The adaptor protein CRK is a pro-apoptotic transducer of endoplasmic reticulum stress.

Excessive demands on the protein-folding capacity of the endoplasmic reticulum (ER) cause irremediable ER stress and contribute to cell loss in a number of cell degenerative diseases, including type 2 diabetes and neurodegeneration. The signals communicating catastrophic ER damage to the mitochondrial apoptotic machinery remain poorly understood. We used a biochemical approach to purify a cytosolic activity induced by ER stress that causes release of cytochrome c from isolated mitochondria. We discovered that the principal component of the purified pro-apoptotic activity is the proto-oncoprotein CRK (CT10-regulated kinase), an adaptor protein with no known catalytic activity. Crk(-/-) cells are strongly resistant to ER-stress-induced apoptosis. Moreover, CRK is cleaved in response to ER stress to generate an amino-terminal M(r)~14K fragment with greatly enhanced cytotoxic potential. We identified a putative BH3 (BCL2 homology 3) domain within this N-terminal CRK fragment, which sensitizes isolated mitochondria to cytochrome c release and when mutated significantly reduces the apoptotic activity of CRK in vivo. Together these results identify CRK as a pro-apoptotic protein that signals irremediable ER stress to the mitochondrial execution machinery

Dissecting the ER stress-induced Apoptotic Pathway

The endoplasmic reticulum (ER) is the main site in the cell for the folding and processing of secreted proteins. Various physiological and pathological processes can overwhelm the protein folding capacity of the ER. This condition, referred to as "ER stress, typically triggers the activation of the Unfolded Protein Response Pathway (UPR). The activation of this pathway results in expansion of the ER, an increase in chaperone proteins, inhibition of cap-dependent translation, and if homeostasis is not reestablished, apoptosis. ER stress-induced apoptosis, secondary to a buildup of misfolded proteins, leads to cell loss in a number of human degenerative diseases including ALS, Parkinson, and type II diabetes. In contrast, cancer cells and viruses manipulate the cytoprotective aspects of the UPR, aberrantly promoting cell survival. Therefore, understanding how to manipulate the UPR's cytoprotective/cytotoxic threshold could lead to new treatment approaches for a wide variety of unrelated diseases.Despite its important role in such a wide variety of diseases, the mechanisms underlying ER stress-induced apoptosis are poorly understood. Utilizing an unbiased biochemical system, I have identified the ER stress-induced pro-apoptotic effectors that signal Bax and Bak at the mitochondrion, BID and CRK, and the mechanisms behind their activation up irremediable ER stress. In addition, the study of viral manipulation of host machinery has historically provided great insight into cellular biology. Viral infection is a biologically relevant system in which to study the cytoprotective/cytotoxic threshold of the UPR. A large lytic virus, such as Kaposi's Sarcoma Herpes Virus (KSHV or HHV-8), must be able to increase the folding capacity of the ER in order to accommodate the acute influx of viral protein during replication, while coordinately inhibiting the apoptotic consequences of intense ER stress. In this study, I have determined that KSHV inhibits UPR signaling through the IRE1 transmembrane kinase/endoribonuclease during lytic infection and is able to expand the ER through a novel function. These findings reveal new insights into the cytoprotective/cytotoxic regulation of the UPR and novel functions of lytic KSHV. These findings have implications for many diseases aside from viral infection, including type II diabetes, cancer, and neurodegeneration

The adaptor protein CRK is a pro-apoptotic transducer of endoplasmic reticulum stress.

Excessive demands on the protein-folding capacity of the endoplasmic reticulum (ER) cause irremediable ER stress and contribute to cell loss in a number of cell degenerative diseases, including type 2 diabetes and neurodegeneration. The signals communicating catastrophic ER damage to the mitochondrial apoptotic machinery remain poorly understood. We used a biochemical approach to purify a cytosolic activity induced by ER stress that causes release of cytochrome c from isolated mitochondria. We discovered that the principal component of the purified pro-apoptotic activity is the proto-oncoprotein CRK (CT10-regulated kinase), an adaptor protein with no known catalytic activity. Crk(-/-) cells are strongly resistant to ER-stress-induced apoptosis. Moreover, CRK is cleaved in response to ER stress to generate an amino-terminal M(r)~14K fragment with greatly enhanced cytotoxic potential. We identified a putative BH3 (BCL2 homology 3) domain within this N-terminal CRK fragment, which sensitizes isolated mitochondria to cytochrome c release and when mutated significantly reduces the apoptotic activity of CRK in vivo. Together these results identify CRK as a pro-apoptotic protein that signals irremediable ER stress to the mitochondrial execution machinery

