Polo-like kinase 3 (Plk3) is an important mediator of the cellular responses to genotoxic stresses. In this study, we examined the physiologic function of Plk3 by generating Plk3-deficient mice. Plk3 À/À mice displayed an increase in weight and developed tumors in various organs at advanced age. Many tumors in Plk3 À/À mice were large in size, exhibiting enhanced angiogenesis. Plk3 À/À mouse embryonic fibroblasts were hypersensitive to the induction of hypoxiainducible factor-1A (HIF-1A) under hypoxic conditions or by nickel and cobalt ion treatments. Ectopic expression of the Plk3-kinase domain (Plk3-KD), but not its Polo-box domain or a Plk3-KD mutant, suppressed the nuclear accumulation of HIF-1A induced by nickel or cobalt ions. Moreover, hypoxiainduced HIF-1A expression was tightly associated with a significant down-regulation of Plk3 expression in HeLa cells. Given the importance of HIF-1A in mediating the activation of the ''survival machinery'' in cancer cells, these studies strongly suggest that enhanced tumorigenesis in Plk3-null mice is at least partially mediated by a deregulated HIF-1 pathway.

Y box-binding protein 1 (YB-1) is a multifunctional protein that can act as a regulator of transcription and of translation. In chicken embryo fibroblasts transformed by the oncoproteins P3k (phosphatidylinositol 3-kinase) or Akt, YB-1 is transcriptionally downregulated. Expression of YB-1 from a retroviral vector induces a strong cellular resistance to transformation by P3k or Akt but does not affect sensitivity to transformation by other oncoproteins, such as Src, Jun, or Qin. The YB-1-expressing cells assume a tightly adherent, flat phenotype, with YB-1 localized in the cytoplasm, and show a greatly reduced saturation density. Both cap-dependent and cap-independent translation is inhibited in these cells, but the activity of Akt remains unaffected, suggesting that YB-1 functions downstream of Akt. A YB-1 protein with a loss-of-function mutation in the RNA-binding motif no longer binds to the mRNA cap structure, is localized in the cell nucleus, does not induce the flat cellular phenotype, and fails to interfere with P3k-or Akt-induced oncogenic transformation. This mutant also does not inhibit capdependent or cap-independent translation. These results suggest that YB-1 acts like a rapamycin mimic, inhibiting translational events that are required in phosphatidylinositol 3-kinase-driven oncogenic transformation.Akt ͉ p3k ͉ cell transformation ͉ TOR P 3k and Akt were originally described as oncoproteins encoded by two highly tumorigenic retroviruses (1, 2). P3k is a homolog of the catalytic subunit p110 of phosphatidylinositol 3-kinase and controls the activity of the Ser͞Thr protein kinase Akt, also referred to as protein kinase B. Both proteins are inherently oncogenic. The phosphatidylinositol 3-kinase-Aktsignaling pathway affects numerous and diverse cellular functions, many related to growth, survival, and differentiation (3-6). A downstream target of Akt is the Ser͞Thr kinase TOR, which regulates translation by targeting two proteins: the translation initiation factor 4E-binding protein 1 (4E-BP1 or PHAS-1) and the p70 S6 kinase (S6K) (7-9). Hypophosphorylated 4E-BP1 binds to the initiation factor 4E and prevents the assembly of the translation initiation complex at the cap structure of mRNAs (10-12). After TOR-dependent phosphorylation, 4E-BP1 no longer binds to 4E, freeing it for assembly of the initiation complex and for cap-binding. Phosphorylation by TOR activates S6K, which then phosphorylates the ribosomal protein S6, controlling the translation of 5ЈTOP mRNAs (mRNAs that contain an oligopyrimidine tract at their 5Ј termini) (13-16). This class of mRNAs encodes ribosomal proteins and translation elongation factors; the oligopyrimidine tract coordinates translation in a growth-dependent fashion. TOR plays a critical role in P3k-and Akt-induced oncogenic transformation. Inhibition of TOR by rapamycin induces cellular resistance to transformation by these two oncoproteins and reduces the growth of tumors that depend on a gain of function in the phosphatidylinositol 3-kinase pathway (17)(18)(19). These...

