Toxic effects of mutant huntingtin in axons are mediated by its proline-rich domain.

Huntington's disease (HD) results from expansion of a polyglutamine tract (polyQ) in mutant huntingtin (mHTT) protein, but mechanisms underlying polyQ expansion-mediated toxic gain-of-mHTT function remain elusive. Here, deletion and antibody-based experiments revealed that a proline-rich domain (PRD) adjacent to the polyQ tract is necessary for mutant huntingtin (mHTT) to inhibit fast axonal transport and promote axonal pathology in cultured mammalian neurons. Further, polypeptides corresponding to subregions of the PRD sufficed to elicit the toxic effect on fast axonal transport, which was mediated by JNK kinases and involved PRD binding to one or more SH3-domain containing proteins. Collectively, these data suggested a mechanism whereby polyQ tract expansion in mHTT promotes aberrant PRD exposure and interactions of this domain with SH3 domain-containing proteins including some involved in activation of JNK kinases. In support, biochemical and immunohistochemical experiments linked aberrant PRD exposure to increased JNK activation in striatal tissues of the zQ175 mouse model and from post-mortem HD patients. Collectively, these findings support a critical role of PRD on mHTT toxicity, suggesting a novel framework for the potential development of therapies aimed to halt or reduce axonal pathology in HD.

CDKN1A-mediated responsiveness of MLL-AF4-positive acute lymphoblastic leukemia to Aurora kinase-A inhibitors.

Overexpression of Aurora kinases is largely observed in many cancers, including hematologic malignancies. In this study, we investigated the effects and molecular mechanisms of Aurora kinase inhibitors in acute lymphoblastic leukemia (ALL). Western blot analysis showed that both Aurora-A and Aurora-B are overexpressed in ALL cell lines and primary ALL cells. Both VE-465 and VX-680 effectively inhibited Aurora kinase activities in nine ALL cell lines, which exhibited different susceptibilities to the inhibitors. Cells sensitive to Aurora kinase inhibitors underwent apoptosis at an IC50 of ∼10-30 nM and displayed a phenotype of Aurora-A inhibition, whereas cells resistant to Aurora kinase inhibitors (with an IC50 more than 10 µM) accumulated polyploidy, which may have resulted from Aurora-B inhibition. Drug susceptibility of ALL cell lines was not correlated with the expression level or activation status of Aurora kinases. Interestingly, RS4;11 and MV4;11 cells, which contain the MLL-AF4 gene, were both sensitive to Aurora kinase-A inhibitors treatment. Complementary DNA (cDNA) microarray analysis suggested that CDKN1A might govern the drug responsiveness of ALL cell lines in a TP53-independent manner. Most importantly, primary ALL cells with MLL-AF4 and CDKN1A expression were sensitive to Aurora kinase inhibitors. Our study suggests CDKN1A could be a potential biomarker in determining the drug responsiveness of Aurora kinase inhibitors in ALL, particularly in MLL-AF4-positive patients.

KIBRA regulates aurora kinase activity and is required for precise chromosome alignment during mitosis.

The Hippo pathway controls organ size and tumorigenesis by inhibiting cell proliferation and promoting apoptosis. KIBRA was recently identified as a novel regulator of the Hippo pathway. Several of the components of the Hippo pathway are important regulators of mitosis-related cell cycle events. We recently reported that KIBRA is phosphorylated by the mitotic kinases Aurora-A and -B. However, the role KIBRA plays in mitosis has not been established. Here, we show that KIBRA activates the Aurora kinases and is required for full activation of Aurora kinases during mitosis. KIBRA also promotes the phosphorylation of large tumor suppressor 2 (Lats2) on Ser(83) by activating Aurora-A, which controls Lats2 centrosome localization. However, Aurora-A is not required for KIBRA to associate with Lats2. We also found that Lats2 inhibits the Aurora-mediated phosphorylation of KIBRA on Ser(539), probably via regulating protein phosphatase 1. Consistent with playing a role in mitosis, siRNA-mediated knockdown of KIBRA causes mitotic abnormalities, including defects of spindle and centrosome formation and chromosome misalignment. We propose that the KIBRA-Aurora-Lats2 protein complexes form a novel axis that regulates precise mitosis.

A novel role for TPX2 as a scaffold and co-activator protein of the Chromosomal Passenger Complex.

