How Lipids Regulate the Cell Surface Localization of HSPA1A, a Stress‐inducible 70‐kDa Heat Shock Protein

HSPA1A is a molecular chaperone that regulates survival stressed and cancer cells. In addition to its cytosolic pro-survival functions, HSPA1A also localizes at the plasma membrane (PM) of stressed and tumor cells. In cancer cells, the presence of HSPA1A at the cell surface allows the latter cells to develop resistance to radiation therapy, show increased invasiveness, and develop distant metastasis. Therefore, abolishing HSPA1A from the surface of tumor cells is a promising therapeutic. However, HSPA1A's pathway to the cell surface remains enigmatic because this protein lacks membrane localization signals. Considering that HSPA1A binds with high selectivity to negatively charged lipids, like phosphatidylserine (PS) and mono-phosphorylated phosphoinositides (PIPs), we hypothesized that the interaction of HSPA1A with these lipids allows the chaperone to translocate and anchor to the PM. To test this hypothesis, we subjected human cell lines to heat shock, depleted specific lipid targets, and quantified HSPA1A's PM localization using confocal microscopy and cell surface biotinylation. These experiments revealed that depletion of PS, PI(4)P, and PI(3)P by co-transfecting HSPA1A with known lipid-biosensors, significantly reduced HSPA1A's surface presentation. In contrast, reducing PI(4,5)P2 availability by overexpression of the PLCδ-PH biosensor had minimal effects on HSPA1A's PM-localization. Next, we manipulated the cellular lipid content using ionomycin (a PLC activator), phenylarsine oxide (PAO) and GSK-A1 (general and a selective PI4-Kinase inhibitors, respectively), wortmannin (a PI3-Kinase inhibitor), and fendiline (a repurposed FDA approved drug that reduces PS) using lipid-biosensors as positive and negative controls. These experiments revealed that HSPA1A's PM localization was unaffected by ionomycin but was greatly reduced in the presence of PAO, GSK-A1, wortmannin, and fendiline, corroborating the findings obtained by the co-transfection experiments. We further verified these results by selectively depleting PI(4,5)P2 and PI(4)P using a well-established rapamycin-induced phosphatase system. Our findings strongly support the notion that HSPA1A's surface presentation is a multifaceted lipid-driven phenomenon controlled by the binding of the chaperone to specific endosomal and plasma membrane lipids. These findings provide the basis for future interventions to render tumor cells sensitive to radiation therapy.