Recently developed methods for fluorescence-activated cell sorting (FACS) of freshly-isolated brain cells from transgenic mice combining fluorescent signals with cell type-specific markers allow cell-type separation. Based upon previous observations in primary cultures of mouse astrocytes we treated transgenic mice tagged with a neuron-specific or an astrocyte-specific marker with fluoxetine, either acute (10 mg/kg for 2 h) or chronic (10 mg/kg daily for 2 weeks). Acute treatment upregulated cfos and fosB mRNA expression in astrocytes and neurons. Chronic effects on astrocytes replicated those demonstrated in cultures, i.e., upregulation of mRNA and/or protein expression of 5-HT(2B) receptors (5-HT(2B)R), and GluK2 receptors, and of cPLA(2a) and ADAR2, together with increased GluK2 and 5-HT(2B)R editing. Neurons showed increased GluK4 and 5-HT(2C) receptor expression. To further correlate these findings with major depression we compared the changes in gene expression with those in a mouse model of anhedonia. Three out of 4 genes up-regulated in astrocytes by fluoxetine were down-regulated, whereas the neuronally upregulated 5-HT(2C) receptor gene showed no change. References are made to recent review papers discussing potential relations between observed fluoxetine effects and clinical effects of SSRIs, emphasizing that all 5 clinically used SSRIs have identical and virtually equipotent effects on cultured astrocytes.

Polo-like kinase 3 (Plk3), an immediate early response gene product, plays an important role in the regulation of mitosis, DNA damage checkpoint activation, and Golgi dynamics. Similar to other members of the Plk family, Plk3 has a conserved kinase domain at the N terminus and a Polo box domain consisting of two Polo boxes at the C terminus. In this study, we demonstrate that the Polo box domain of Plk3 is sufficient for subcellular localization of this kinase to the centrosomes, the spindle poles, and the midbody when ectopically expressed in HeLa and U2OS cells. Both Polo boxes are required for the subcellular localization. Overexpression of the Polo box domain, not the kinase domain, of Plk3 causes significant cell cycle arrest and cytokinesis defects, eventually leading to mitotic catastrophe/apoptosis. Interestingly, the Polo box domain of Plk3 is more potent in inhibiting cell proliferation and inducing apoptosis than that of Plk1, suggesting that this domain can provide an additional structural basis for discovery of new anticancer drugs given the current emphasis on Plk1 as a therapeutic target.Reversible phosphorylation and protein degradation are two major molecular mechanisms that control progression of the cell cycle. Among known gene products that regulate the cell cycle, Plks are a group of protein serine/threonine kinases important for monitoring various transitions during the cell cycle (1, 2). Extensive research in the past demonstrates that Plks have multiple functions in mitosis including modulating the activity of Cdk1/cyclinB (3), mediating fragmentation of the Golgi complex (4 -6), and regulating sister chromatid segregation by phosphorylation of cohesin and components of the anaphase-promoting complex (7,8).Plks are highly conserved in terms of their structures and functions (1, 2, 9). In mammals, The Plk family consists of four members, namely, Plk1, Plk2, Plk3, and Plk4 (9). In addition to the kinase domain at the N terminus, Plks share one or two stretches of conserved amino acid sequences termed Polo box (9). Without affecting the kinase activity, mutations in the Polo boxes of Cdc5, a Plk ortholog in the budding yeast, result in its inability to complement temperature-sensitive phenotypes (10). Further studies showed that Polo box domains (PBDs) 2 of yeast Cdc5 and mammalian Plk1 play an essential role in subcellular localization (11,12). Through an elegant peptide library screening approach, Yaffe and co-workers (13, 14) demonstrate that PBDs from yeast, Xenopus, and human Plks recognize similar phosphoserine/threonine-containing motifs, functioning as an intact domain that bind to phosphoserine or phosphothreonine. Specific phosphopeptides fit into a conserved and positively charged pocket formed at the edge of the Polo box interface; mutations that interfere or disrupt the phosphodependent interaction impair cell cycle-dependent localization, thus strongly suggesting that the binding of phospholigands to the PBD is essential for mitotic progression (13). Given that overexpression o...