RESUMO
Failure in cytokinesis, the final step in cell division, by generating tetra- and polyploidization promotes chromosomal instability, a hallmark of cancer. Here we show that HIPK2, a kinase involved in cell fate decisions in development and response to stress, controls cytokinesis and prevents tetraploidization through its effects on histone H2B. HIPK2 binds and phosphorylates histone H2B at S14 (H2B-S14(P)), and the two proteins colocalize at the midbody. HIPK2 depletion by targeted gene disruption or RNA interference results in loss of H2B-S14(P) at the midbody, prevention of cell cleavage, and tetra- and polyploidization. In HIPK2 null cells, restoration of wild-type HIPK2 activity or expression of a phosphomimetic H2B-S14D derivative abolishes cytokinesis defects and rescues cell proliferation, showing that H2B-S14(P) is required for a faithful cytokinesis. Overall, our data uncover mechanisms of a critical HIPK2 function in cytokinesis and in the prevention of tetraploidization.
Assuntos
Proteínas de Transporte/metabolismo , Citocinese , Histonas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Western Blotting , Proteínas de Transporte/genética , Divisão Celular , Linhagem Celular , Linhagem Celular Tumoral , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Células HeLa , Histonas/genética , Humanos , Camundongos , Camundongos Knockout , Microscopia de Fluorescência , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , TetraploidiaRESUMO
Foot-and-mouth disease virus (FMDV) induces a very rapid inhibition of host cell protein synthesis within infected cells. This is accompanied by the cleavage of the eukaryotic translation initiation factor 4GI (eIF4GI). The cleavage of the related protein eIF4GII has now been analyzed. Within FMDV-infected cells, cleavage of eIF4GI and eIF4GII occurs with similar kinetics. Cleavage of eIF4GII is induced in cells and in cell extracts by the FMDV leader protease (L(pro)) alone, generating cleavage products similar to those induced by enterovirus and rhinovirus 2A protease (2A(pro)). By the use of a fusion protein containing residues 445 to 744 of human eIF4GII, it was demonstrated that the FMDV L(pro) specifically cleaves this protein between residues G700 and S701, immediately adjacent to the site (V699/G700) cleaved by rhinovirus 2A(pro) in vitro. The G700/S701 cleavage site does not correspond, by amino acid sequence alignment, to that cleaved in eIF4GI by the FMDV L(pro) in vitro. Knowledge of the cleavage sites and the three-dimensional structures of the FMDV L(pro) and rhinovirus 2A(pro) enabled mutant forms of the eIF4GII sequence to be generated that are differentially resistant to either one of these proteases. These results confirmed the specificity of each protease and showed that the mutant forms of the fusion protein substrate retained their correct sensitivity to other proteases.
Assuntos
Endopeptidases/metabolismo , Fator de Iniciação Eucariótico 4G , Vírus da Febre Aftosa/enzimologia , Vírus da Febre Aftosa/fisiologia , Fatores de Iniciação de Peptídeos/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular , Cricetinae , Endopeptidases/genética , Vírus da Febre Aftosa/genética , Humanos , Dados de Sequência Molecular , Mutação , Fragmentos de Peptídeos/metabolismo , Fatores de Iniciação de Peptídeos/genética , Especificidade por SubstratoRESUMO
Several picornaviruses shut down host cellular protein synthesis by proteolytic cleavage of the eukaryotic initiation factor (eIF) 4GI and eIF4GII isoforms. Viral RNA translation is maintained by a cap-independent mechanism. Here, we identify the human rhinovirus 2 2A(pro) cleavage site in eIF4GII in vitro as PLLNV(699)*GSR; this sequence lies seven amino acids C-terminal to the cleavage site previously identified in eIF4GI (LSTR681*GPP).
Assuntos
Cisteína Endopeptidases/metabolismo , Fator de Iniciação Eucariótico 4G , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fatores de Iniciação de Peptídeos/química , Fatores de Iniciação de Peptídeos/metabolismo , Rhinovirus/enzimologia , Proteínas Virais , Sequência de Aminoácidos , Células HeLa , Humanos , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , Fatores de Iniciação de Peptídeos/genética , Alinhamento de SequênciaRESUMO
Infection of mammalian cells with picornaviruses like entero-, rhino-, and aphthoviruses leads to an inhibition of cap-dependent cellular protein synthesis by the cleavage of both translation initiation factors, eIF4GI and eIF4GII. In entero- and rhinovirus infection this cleavage process is mediated by the viral 2A proteinase (2A(pro)). In order to discriminate between a direct mode of eIF4G cleavage and an indirect cleavage via activation of a cellular proteinase, a thermosensitive 2A(pro) mutant (ts-2A(pro)) of human rhinovirus 2 was employed. Temperature shift experiments of cytoplasmic HeLa cell extracts incubated with ts-2A(pro) strongly support a direct mode of cleavage of eIF4GI and eIF4GII by the viral 2A(pro).