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.

Protein acetylation and deacetylation play key roles in multiple physiological functions. Histone deacetylase 3 (HDAC3) is a highly conserved, ubiquitously expressed protein that forms multiprotein corepressor complexes to repress gene transcription. Recent studies show that HDAC3 may play a role in cell proliferation. Altered HDAC3 level increases G(2)/M cells, but the mechanism remains unknown. Here we show for the first time, to our knowledge, that the HDAC3 complex, including nuclear receptor corepressor (N-CoR), transducin-beta-like protein 1 (TBL1), and TBL1-related protein 1 (TBLR1), is localized on the mitotic spindle. Knockdown of HDAC3 or N-CoR resulted in a collapsed mitotic spindle that was surrounded by chromosomes arranged in a dome-like configuration. Treatment of mitotic cells with Trichostatin A, an HDAC inhibitor, resulted in similar spindle defects independent of transcriptional regulation. In addition, wild-type HDAC3 but not a deacetylase-dead mutant HDAC3 rescued the phenotypes of HDAC3-depleted cells, suggesting that the enzymatic activity of HDAC3 is important for proper spindle function. Whereas the kinetochores and the spindle assembly checkpoint appeared intact in HDAC3-deficient cells, kinetochore-microtubule attachments were impaired because spindle microtubules were unstable in response to cold treatment. These data suggest that the HDAC3 complex is involved in the formation of functional mitotic spindles and proper kinetochore-microtubule attachment. The level or distribution of acetylated alpha-tubulin was not altered in HDAC3-deficient cells. Taken together, our studies raise the interesting possibility that acetylation-deacetylation of mitotic spindle components may be essential for mitotic spindle function.

Bone metastasis from prostate cancer can occur years after prostatectomy, due to reactivation of dormant disseminated tumor cells (DTC) in the bone, yet the mechanism by which DTCs are initially induced into a dormant state in the bone remains to be elucidated. We show here that the bone microenvironment confers dormancy to C4-2B4 prostate cancer cells, as they become dormant when injected into mouse femurs but not under the skin. Live-cell imaging of dormant cells at the single-cell level revealed that conditioned medium from differentiated, but not undifferentiated, osteoblasts induced C4-2B4 cellular quiescence, suggesting that differentiated osteoblasts present locally around the tumor cells in the bone conferred dormancy to prostate cancer cells. Gene array analyses identified GDF10 and TGFβ2 among osteoblast-secreted proteins that induced quiescence of C4-2B4, C4-2b, and PC3-mm2, but not 22RV1 or BPH-1 cells, indicating prostate cancer tumor cells differ in their dormancy response. TGFβ2 and GDF10 induced dormancy through TGFβRIII to activate phospho-p38MAPK, which phosphorylates retinoblastoma (RB) at the novel N-terminal S249/T252 sites to block prostate cancer cell proliferation. Consistently, expression of dominant-negative p38MAPK in C4-2b and C4-2B4 prostate cancer cell lines abolished tumor cell dormancy both and Lower TGFβRIII expression in patients with prostate cancer correlated with increased metastatic potential and decreased survival rates. Together, our results identify a dormancy mechanism by which DTCs are induced into a dormant state through TGFβRIII-p38MAPK-pS249/pT252-RB signaling and offer a rationale for developing strategies to prevent prostate cancer recurrence in the bone. These findings provide mechanistic insights into the dormancy of metastatic prostate cancer in the bone and offer a rationale for developing strategies to prevent prostate cancer recurrence in the bone. .

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.