Effect of the actin- and calcium-regulating activities of ITPKB on the metastatic potential of lung cancer cells.

Inositol-1,4,5-trisphosphate 3-kinase-A (ITPKA) exhibits oncogenic activity in lung cancer cells by regulating Ins(1,4,5)P3-mediated calcium release and cytoskeletal dynamics. Since, in normal cells, ITPKA is mainly expressed in the brain, it is an excellent target for selected therapy of lung cancer. However, ITPKB is strongly expressed in normal lung tissues, but is down-regulated in lung cancer cells by miR-375, assuming that ITPKB might have tumor suppressor activity. In addition, ITPKB binds to F-actin making it likely that, similar to ITPKA, it controls actin dynamics. Thus, the treatment of ITPKA-expressing lung cancer with ITPKA inhibitors simultaneously inhibiting ITPKB may counteract the therapy. Based on these considerations, we analyzed if ITPKB controls actin dynamics and if the protein reduces aggressive progression of lung cancer cells. We found that ITPKB bundled F-actin in cell-free systems. However, the stable expression of ITPKB in H1299 lung cancer cells, exhibiting very low endogenous ITPKB expression, had no significant effect on the actin structure. In addition, our data show that ITPKB negatively controls transmigration of H1299 cells in vitro by blocking Ins(1,4,5)P3-mediated calcium release. On the other hand, colony formation was stimulated by ITPKB, independent of Ins(1,4,5)P3-mediated calcium signals. However, dissemination of H1299 cells from the skin to the lung in NOD scid gamma mice was not significantly affected by ITPKB expression. In summary, ITPKB does not affect the cellular actin structure and does not suppress dissemination of human lung cancer cells in mice. Thus, our initial hypotheses that ITPKB exhibits tumor suppressor activity could not be supported.

A Cell-Based Assay for Mitotic Spindle Orientation.

The regulation of mitotic spindle orientation is essential to ensure proper cell division and development (Kiyomitsua and Cheeseman Nat Cell Biol 14:311-317, 2012). For identification of potential spindle orientation regulators, determination of the mitotic spindle angle is a well-known but time-consuming procedure. Here we describe a simple and time-saving phenotypic screening assay for the identification of potential spindle orientation regulators. This screen is based on the analysis of monopolar mitotic spindle structures, which form upon inhibition of the mitotic kinesin Eg5/KSP by the small-molecule inhibitor dimethylenastron (DME) or similar compounds.

The Vps39-like TRAP1 is an effector of Rab5 and likely the missing Vps3 subunit of human CORVET.

Membrane fusion in the endocytic pathway is mediated by a protein machinery consistent of Rab GTPases, tethering factors and SNAREs. In yeast, the endosomal CORVET and lysosomal HOPS tethering complexes share 4 of their 6 subunits. The 2 additional subunits in each complex - Vps3 and Vps8 for CORVET, and the homologous Vps39 and Vps41 for HOPS - bind directly to Rab5 and Rab7, respectively. In humans, all subunits for HOPS have been described. However, human CORVET remains poorly characterized and a homolog of Vps3 is still missing. Here we characterize 2 previously identified Vps39 isoforms, hVps39-1/hVam6/TLP and hVps39-2/TRAP1, in yeast and HEK293 cells. None of them can compensate the loss of the endogenous yeast Vps39, though the specific interaction of hVps39-1 with the virus-specific LT protein was reproduced. Both human Vps39 proteins show a cytosolic localization in yeast and mammalian cells. However, hVps39-2/TRAP1 strongly co-localizes with co-expressed Rab5 and interacts directly with Rab5-GTP in vitro. We conclude that hVps39-2/TRAP1 is an endosomal protein and an effector of Rab5, suggesting a role of the protein as a subunit of the putative human CORVET complex.