Human Dynactin-Associated Protein Transforms NIH3T3 Cells to Generate Highly Vascularized Tumors with Weak Cell-Cell Interaction.

Human dynactin-associated protein (dynAP) is a transmembrane protein that promotes AktSer473 phosphorylation. Here, we report the oncogenic properties of dynAP. In contrast to control NIH3T3 cells expressing LacZ (NIH3T3LacZ), NIH3T3dynAP cells vigorously formed foci in two-dimensional culture, colonies on soft agar, and spheroids in anchorage-deficient three-dimensional culture. NIH3T3dynAP cells injected into nude mice produced tumors with abundant blood vessels and weak cell-cell contacts. Expression of dynAP elevated the level of rictor (an essential subunit of mTORC2) and promoted phosphorylation of FOXO3aSer253. FOXO3a is a transcriptional factor that stimulates expression of pro-apoptotic genes and phosphorylation of FOXO3a abrogates its function, resulting in promoted cell survival. Knockdown of rictor in NIH3T3dynAP cells reduced AktSer473 phosphorylation and formation of foci, colony in soft agar and spheroid, indicating that dynAP-induced activation of the mTORC2/AktSer473 pathway for cell survival contributes to cell transformation. E-cadherin and its mRNA were markedly reduced upon expression of dynAP, giving rise to cells with higher motility, which may be responsible for the weak cell-cell adhesion in tumors. Thus, dynAP could be a new oncoprotein and a target for cancer therapy.

A novel human dynactin-associated protein, dynAP, promotes activation of Akt, and ergosterol-related compounds induce dynAP-dependent apoptosis of human cancer cells.

There are several human genes that may encode proteins whose functions remain unknown. To find clues to their functions, we used the mutant yeast defective in Mad2, a component of the spindle checkpoint complex. Phenotypes that were provoked by the expression of a human C18orf26 protein in the mutant yeast encouraged further characterization of this protein in human cells. This protein was designated dynAP (dynactin-associated protein) because of its interaction with dynactin subunits that comprised a microtubule-based motor protein complex. The dynAP is a transmembrane protein localizing to Golgi apparatus and plasma membrane in a microtubule-dependent manner. This protein was expressed in half of human cancer cell lines but barely in normal human fibroblasts tested. The SV40-transformed fibroblasts expressed dynAP. Importantly, the expression of dynAP activated Akt (also known as protein kinase B) by promoting Ser47³ phosphorylation required for the full activation, whereas knockdown of dynAP abolished this activation. The ergosterol-related compounds identified by the yeast cell-based high-throughput screen abrogated activation of Akt and induced apoptosis in a dynAP-dependent manner. We propose a possible advantage of dynAP expression in cancer cells; the survival of cancer cells that express dynAP is supported by dynAP-induced activation of Akt, sustaining high rates of proliferation. The inactivation of dynAP by the selected compounds nullifies this advantage, and thereby, the apoptotic machinery is allowed to operate. Taken together, dynAP can be a new target for cancer therapy, and the selected chemicals are useful for developing a new class of anticancer drugs.

Comprehensive screening of human genes with inhibitory effects on yeast growth and validation of a yeast cell-based system for screening chemicals.

To evaluate yeast as a high-throughput cell-based system for screening chemicals that may lead to drug development, 10,302 full-length human cDNAs (~50% of the total cDNAs) were introduced into yeast. Approximately 5.6% (583 clones) of the cDNAs repressed the growth of yeast. Notably, ~25% of the repressive cDNAs encoded uncharacterized proteins. Small chemicals can be readily surveyed by monitoring their restorative effects on the growth of yeast. The authors focused on protein kinases because protein kinases are involved in various diseases. Among 263 protein kinase cDNAs (~50% of the total) expressed in yeast, 60 cDNAs (~23%), including c-Yes, a member of the Src tyrosine kinase family, inhibited the growth of yeast. Known inhibitors for protein kinases were examined for whether they reversed the c-Yes-induced inhibition of the yeast growth. Among 85 inhibitors tested, 6 compounds (PP2, PP1, SU6656, purvalanol, radicicol, and geldanamycin) reversed the inhibition, indicating a high specificity sufficient for validating this screening system. Human c-Yes was found to interact with Hsc82, one of the yeast chaperones. Radicicol and geldanamycin probably exerted their actions through interactions with Hsc82. These results indicate that when human proteins requiring molecular chaperones for their activities are subjected to the yeast screening system, 2 groups of chemicals may be found. The actions of one group are exerted through direct interactions with the human proteins, whereas those of the other group are mediated through interactions with chaperones.