A Novel Human Dynactin-Associated Protein, dynAP, Promotes Activation of Akt, and Ergosterol-Related Compounds Induce dynAP-Dependent Apoptosis of Human Cancer Cells

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 Ser 473 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.

Loss of CAMSAP3 promotes EMT via the modification of microtubule–Akt machinery

“…Furthermore, αTAT1 overexpression was sufficient for Akt activation and mesenchymal marker upregulation. In addition, dynactin was involved in CAMSAP3 knockout-mediated processes for Akt activation and EMT induction, which agreed with the previous finding that microtubule-dependent Akt activation is mediated by dynactin or its associated proteins (Jo et al, 2014;Kunoh et al, 2010). These observations indicate that Akt activity is elevated via CAMSAP3 knockout-induced increased tubulin acetylation, thus prompting cells to undergo EMT.…”

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

“…Parental NIH3T3 cells expressing EGFP and NIH3T3H-Ras cells (NIH3T3 cells expressing EGFP and mutant H-RasG12V) were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 4.5g/l glucose (Nacalai Tesque, Kyoto, Japan) and 10% fetal calf serum (FCS) (JRH Biosciences, St. Louis, MO, USA). The human cell lines and media used in this study have been described previously [ 11 ].…”

“…Antibodies were purchased from indicated sources: anti-Akt, anti-phospho-AktSer47, anti-S6K, anti-sphopho-S6KThr389, anti-4E-BP1, and anti-FOXO3a from Abcam (Cambridge, MA, USA); anti-phospho-4E-BP1Ser65, and anti-phospho-FOXO3Ser253 from Cell Signaling Technology (Danvers, MA, USA); anti-p53, anti-rictor, anti-β-actin, and anti-E-cadherin from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The anti-dynAP antibody was prepared as described previously [ 11 ]. A goat anti-rabbit IgG conjugated to horseradish peroxidase (MBL, Nagoya, Japan) was used as a secondary antibody.…”

“…RT-PCR was performed as described previously [ 11 ]. Briefly, total RNA was extracted using an RNeasy kit (Qiagen, Chatsworth, CA, USA) according to the manufacturer's protocol.…”

“…Previously, we reported that the human C18orf26 gene encodes a protein that is expressed in half of the tested human cancer cell lines but barley in normal cells [ 11 ]. This protein was designated as dynAP (dynactin-associating protein) because of its interaction with dynactin subunits that compose a microtubule-based motor protein complex.…”