Caspase-2 Cleavage of BID Is a Critical Apoptotic Signal Downstream of Endoplasmic Reticulum Stress▿

The accumulation of misfolded proteins stresses the endoplasmic reticulum (ER) and triggers cell death through activation of the multidomain proapoptotic BCL-2 proteins BAX and BAK at the outer mitochondrial membrane. The signaling events that connect ER stress with the mitochondrial apoptotic machinery remain unclear, despite evidence that deregulation of this pathway contributes to cell loss in many human degenerative diseases. In order to “trap” and identify the apoptotic signals upstream of mitochondrial permeabilization, we challenged Bax−/− Bak−/− mouse embryonic fibroblasts with pharmacological inducers of ER stress. We found that ER stress induces proteolytic activation of the BH3-only protein BID as a critical apoptotic switch. Moreover, we identified caspase-2 as the premitochondrial protease that cleaves BID in response to ER stress and showed that resistance to ER stress-induced apoptosis can be conferred by inhibiting caspase-2 activity. Our work defines a novel signaling pathway that couples the ER and mitochondria and establishes a principal apoptotic effector downstream of ER stress

Immune evasion mediated by tumor-derived lactate dehydrogenase induction of NKG2D ligands on myeloid cells in glioblastoma patients.

Myeloid cells are key regulators of the tumor microenvironment, governing local immune responses. Here we report that tumor-infiltrating myeloid cells and circulating monocytes in patients with glioblastoma multiforme (GBM) express ligands for activating the Natural killer group 2, member D (NKG2D) receptor, which cause down-regulation of NKG2D on natural killer (NK) cells. Tumor-infiltrating NK cells isolated from GBM patients fail to lyse NKG2D ligand-expressing tumor cells. We demonstrate that lactate dehydrogenase (LDH) isoform 5 secreted by glioblastoma cells induces NKG2D ligands on monocytes isolated from healthy individuals. Furthermore, sera from GBM patients contain elevated amounts of LDH, which correlate with expression of NKG2D ligands on their autologous circulating monocytes. NKG2D ligands also are present on circulating monocytes isolated from patients with breast, prostate, and hepatitis C virus-induced hepatocellular carcinomas. Together, these findings reveal a previously unidentified immune evasion strategy whereby tumors produce soluble factors that induce NKG2D ligands on myeloid cells, subverting antitumor immune responses

The adaptor protein CRK is a pro-apoptotic transducer of endoplasmic reticulum stress

Excessive demands on the protein-folding capacity of the endoplasmic reticulum (ER) cause irremediable ER stress and contribute to cell loss in a number of cell degenerative diseases, including type 2 diabetes and neurodegeneration. The signals communicating catastrophic ER damage to the mitochondrial apoptotic machinery remain poorly understood. We used a biochemical approach to purify a cytosolic activity induced by ER stress that causes release of cytochrome c from isolated mitochondria. We discovered that the principal component of the purified pro-apoptotic activity is the proto-oncoprotein CRK (CT10-regulated kinase), an adaptor protein with no known catalytic activity. Crk(-/-) cells are strongly resistant to ER-stress-induced apoptosis. Moreover, CRK is cleaved in response to ER stress to generate an amino-terminal M(r)~14K fragment with greatly enhanced cytotoxic potential. We identified a putative BH3 (BCL2 homology 3) domain within this N-terminal CRK fragment, which sensitizes isolated mitochondria to cytochrome c release and when mutated significantly reduces the apoptotic activity of CRK in vivo. Together these results identify CRK as a pro-apoptotic protein that signals irremediable ER stress to the mitochondrial execution machinery