Abstract
Heat shock protein 90 (HSP90) is a molecular chaperone that is up-regulated in cancer and is required for the folding of numerous signaling proteins. Consequently, HSP90 represents an ideal target for the development of new anti-cancer agents. The human HSP90 isoform, glucose-regulated protein 94 (GRP94), resides in the endoplasmic reticulum and regulates secretory pathways, integrins, and Toll-like receptors, which contribute to regulating immunity and metastasis. However, the cellular function of GRP94 remains underinvestigated. We report that GRP94 knockdown cells are defective in intracellular transport and, consequently, negatively impact the trafficking of F-actin toward the cellular cortex, integrin α2 and integrin αL toward the cell membrane and filopodia, and secretory vesicles containing the HSP90α-AHA1-survivin complex toward the leading edge. As a result, GRP94 knockdown cells form a multipolar spindle instead of bipolar morphology and consequently manifest a defect in cell migration and adhesion.
Highlights
Cell migration is the process by which cells move during developmental morphogenesis, tissue repair and regeneration, and cancer metastasis
We report that glucose-regulated protein 94 (GRP94) knockdown cells are defective in intracellular transport and, negatively impact the trafficking of F-actin toward the cellular cortex, integrin ␣2 and integrin ␣L toward the cell membrane and filopodia, and secretory vesicles containing the Heat shock protein 90 (HSP90)␣-AHA1-survivin complex toward the leading edge
Whereas wild type and control cells migrated toward the wound and closed the gap within 24 h, the GRP94 knockdown cells manifested a defect in cell migration and were unable to move efficiently toward the wound (Fig. 1A)
Summary
The novobiocin analogue, KU-135, was synthesized as described previously, and purity was verified as Ͼ95% by NMR and mass spectrometry [14]. AHA1-GFP plasmid was obtained from Origene (Rockville, MD) Canine GRP94-KDEL-GFP plasmid was a kind gift from Dr Yair Argon [15]. The plasmids were transfected into PC3-MM2 wild type and GRP94 knockdown cell lines using Lipofectamine 2000 as described by the manufacturer. 2808) (Cell Signaling Technology, Danvers, MA); goat anti-HSP90 Sc1616-R) (Santa Cruz Biotechnology, Inc.); mouse anti-TRAP1 Ab13048) (detects RAC1 with slight cross-reactivity with RAC2), and rabbit anti-integrin ␣2
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