Aurora B kinase forms the enzymatic core of the Chromosomal Passenger Complex (CPC) and is a master regulator of mitosis. Understanding the regulation of Aurora B is critical to illuminate its role in mitosis. INCENP, Survivin and Borealin have all been known to promote Aurora B activation. In this study, we have identified the Aurora A activator protein TPX2 as a novel scaffold and co-activator protein of the CPC. Studies utilizing M-phase Xenopus egg extracts (XEE) revealed that the immunodepletion of endogenous TPX2 from XEE decreases Aurora B-Survivin and Aurora B-INCENP interactions, leading to a consequent reduction in Aurora B activity. Further, residues 138 to 328 of Xenopus TPX2 (TPX2 B) are sufficient to enhance Aurora B-Survivin association and Aurora B kinase activity in vitro. Importantly, experiments with pancreatic cancer cell lines suggest that this mechanism of Aurora B activation by TPX2 is likely to be conserved in human cells. Strikingly, the overexpression of human TPX2 B in HeLa cells causes defects in metaphase chromosome alignment and INCENP localization. Thus, in addition to its already established role as an Aurora A activator, our data support the role of TPX2 as a novel co-activator of Aurora kinase B.

A novel Aurora kinase A inhibitor MK-8745 predicts TPX2 as a therapeutic biomarker in non-Hodgkin lymphoma cell lines.

Selective small-molecule kinase inhibitors have encouraging clinical efficacy in several malignancies. These agents are still limited to a subset of patients, indicating the need to develop therapeutic biomarkers that influence clinical benefit. In this study, we demonstrate that treatment with MK-8745, a novel Aurora-A specific inhibitor, leads to cell cycle arrest at the G2/M phase with accumulation of tetraploid nuclei followed by cell death in non-Hodgkin lymphoma (NHL) cell lines. The sensitivity of the cell lines to MK-8745 is correlated with the expression level of Aurora-A activator. The siRNA knockdown of Aurora-A activator TPX2 (targeting protein for Xenopus kinase-like protein 2) increased MK-8745 sensitivity in less-MK-8745-sensitive NHL cell lines, whereas overexpression of TPX2 in high-MK-8745-sensitive NHL cell lines increased drug resistance. Our results indicate that TPX2 may serve as a biomarker for identifying subpopulations of patients sensitive to Aurora-A inhibitor treatment.

KIBRA protein phosphorylation is regulated by mitotic kinase aurora and protein phosphatase 1.

Recent genetic studies in Drosophila identified Kibra as a novel regulator of the Hippo pathway, which controls tissue growth and tumorigenesis by inhibiting cell proliferation and promoting apoptosis. The cellular function and regulation of human KIBRA remain largely unclear. Here, we show that KIBRA is a phosphoprotein and that phosphorylation of KIBRA is regulated in a cell cycle-dependent manner with the highest level of phosphorylated KIBRA detected in mitosis. We further demonstrate that the mitotic kinases Aurora-A and -B phosphorylate KIBRA both in vitro and in vivo. We identified the highly conserved Ser(539) as the primary phosphorylation site for Aurora kinases. Moreover, we found that wild-type, but not catalytically inactive, protein phosphatase 1 (PP1) associates with KIBRA. PP1 dephosphorylated Aurora-phosphorylated KIBRA. KIBRA depletion impaired the interaction between Aurora-A and PP1. We also show that KIBRA associates with neurofibromatosis type 2/Merlin in a Ser(539) phosphorylation-dependent manner. Phosphorylation of KIBRA on Ser(539) plays a role in mitotic progression. Our results suggest that KIBRA is a physiological substrate of Aurora kinases and reveal a new avenue between KIBRA/Hippo signaling and the mitotic machinery.

What's Nu(SAP) in mitosis and cancer?

Unperturbed mitosis is a prerequisite for the generation of two genetically identical daughter cells. Nucleolar-spindle associated protein (NuSAP) is an important mitotic regulator. The activity of NuSAP is essential for a variety of cellular events that occur during mitosis starting from spindle assembly to cytokinesis. In addition to playing crucial roles during mitosis, NuSAP has been in the spotlight recently due to different studies exhibiting its importance in embryogenesis and cancer. In this review, we have extensively mined the current literature and made connections between different studies involving NuSAP. Importantly, we have assembled data pertaining to NuSAP from several proteomic studies and analyzed it thoroughly. Our review focuses on the role of NuSAP in mitosis and cancer, and brings to light several unanswered questions regarding the regulation of NuSAP in mitosis and its role in carcinogenesis.

Arpc1b, a centrosomal protein, is both an activator and substrate of Aurora A.

