Abstract
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.
Highlights
As the simplest eukaryotic cells, yeast cells have been used to identify the functions of human proteins and to screen chemicals that can act as lead compounds for the development of therapeutic agents
We extended this screening to the mutant yeast defective in Mad2, which is an essential member of spindle checkpoint proteins that monitor the attachment of spindles to kinetochores with sufficient tension to avoid the missegregation of sister chromatids [6, 7]
Among several human proteins that inhibited the growth of the mutant yeast but not the wild-type yeast, C18orf26 protein, which we dubbed “dynAP,” repressed the growth of mutant yeasts deficient in other spindle checkpoint factors, Mad1, Mad3, and Bub3 (Supplementary Fig. S1A)
Summary
As the simplest eukaryotic cells, yeast cells have been used to identify the functions of human proteins and to screen chemicals that can act as lead compounds for the development of therapeutic agents. Many proteins linked with accelerated proliferation of human cancer cells repress the growth of yeast [1,2,3]. These observations have provided a cell-based high-throughput screen-. Ing system for anticancer drugs because inhibitors of human proteins can be screened by monitoring the restoration of yeast proliferation [4]. We speculate that if the growth of the Mad2-defective yeast is inhibited by expressing a human protein, this protein may be involved in cell proliferation events. Small chemicals that act on this protein and their properties are presented
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