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Maternal embryonic leucine zipper kinase (MELK) belongs to the subfamily of AMP-activated Ser/Thr protein kinases. The expression of MELK is very high in glioblastoma-type brain tumors, but it is not clear how this contributes to tumor growth. Here we show that the siRNA-mediated loss of MELK in U87 MG glioblastoma cells causes a G1/S phase cell cycle arrest accompanied by cell death or a senescence-like phenotype that can be rescued by the expression of siRNA-resistant MELK. This cell cycle arrest is mediated by an increased expression of p21WAF1/CIP1, an inhibitor of cyclin-dependent kinases, and is associated with the hypophosphorylation of the retinoblastoma protein and the down-regulation of E2F target genes. The increased expression of p21 can be explained by the consecutive activation of ATM(ataxia telangiectasia mutated), Chk2, and p53. Intriguingly, the activation of p53 in MELK-deficient cells is not due to an increased stability of p53 but stems from the loss of MDMX (mouse double minute-X), an inhibitor of p53 transactivation. The activation of the ATM-Chk2 pathway in MELK-deficient cells is associated with the accumulation of DNA double-strand breaks during replication, as demonstrated by the appearance of γH2AX foci. Replication stress in these cells is also illustrated by an increased number of stalled replication forks and a reduced fork progression speed. Our data indicate that glioblastoma cells have elevated MELK protein levels to better cope with replication stress during unperturbed S phase. Hence, MELK inhibitors hold great potential for the treatment of glioblastomas as such or in combination with DNA-damaging therapies. [ABSTRACT FROM AUTHOR]

Recombinant muscle GYS1 (glycogen synthase 1) and recombinant liver GYS2 were phosphorylated by recombinant AMPK (AMP-activated protein kinase) in a time-dependent manner and to a similar stoichiometry. The phosphorylation site in GYS2 was identified as Ser7, which lies in a favourable consensus for phosphorylation by AMPK. Phosphorylation of GYS1 or GYS2 by AMPK led to enzyme inactivation by decreasing the affinity for both UDP-Glc (UDP-glucose) [assayed in the absence of Glc-6-P (glucose-6-phosphate)] and Glc- 6-P (assayed at low UDP-Glc concentrations). Incubation of freshly isolated rat hepatocytes with the pharmacological AMPK activators AICA riboside (5-aminoimidazole-4-carboxamide- 1-β-D-ribofuranoside) or A769662 led to persistent GYS inactivation and Ser7 phosphorylation, whereas inactivation by glucagon treatment was transient. In hepatocytes from mice harbouring a liver-specific deletion of the AMPK catalytic α1/α2 subunits, GYS2 inactivation by AICA riboside and A769662 was blunted, whereas inactivation by glucagon was unaffected. The results suggest that GYS inactivation by AMPK activators in hepatocytes is due to GYS2 Ser7 phosphorylation. [ABSTRACT FROM AUTHOR]

Maternal embryonic leucine zipper kinase (MELK) is a protein Ser/Thr kinase that has been implicated in stem cell renewal, cell cycle progression, and pre-mRNA splicing, but its substrates and regulation are not yet known. We show here that MELK has a rather broad substrate specificity and does not appear to require a specific sequence surrounding its (auto)phosphorylation sites. We have mapped no less than 16 autophosphorylation sites including serines, threonines, and a tyrosine residue and show that the phosphorylation of Thr167 and Ser171 is required for the activation of MELK. The expression of MELK activity also requires reducing agents such as dithiothreitol or reduced glutathione. Furthermore, we show that MELK is a Ca2+-binding protein and is inhibited by physiological Ca2+ concentrations. The smallest MELK fragment that was still catalytically active comprises the N-terminal catalytic domain and the flanking ubiquitin-associated domain. A C-terminal fragment of MELK functions as an autoinhibitory domain. Our data show that the activity of MELK is regulated in a complex manner and offer new perspectives for the further elucidation of its biological function. [ABSTRACT FROM AUTHOR]