Here we provide evidence in support of an inherent role for Arpc1b, a component of the Arp2/3 complex, in regulation of mitosis and demonstrate that its depletion inhibits Aurora A activation at the centrosome and impairs the ability of mammalian cells to enter mitosis. We discovered that Arpc1b colocalizes with gamma-tubulin at centrosomes and stimulates Aurora A activity. Aurora A phosphorylates Arpc1b on threonine 21, and expression of Arpc1b but not a nonphosphorylatable Arpc1b mutant in mammalian cells leads to Aurora A kinase activation and abnormal centrosome amplification in a Pak1-independent manner. Together, these findings reveal a new function for Arpc1b in centrosomal homeostasis. Arpc1b is both a physiological activator and substrate of Aurora A kinase and these interactions help to maintain mitotic integrity in mammalian cells.

Inhibition of protein phosphorylation in MIA pancreatic cancer cells: confluence of metabolic and signaling pathways.

Oxythiamine (OT), a transketolase inhibitor, is known to inhibit pancreatic cancer cell proliferation. In this study, we investigated the effect of inhibition of the transketolase pathway on signaling pathways in MIA PaCa cancer cells using in-house proteomic techniques. We hypothesized that OT alter protein phosphorylation thus affecting cell cycle arrest and cell proliferation. MIA PaCa-2 cells were cultured in media containing an algal (15)N amino acid mixture at 50% enrichment, with and without OT, to determine protein expression and synthesis. Analysis of cell lysates using two-dimensional gel electrophoresis matrix assisted laser desorption and ionization time-of-flight and time-of-flight mass spectrometry (2-DE-MALDI-TOF/TOF MS) identified 12 phosphor proteins that were significantly suppressed by OT treatment. Many of these proteins are involved in regulation of cycle activities and apoptosis. Among the proteins identified, expression of the phosphor heat shock protein 27 (Hsp27) was dramatically inhibited by OT treatment while the level of its total protein remained unchanged. Hsp27 expression and phosphorylation is known to be associated with drug resistance and cancer cell survival. The changes in phosphorylation of key proteins of cancer proliferation and survival suggest that protein phosphorylation is the confluence of the effects of OT on metabolic and signaling pathways.

Requirement for Nudel and dynein for assembly of the lamin B spindle matrix.

The small guanosine triphosphatase Ran loaded with GTP (RanGTP) can stimulate assembly of the type V intermediate filament protein lamin B into a membranous lamin B spindle matrix, which is required for proper microtubule organization during spindle assembly. Microtubules in turn enhance assembly of the matrix. Here we report that the isolated matrix contains known spindle assembly factors such as dynein and Nudel. Using spindle assembly assays in Xenopus egg extracts, we show that Nudel regulates microtubule organization during spindle assembly independently of its function at kinetochores. Importantly, Nudel interacts directly with lamin B to facilitate the accumulation and assembly of lamin-B-containing matrix on microtubules in a dynein-dependent manner. Perturbing either Nudel or dynein inhibited the assembly of lamin B matrix. However, depleting lamin B still allowed the formation of matrices containing dynein and Nudel. Therefore, dynein and Nudel regulate assembly of the lamin B matrix. Interestingly, we found that whereas depleting lamin B resulted in disorganized spindle and spindle poles, disrupting the function of Nudel or dynein caused a complete lack of spindle pole focusing. We suggest that Nudel regulates microtubule organization in part by facilitating assembly of the lamin B spindle matrix in a dynein-dependent manner.

Aurora A kinase-coated beads function as microtubule-organizing centers and enhance RanGTP-induced spindle assembly.

The roles of the kinase Aurora A (AurA) in centrosome function and spindle assembly have been established in Drosophila, C. elegans, and Xenopus egg extracts . Recently, we have shown that AurA acts downstream of the RanGTPase signaling pathway to stimulate spindle assembly in mitosis . However, it is still not clear whether AurA can stimulate the formation of microtubule organizing centers (MTOC) on its own. Moreover, whether AurA is essential for spindle assembly in the absence of centrosomes has remained unclear . Here, we report the development of functional assays that allow us to show that activation of AurA by TPX2 is essential for Ran-stimulated spindle assembly in the presence or absence of centrosomes. Furthermore, AurA-coated magnetic beads function as MTOCs in the presence of RanGTP in Xenopus egg extracts and RanGTP stimulates AurA to recruit activities responsible for both MT nucleation and organization to the beads. The MTOC function of AurA-coated beads require both MT nucleators and motors. Compared to XMAP215-coated beads , AurA-coated beads increase the rate of bipolar spindle assembly in the presence of RanGTP, and the kinase activity of AurA is essential for the beads to function as MTOCs.