from the nucleus to the cytoplasm in response to Hsp90 interference leads to increased ENaCalpha expression. This is accompanied by a decrease in AF9 occupancy at the ENaCalpha promoter. Our data suggest that the interaction of Hsp90, Hsp70, and p60/Hop with AF9 is necessary for the proper subnuclear localization and activity of AF9. AF9 is among a growing number of nuclear proteins recognized to rely on the Hsp90 complex for nuclear targeting.
Assuntos
Canais Epiteliais de Sódio/genética , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas Nucleares/metabolismo , Animais , Sequência de Bases , Benzoquinonas/farmacologia , Sítios de Ligação/genética , Linhagem Celular , Expressão Gênica , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/genética , Células HeLa , Humanos , Técnicas In Vitro , Lactamas Macrocíclicas/farmacologia , Camundongos , Complexos Multiproteicos , Novobiocina/farmacologia , Proteínas Nucleares/química , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , Ligação Proteica , RNA Interferente Pequeno/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Frações Subcelulares/metabolismoRESUMO
Acute leukemias caused by translocations of the MLL gene at chromosome 11 band q23 (11q23) are characterized by a unique gene expression profile. More recently, data from several laboratories indicate that the most commonly encountered MLL fusion proteins, MLLT1, MLLT3, and AFF1 are found within a molecular complex that facilitates the elongation phase of mRNA transcription. Mutational analyses suggest that interaction between the MLLT1/3 proteins and AFF family proteins are required for experimental transformation of hematopoietic progenitor cells (HPCs). Here, we define a specific pairing of two amino acids that creates a salt bridge between MLLT1/3 and AFF proteins that is critically important for MLL-mediated transformation of HPCs. Our findings, coupled with the newly defined structure of MLLT3 in complex with AFF1, should facilitate the development of small molecules that block this amino acid interaction and interfere with the activity of the most common MLL oncoproteins.
Assuntos
Aminoácidos/genética , Proteínas de Ligação a DNA/genética , Leucemia Experimental/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Proteínas de Ligação a DNA/química , Células HEK293 , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas Nucleares/química , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/química , Fatores de Elongação da TranscriçãoRESUMO
OBJECTIVES: To examine the usefulness of the MYD88 L265P somatic mutation in identifying cases of lymphoplasmacytic lymphoma (LPL) from other lymphoplasmacytic neoplasms in bone marrow biopsy specimens. METHODS: We studied 64 bone marrow biopsy specimens with involvement by various small B-cell lymphomas or plasma cell myeloma. RESULTS: The MYD88 L265P somatic mutation was present in 13/13 cases of LPL, 1/13 cases of hairy cell leukemia, and absent in the other mature B-cell neoplasms tested. A test set of diagnostically challenging bone marrow cases with lymphoplasmacytoid morphology (B-cell lymphoma, not otherwise specified) was selected for additional review and reclassified, without knowledge of the MYD88 L265P status. Of those 16 cases, 7 were positive for MYD88, including 4/4 cases that were reclassified as LPL during the review. CONCLUSIONS: Although not entirely specific, MYD88 L265P is a useful adjunct for bone marrow diagnosis in separating LPL from other small B-cell lymphomas and plasma cell myeloma.