Cell surface translocation of the chaperone glucose‐regulated protein 78 kDa (GRP78) is a key event that promotes cancer cell survival during endoplasmic reticulum (ER) stress. Here, we identify Gα‐interacting vesicle‐associated protein (GIV) – an enhancer of prosurvival signaling during ER stress – as a binding partner of GRP78. We show that GIV and GRP78 interact in an ER stress‐dependent manner through their respective carboxyl terminal domains and that GIV aids in the localization of GRP78 to the plasma membrane. Kaplan–Meier analysis of disease‐free survival in cancer patients shows poor prognosis for patients with high expression of both GIV and GRP78, further suggesting a vital role for these two proteins in enhancing cancer cell viability.

Cyclin-dependent kinase 5 (CDK5) is a serine/threonine kinase essential for embryonic development whose overactivation has been implicated in several pathologies including neurodegeneration, cancer cell metastasis and type II diabetes. Therefore, it is important to investigate molecular mechanism(s) that mediate regulation of CDK5 activity. Here we identify and characterize a novel phosphoregulatory site on CDK5. Our mass spectrometry analysis identified seven putative phosphorylation sites on CDK5. Using phosphomimetic and non-phosphorylatable mutants, we determined that phosphorylation of S47, one of the identified sites, renders the kinase catalytically inactive. The inactivation of the kinase due to the phosphomimetic change at S47 results from inhibition of its interaction with its cognate activator, p35. We connect the effect of this regulatory event to a cellular phenotype by showing that the S47D CDK5 mutant inhibits cell migration and promotes cell proliferation. Together, these results have uncovered a potential physiological mechanism to regulate CDK5 activity. The evolutionary placement of a phosphorylatable residue (S/T) at this position not only in CDK5 but also in the majority of other CDK family members suggests that this phosphosite may represent a shared regulatory mechanism across the CDK family.

Exosomes are a subtype of extracellular vesicles (EV) that are secreted upon fusion of multivesicular bodies (MVBs) with the plasma membrane. In order to identify proteins responsible for this fusion event, we developed sensitive cellular and cell-free assays to measure exosome secretion. Our results suggest that exosome secretion is coupled to Ca2+-dependent plasma membrane repair. Using a targeted proteomics approach, we identify annexin A6 (ANXA6), a well-known plasma membrane repair protein, as a MVB binding protein and demonstrate that it is required for Ca2+-dependent exosome secretion both in intact and in permeabilized cells. Our results suggest that cells employ MVBs as a membrane source for plasma membrane lesion repair during physiological mechanical stress or treatment with a bacterial pore-forming toxin, and that this repair process results in the coincident secretion of exosomes. We propose that this unconventional secretion process may contribute significantly to the heterogeneity of EVs in biological fluids.

Endoplasmic Reticulum (ER) stress occurs when misfolded proteins accumulate in the ER. Cells facing ER stress initially attempt to restore cellular homeostasis, but if the stress becomes chronic and homeostasis is not achieved within a reasonable timeframe, cells initiate programmed cell death. Cancer cells are able to withstand and survive ER stress better than regular cells. Overexpression of the chaperone Glucose Regulated Protein 78 kDa (GRP78) is one of the key factors that promote cancer cell survival during ER stress. Although it is an ER resident protein, recent studies have shown that when overexpressed, GRP78 can translocate to other cellular locations including the cell surface from where it can activate cytoprotective signals; however, the mechanism by which it does so remains elusive. Our laboratory has recently identified Gα‐Interacting Vesicle associated protein (GIV) ‐ a known enhancer of the pro‐survival Akt signaling ‐ as a novel binding partner of GRP78. We hypothesize that this interaction may be the missing link in understanding the mechanism by which cell surface GRP78 increases cell viability. Because GIV and GRP78 are normally localized in different cellular compartments, we characterized the spatiotemporal dynamics of this interaction by analyzing the sub‐cellular localization of these proteins in HeLa cells treated with Tunicamycin (an ER stress inducer) through differential centrifugation and immunofluorescence microscopy. Our results showed that GIV is present in both membrane and cytosolic fractions during normal as well as ER stress conditions. GRP78, on the other hand, is in the membrane fraction under normal cellular conditions but appears in the cytosolic fraction upon ER stress. GIV and GRP78 co‐localized in a perinuclear region as well as at the cell surface of the ER stressed cells. Finally, we also mapped the interaction between GIV and GRP78 to the carboxyl terminus domains on both proteins by performing pull‐down assays. Taken together, this work has characterized GIV‐GRP78 interaction in the face of ER stress. Our current and future goals include studying the role of this novel interaction in mediating cell survival during chronic ER stress.Support or Funding InformationThis project is supported by the NIH‐NIGMS grant # SC2GM121246 (DB). JN is supported by the NIH MARC program award number: T34GM008074.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.