Inhibition of Spliceosome Assembly by the Cell Cycle-regulated Protein Kinase MELK and Involvement of Splicing Factor NIPP1

Abstract

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.The nuclear protein NIPP1 1 (39 kDa) was originally discovered as a potent and specific inhibitor of protein Ser/Thr phosphatase-1 (PP1), hence its name, nuclear inhibitor of PP1 (1-7).More recently, we have demonstrated that NIPP1 is also implicated in transcription as well as in pre-mRNA splicing by mechanisms that do not involve PP1 (8, 9). In transient transfection experiments, NIPP1 acted as a transcriptional repressor, which may be accounted for by the binding of the central and C-terminal domains of NIPP1 to the Polycomb protein, EED (embryonic ectoderm development) (9). The latter promotes transcriptional repression by the recruitment of a histone methyltransferase and histone deacetylases. NIPP1 also appears to be required for the assembly of spliceosomes, the protein-RNA complexes that catalyze pre-mRNA splicing (8). The spliceosomal function of NIPP1 requires its C-terminal domain as well as its N-terminal Forkhead-associated (FHA) domain, an established phosphothreonine-binding module. The FHA domain of NIPP1 mediates targeting to both the spliceosomes and the nuclear storage sites for splicing factors, known as "speckles" (8, 10). The targeting function of the FHA domain of NIPP1 is likely explained by its ability to bind to phosphorylated forms of the essential splicing factors CDC5L (11) and SAP155 (12).Here we show that the protein kinase MELK, which is structurally related to the AMP-activated protein kinases, also interacts in a phosphorylation-dependent manner with the FHA domain of NIPP1 and that this interaction is increased during mitosis. Furthermore, we demonstrate that recombinant MELK blocks spliceosome assembly by a mechanism that involves NIPP1. Our data suggest a novel link between pre-mRNA processing and cell cycle progression. EXPERIMENTAL PROCEDURESYeast , cloned into the pEG202 vector in-frame with the LexA DNA-binding domain, was used as bait for the screening of a HeLa cell cDNA library (11). In this library, the cDNAs are subcloned behind a galactose-inducible promoter in the pJG4 -5 vector in-frame with the B42 activation domain. Interacting proteins were identified by growth of the yeast strain EGY188 in a Ϫleucine/ϩ galactose medium. The use of a plasmid-borne LacZ reporter gene (pSH18 ...

Maternal Embryonic Leucine Zipper Kinase (MELK) Reduces Replication Stress in Glioblastoma Cells

“…These studies have implicated MELK in various cellular processes, including (a)symmetric cell division (6 -10), transcription (11), pre-mRNA splicing (12), DNA repair (13), and apoptosis (14 -15). However, with some exceptions (15)(16)(17), the relevant MELK substrates are unknown, and a unifying hypothesis on the biological function of MELK is missing.…”

Characterization of the Condensin Component Cnap1 and Protein Kinase Melk as Novel E2F Target Genes Down-regulated by 1,25-Dihydroxyvitamin D3

“…Cnap1 is an essential component of the highly conserved condensin complex required for mitotic chromosome condensation (29) and for the correct attachment between chromosome kinetochores and microtubules of the mitotic spindle (30). The cell cycle-regulated protein Ser/ Thr kinase Melk is involved in pre-mRNA processing (31) and is hypothesized to play a key role during preimplantation embryonic development (32). The histone methyltransferase Ezh2 belongs to the Polycomb group (PcG) genes that modify chromatin structure and play an important role in maintaining the silent state of HOX genes during embryonic development (33).…”

The transcriptional repressor NIPP1 is an essential player in EZH2-mediated gene silencing

“…The knockout of NIPP1 in mice is embryonic lethal before gastrulation and NIPP1 À/À cell lines are not viable . NIPP1 binds in vivo to protein Ser/Thr kinase MELK (Vulsteke et al, 2004) and protein Ser/Thr phosphatase-1 , but the physiological implication of these interactions is not known. In addition, NIPP1 interacts with RNA and the essential pre-mRNA splicing factors CDC5L and SAP155 (Jagiello et al, 1997;Jin et al, 1999;Boudrez et al, 2000Boudrez et al, , 2002.…”