RAR and AML1 transcription factors are found in leukemias as fusion proteins with PML and ETO, respectively. Association of PML-RAR and AML1-ETO with the nuclear corepressor (N-CoR)/histone deacetylase (HDAC) complex is required to block hematopoietic differentiation. We show that PML-RAR and AML1-ETO exist in vivo within high molecular weight (HMW) nuclear complexes, reflecting their oligomeric state. Oligomerization requires PML or ETO coiled-coil regions and is responsible for abnormal recruitment of N-CoR, transcriptional repression, and impaired differentiation of primary hematopoietic precursors. Fusion of RAR to a heterologous oligomerization domain recapitulated the properties of PML-RAR, indicating that oligomerization per se is sufficient to achieve transforming potential. These results show that oligomerization of a transcription factor, imposing an altered interaction with transcriptional coregulators, represents a novel mechanism of oncogenic activation.

Vesicles (exosomes) released during sheep reticulocyte maturation contain a number of plasma membrane functions. Using an antibody coated, magnetic core bead, it has been shown unequivocally that vesicles that contain the transferrin receptor also contain other plasma membrane activities, such as the nucleoside transporter and acetylcholinesterase. Lysosomal activities, normally found in the same pellet, are excluded from the transferrin receptor-containing exosomes, suggesting that there is a common mechanism to segregate and externalize specific plasma membrane proteins. In addition to the sheep, electron micrographic studies show that exosomes can be retrieved from the circulation of anemic pigs, rats, and rabbits.

Deregulation of the phosphatidyl inositol trisphosphate kinase/AKT/mammalian target of rapamycin (mTOR) and RAS/mitogen-activated protein kinase (MAPK)/MNK pathways frequently occurs in human prostate carcinomas (PCas) and leads to aberrant modulation of messenger RNA (mRNA) translation. We have investigated the relative contribution of these pathways to translational regulation and proliferation of PCa cells. MNK-dependent phosphorylation of eIF4E is elevated in DU145 cells, which have low basal levels of AKT/mTOR activity due to the expression of the tumor suppressor PTEN. In contrast, eIF4E phosphorylation is low in PC3 and LNCaP cells with mutated PTEN and constitutively active AKT/mTOR pathway, but it can be strongly induced through inhibition of mTOR activity by rapamycin or serum depletion. Remarkably, we found that inhibition of MNKs strongly reduced the polysomal recruitment of terminal oligopyrimidine messenger RNAs (TOP mRNAs), which are known targets of mTOR-dependent translational control. Pull-down assays of the eIF4F complex indicated that translation initiation was differently affected by inhibition of MNKs and mTOR. In addition, concomitant treatment with MNK inhibitor and rapamycin exerted additive effects on polysomal recruitment of TOP mRNAs and protein synthesis. The MNK inhibitor was more effective than rapamycin in blocking proliferation of PTEN-expressing cells, whereas combination of the two inhibitors suppressed cell cycle progression in both cell lines. Microarray analysis showed that MNK affected translation of mRNAs involved in cell cycle progression. Thus, our results indicate that a balance between the activity of the AKT/mTOR and the MAPK/MNK pathway in PCa cells maintains a defined translational level of specific mRNAs required for ribosome biogenesis, cell proliferation and stress response and might confer to these cells the ability to overcome negative insults.

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Okadaic acid (OA) causes meiotic progression and chromosome condensation in cultured pachytene spermatocytes and an increase in maturation promoting factor (cyclin B1/cdc2 kinase) activity, as evaluated by H1 phosphorylative activity in anti-cyclin B1 immunoprecipitates. OA also induces a strong increase of phosphorylative activity toward the mitogen-activated protein kinase substrate myelin basic protein (MBP). Immunoprecipitation experiments with anti-extracellular signal-regulated kinase 1 (ERK1) or anti-ERK2 antibodies followed by MBP kinase assays, and direct in-gel kinase assays for MBP, show that p44/ERK1 but not p42/ERK2 is stimulated in OA-treated spermatocytes. OA treatment stimulates phosphorylation of ERK1, but not of ERK2, on a tyrosine residue involved in activation of the enzyme. ERK1 immunoprecipitated from extracts of OAstimulated spermatocytes induces a stimulation of H1 kinase activity in extracts from control pachytene spermatocytes, whereas immunoprecipitated ERK2 is uneffective. We also show that natural G 2 /M transition in spermatocytes is associated to intracellular redistribution of ERKs, and their association with microtubules of the metaphase spindle. Preincubation of cultured pachytene spermatocytes with PD98059 (a selective inhibitor of ERK-activating kinases MEK1/2) completely blocks the ability of OA to induce chromosome condensation and progression to meiotic metaphases. These results suggest that ERK1 is specifically activated during G 2 /M transition in mouse spermatocytes, that it contributes to the mechanisms of maturation promoting factor activation, and that it is essential for chromosome condensation associated with progression to meiotic metaphases.Male meiosis is a process in which a dyploid spermatocyte gives rise to four haploid round spermatids after a single round of DNA replication followed by two subsequent cell divisions. The homologous chromosomes are segregated in the two daughter cells (secondary spermatocytes) during the first meiotic division, whereas the sister chromatids, through a process resembling mitosis, are segregated during the second division to generate haploid cells. The prophase of male first meiotic division is a lengthy process which ensures the correct pairing and the crossing over between homologous chromosomes. In the mouse, this process lasts approximately 10 days, during which the homologous chromosomes become partially condensed, anneal to each other, and are maintained in proximity by a structure called the synaptonemal complex. After genetic exchange (crossing over) has occurred, the synaptonemal complex disappears and chromosome condensation allows the sites of DNA exchange to become visible as chiasmata. As for mitotic division, the nuclear envelope breaks down and microtubules are assembled into a spindle. The chromosomes become aligned to the equator of the meiotic spindle and the two sets of homologous chromosomes are separated during the anaphase. While the meiotic prophase is considerably long, metaphase and anaphase transitions ...