Polo-like kinase 1 (Plk1) plays essential roles at multiple events during cell division, yet little is known about its physiological substrates. In a cDNA phage display screen using Plk1 C-terminal affinity columns, we identified NudC (nuclear distribution gene C) as a Plk1 binding protein. Here, we characterize the interaction between Plk1 and NudC, show that Plk1 phosphorylates NudC at conserved S274 and S326 residues in vitro, and present evidence that NudC is also a substrate for Plk1 in vivo. Downregulation of NudC by RNA interference results in multiple mitotic defects, including multinucleation and cells arrested at the midbody stage, which are rescued by ectopic expression of wild-type NudC, but not by NudC with mutations in the Plk1 phosphorylation sites. These results suggest that Plk1 phosphorylation of NudC may influence cytokinesis.

NudC, a nuclear movement protein that associates with dynein, was originally cloned as a mitogen-inducible early growth response gene. NudC forms a biochemical complex with components of the dynein/dynactin complex and is suggested to play a role in translocation of nuclei in proliferating neuronal progenitors as well as in migrating neurons in culture. Here, we show that NudC plays multiple roles in mitosis and cytokinesis in cultured mammalian cells. Altering NudC levels by either small interfering RNA-mediated gene silencing or adenovirus-mediated overexpression resulted in multinucleated cells and cells with persistent intercellular connections and disorganized midzone and midbody matrix. These phenotypes suggest a failure in cytokinesis in NudC altered cells. Further, a key mitotic enzyme, polo-like kinase, is mislocalized from the centrosomes and the midbody in NudC altered cells. Gene silencing of nud-1, the Caenorhabditis elegansortholog of NudC, led to a loss of midzone microtubules and the rapid regression of the cleavage furrow, which resulted in one-celled embryos containing two nuclei. The loss of midzone microtubule organization owing to silencing of the NudC/nud-1 gene in two systems, coupled with the loss of Plk1 from mitotic structures in mammalian cells, provide clues to the cytokinesis defect and the multinucleation phenotype. Our findings suggest that NudC functions in mitosis and cytokinesis, in part by regulating microtubule organization at the midzone and midbody.

NUDC is a highly conserved protein important for nuclear migration and viability in Aspergillus nidulans. Mammalian NudC interacts with Lis1, a neuronal migration protein important during neocorticogenesis, suggesting a conserved mechanism of nuclear movement in A. nidulans and neuronal migration in the developing mammalian brain (S. M. Morris et al., 1998). To further investigate this possibility, we show for the first time that NudC, Lis1, and cytoplasmic dynein intermediate chain (CDIC) colocalize at the microtubule organizing center (MTOC) around the nucleus in a polarized manner facing the leading pole of cerebellar granule cells with a migratory morphology. In neurons with stationary morphology, NudC is distributed throughout the soma and colocalizes with CDIC and tubulin in neurites as well as at the MTOC. At the subcellular level, NudC, CDIC, and p150 dynactin colocalize to the interphase microtubule array and the MTOC in fibroblasts. The observed colocalization is confirmed biochemically by coimmunoprecipitation of NudC with CDIC and cytoplasmic dynein heavy chain (CDHC) from mouse brain extracts. Consistent with its expression in individual neurons, a high level of NudC is detected in regions of the embryonic neocortex undergoing extensive neurogenesis as well as neuronal migration. These data suggest a biochemical and functional interaction of NudC with Lis1 and the dynein motor complex during neuronal migration in vivo.

The mouse mammary tumor virus promoter has been shown to be inducible by glucocorticoids and progesterone. Although steroid hormone receptors bind with high affinity to palindromic response elements, the hormoneresponsive region of the mouse mammary tumor virus promoter contains a pair of directly repeated half-sites that are important for hormone inducibility. Recent experiments have also indicated that direct repeats can function as estrogen response elements. Here, we have investigated DNA binding by steroid receptors to direct repeats and provide evidence using gel retardation assays, methylation interference, and gene transfer experiments that direct repeats of TGTTCT or RGGTCA motifs function as response elements for glucocorticoid (GR) or estrogen receptors (ER), respectively, by binding receptor homodimers. Specific GR-or ER-DNA complexes were observed on direct repeats with different spacings between half-sites, indicating that binding of steroid receptors to direct repeats is more flexible than binding to palindromic elements. This flexibility was further emphasized by the observation that the GR could also bind to everted repeats of TGTTCT motifs separated by 9 base pairs. The isolated DNA binding domains of the GR and ER bound cooperatively to palindromes, but no evidence was observed for cooperative binding to direct repeats. Under similar conditions the DNA binding domains of retinoid receptors retinoid X receptor and retinoic acid receptor bound to direct repeats cooperatively as heterodimers. Similarly, ER derivative HE15, which lacks a functional ligand binding domain, bound palindromic response elements but failed to bind direct repeats. These results indicate that the dimerization domain in the ligand binding domain is essential for binding of steroid receptors to direct repeats and that the dimerization domain in the D-box of the DNA binding domain is not functional under these conditions. Moreover, the results suggest that steroid receptor DNA binding domains may lack dimerization domains outside the D-box, which would function in binding to direct repeats, in contrast to receptors for retinoids and thyroid hormone. A comparison of the mechanisms of binding of steroid receptors and retinoid and thyroid hormone receptors to direct repeats is presented.The nuclear receptors are a family of transcriptional enhancer factors that bind to specific DNA sequences in target promoters known as response elements (1-5). Specific members of the nuclear receptor family represent the primary intracellular targets for small lipid-soluble molecules, such as steroid and thyroid hormones, retinoids, and vitamin D 3 , and act as ligand-inducible transcriptional regulators. These ligand-inducible receptors control a wide spectrum of developmental and physiological processes through modulating the transcription of target genes.Sequence analyses of nuclear receptors have shown that they are composed of a series of conserved domains (1-6). The DNA binding domain (region C) is the most highly conserved and contains ...