NIPP1 (nuclear inhibitor of protein phosphatase 1) is a ubiquitously expressed nuclear scaffold protein that has been implicated in both transcription and RNA processing. Among its protein ligands are a protein kinase, a protein phosphatase, two splicing factors, and a transcriptional regulator, and the binding of these proteins to NIPP1 is tightly regulated by phosphorylation. To study the function of NIPP1 in vivo, we have used homologous recombination to generate mice that are deficient in NIPP1. NIPP1-/+ mice developed normally. However, NIPP1-/- embryos showed severely retarded growth at embryonic day 6.5 (E6.5) and were resorbed by E8.5. This early embryonic lethality was not associated with increased apoptosis but correlated with impaired cell proliferation. Blastocyst outgrowth experiments and the RNA interference-mediated knockdown of NIPP1 in cultured cells also revealed an essential role for NIPP1 in cell proliferation. In further agreement with this function, no viable NIPP1-/- cell lines were obtained by derivation of embryonic stem (ES) cells from blastocysts of NIPP1-/+ intercrosses or by forced homogenotization of heterozygous ES cells at high concentrations of Geneticin. We conclude that NIPP1 is indispensable for early embryonic development and cell proliferation. [ABSTRACT FROM AUTHOR]

Summary: The transient mitotic histone H3 phosphorylation by various protein kinases regulates chromosome condensation and segregation, but the counteracting phosphatases have been poorly characterized []. We show here that PP1γ is the major histone H3 phosphatase acting on the mitotically phosphorylated (ph) residues H3T3ph, H3S10ph, H3T11ph, and H3S28ph. In addition, we identify Repo-Man, a chromosome-bound interactor of PP1γ [], as a selective regulator of H3T3ph and H3T11ph dephosphorylation. Repo-Man promotes H3T11ph dephosphorylation by an indirect mechanism but directly and specifically targets H3T3ph for dephosphorylation by associated PP1γ. The PP1γ/Repo-Man complex opposes the protein kinase Haspin-mediated spreading of H3T3ph to the chromosome arms until metaphase and catalyzes the net dephosphorylation of H3T3ph at the end of mitosis. Consistent with these findings, Repo-Man modulates in a PP1-dependent manner the H3T3ph-regulated chromosomal targeting of Aurora kinase B and its substrate MCAK. Our study defines a novel mechanism by which PP1 counteracts Aurora B. [ABSTRACT FROM AUTHOR]

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) has potent antiproliferative effects characterized by a hampered G1/S transition, cDNA microarrays were used to monitor expression of 21,492 genes in MC3T3-E1 mouse osteoblasts at 1, 6, 12, 24, and 36 h after treatment with 1,25(OH)2D3. Statistical analysis revealed a cluster of genes that were strongly down-regulated by 1,25(OH)2D3 and which not only function in cell cycle regulation and DNA replication but also mediate checkpoint control, DNA repair, chromosome modifications, and mitosis. Because many of these genes were shown earlier to be regulated by the transcriptional repressor E2F4, the intergenic regions of these 1,25(OH)2D3-down-regulated genes were searched for the presence of E2F binding sites. This led to the characterization of two novel E2F target genes, chromosome condensation-related SMC-associated protein 1 (Chap1) and maternal embryonic leucine zipper kinase (Melk). Transfection studies and site-directed mutagenesis confirmed Cnap1 and Melk to be bona fide E2F targets. Repression of Cnap1 and Melk by 1,25(OH)2D3 was confirmed not only in MC3T3-E1 cells but also in several other bone-unrelated cell types. This down-regulation as well as the antiproliferative effect of 1,25(OH)2D3 depended on the pocket proteins p107 and p130 because 1,25(OH)2D3 failed to repress these E2F target genes and lost its antiproliferative action in p107-/-;p130-/- cells but not in pRb-/- cells. [ABSTRACT FROM AUTHOR]