RESUMO
NIPP1 is a ubiquitously expressed nuclear protein that functions both as a regulator of protein Ser/Thr phosphatase-1 and as a splicing factor. The N-terminal part of NIPP1 consists of a phosphothreonine-interacting Forkhead-associated (FHA) domain. We show here that the FHA domain of NIPP1 interacts in vitro and in vivo with a TP dipeptide-rich fragment of the splicing factor SAP155/SF3b(155), a component of the U2 small nuclear ribonucleoprotein particle. The NIPP1-SAP155 interaction was entirely dependent on the phosphorylation of specific TP motifs in SAP155. Mutagenesis and competition studies revealed that various phosphorylated TP motifs competed for binding to the same site in the FHA domain. The SAP155 kinases in cell lysates were blocked by the Ca(2+) chelator EGTA and by the cyclin-dependent protein kinase inhibitor roscovitine. The phosphorylation level of SAP155 was dramatically increased during mitosis, and accordingly the activity of SAP155 kinases was augmented in mitotic lysates. We discuss how the interaction between NIPP1 and SAP155 could contribute to spliceosome (dis)assembly and the catalytic steps of splicing.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Proteínas de Transporte , Endorribonucleases , Peptídeos e Proteínas de Sinalização Intracelular , Fragmentos de Peptídeos/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Proteínas de Saccharomyces cerevisiae , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Clonagem Molecular , Quinase 2 Dependente de Ciclina , Quinases Ciclina-Dependentes/metabolismo , Feminino , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Fígado/metabolismo , Dados de Sequência Molecular , Oócitos/fisiologia , Fragmentos de Peptídeos/química , Fosfoproteínas Fosfatases , Fosforilação , Proteína Fosfatase 1 , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Spliceossomos/metabolismo , Proteínas de Xenopus , Xenopus laevisRESUMO
NIPP1 is a ubiquitous nuclear protein that is required for spliceosome assembly. We report here that the phosphothreonine-binding Forkhead-associated domain of NIPP1 interacts with the cell cycle-regulated protein Ser/Thr kinase MELK (maternal embryonic leucine zipper kinase). The NIPP1-MELK interaction was critically dependent on the phosphorylaton of Thr-478 of MELK and was increased in lysates from mitotically arrested cells. Recombinant MELK was a potent inhibitor of an early step of spliceosome assembly in nuclear extracts. This splicing defect was also seen with a kinase-dead mutant but was absent after mutation (T478A) of the NIPP1 binding site of MELK, indicating a mediatory role for NIPP1. Our data suggest that MELK has a role in the cell cycle-regulated control of pre-mRNA splicing.