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The 90-kDa heat shock protein (hsp90) has been implicated in modulating steroid receptor function in vitro and in vivo. Previous studies have suggested that hsp90 interacts with large portions of the estrogen receptor (ER) ligand-binding domain and sequences of the receptor required for stable DNA binding. To characterize the interaction of the ER ligand-binding domain with hsp90, we have compared the properties of chimeras created by coupling the ligand-binding domain to the constitutive transactivator VP16-GAL. Two types of chimeras were created: VP16-GAL-ER G , containing the wild-type ligand-binding domain derived from the cDNA HEG0, and VP16-GAL-ER V , containing the substitution G400V derived from the ligand-binding domain of the original ER cDNA isolate, HE0. The G400V mutation alters the physical properties of VP16-GAL-ER V by rendering it hormone-dependent for DNA binding and more strongly dependent on estradiol for transactivation compared with VP16-GAL-ER G . Glycerol gradient analyses and chemical cross-linking/coimmunoprecipitation showed that, unlike VP16-GAL-ER G , VP16-GAL-ER V formed stable complexes with hsp90 in vitro. These data show that hsp90 selectively recognizes the altered ER ligand-binding domain containing the G400V substitution and indicate that the wild-type ER ligand-binding domain of VP16-GAL-ER G does not interact with hsp90 in vitro. Hormone binding studies showed that the ligand-binding domain of VP16-GAL-ER V was destabilized by incubation in the presence of high concentrations of salt or in the absence of sodium molybdate, conditions that disrupt its interaction with hsp90. The ligand-binding domain of the Val-400 ER thus behaves similarly to that of the wild-type glucocorticoid receptor, which has previously been shown to interact with hsp90 in vitro. These results provide evidence for the action of hsp90 as a molecular chaperone by selectively recognizing destabilized proteins.In the absence of hormone, the estrogen receptor (ER), 1 similar to all steroid receptors, has been shown to form heteromeric complexes with accessory proteins both in vivo and in vitro (1-10). Among these accessory proteins are at least two members of the heat shock protein (hsp) family, hsp90 and hsp70, as well as members of the immunophilin family and several other receptor-associated proteins. It is thought that ligand-free steroid receptors interact directly with hsp90, stabilizing the receptor in an inactive state.The ER is a member of a large family of nuclear receptors and, as such, is a hormone-dependent transcriptional regulator. The ER has been subdivided into six functionally distinct domains on the basis of sequence homology between receptors from different species (see Fig. 1) (11,12). The highly conserved DNA-binding domain, encoded by region C, is composed of two zinc finger motifs, each with four conserved cysteine residues (13,14). In addition to amino acids controlling site-specific DNA binding, region C contains a dimerization interface (15). Region D contains amino acids required ...

The ligand-free estrogen receptor (ER), like other steroid receptors, interacts with the 90-kDa heat shock protein hsp90 in vitro. Analysis of the effect of potential ER-hsp90 interactions in vivo on receptor function is complicated by the fact that hsp90 binds to ER domains required for hormone binding and stable DNA binding. ER chimeras were therefore created by replacing the ER DNA binding domain with that of GAL4. In addition, the N-terminal AF-1 domain of the ER was replaced with the strong constitutive activation domain of VP16 to create VP16-GAL-ERs. These chimeras bind DNA in a ligandindependent manner, but, importantly, are ligand-dependent transactivators, unlike VP16-GAL, which displays strong constitutive activity under the same conditions. Hormone induces transactivation by VP16-GAL-ERs to levels similar to the constitutive activity of VP16-GAL. Glycerol gradient and coimmunoprecipitation experiments showed that, unlike the wild-type ER, VP16-GAL-ER chimeras do not interact with hsp90. Deletion analyses indicate that a region of the ER, primarily between amino acids 370 and 470, is responsible for repressed transcription. Our results suggest that inter-actionwithhsp90isnotnecessaryforcontrollinghormonedependent transcription by the ER and provide evidence for repressor factors that interact with the N-terminal portion of the receptor's ligand binding domain in the absence of hormone.