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Depletion of the adaptor protein NCK increases UV-induced p53 phosphorylation and promotes apoptosis.

ABSTRACT: The cellular response to DNA damage requires the coordination of many proteins involved in diverse molecular processes. Discrete molecular pathways are becoming increasingly well understood, but the interconnectivity and coordination of multiple pathways remains less clear. We now show that NCK, an adapter protein involved in cytoskeletal responses to tyrosine kinase receptor signaling, accumulates in the nucleus in response to DNA damage and this translocation can be blocked by specific inhibition of the ATR protein kinase. Strikingly, HeLa cells depleted of NCK undergo apoptosis shortly after UV irradiation, as monitored by caspase-3 cleavage and PARP cleavage. This rapid, hyperactive apoptosis in NCK depleted cells might be p53 dependent, because loss of NCK also increased UV-induced p53 phosphorylation. Importantly, depletion of SOCS7, which is necessary for NCK nuclear translocation, phenocopies NCK depletion, indicating the nuclear accumulation of NCK is responsible for these molecular events. There are two NCK isoforms that have mostly redundant functions, and although NCK2 appears to have a greater contribution, depletion of NCK1 or NCK2, led to increased p53 phosphorylation and early apoptosis after UV exposure. These data reveal a novel function for NCK in regulating p53 phosphorylation and apoptosis, and provide evidence for interconnectedness of growth factor signaling proteins and the DNA damage response.

SUBMITTER: Errington TM

PROVIDER: S-EPMC3781058 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Depletion of the adaptor protein NCK increases UV-induced p53 phosphorylation and promotes apoptosis.

Errington Timothy M TM Macara Ian G IG

PloS one 20130923 9

The cellular response to DNA damage requires the coordination of many proteins involved in diverse molecular processes. Discrete molecular pathways are becoming increasingly well understood, but the interconnectivity and coordination of multiple pathways remains less clear. We now show that NCK, an adapter protein involved in cytoskeletal responses to tyrosine kinase receptor signaling, accumulates in the nucleus in response to DNA damage and this translocation can be blocked by specific inhibit ...[more]

PMID: 24086708

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Project description:Background & aimsPancreatic acinar cells are polarized epithelial cells that store enzymes required for digestion as inactive zymogens, tightly packed at the cell apex. Stimulation of acinar cells causes the zymogen granules to fuse with the apical membrane, and the cells undergo exocytosis to release proteases into the intestinal lumen. Autophagy maintains homeostasis of pancreatic acini. Syntaxin 2 (STX2), an abundant soluble N-ethyl maleimide sensitive factor attachment protein receptor in pancreatic acini, has been reported to mediate apical exocytosis. Using human pancreatic tissues and STX2-knockout (KO) mice, we investigated the functions of STX2 in zymogen granule-mediated exocytosis and autophagy.MethodsWe obtained pancreatic tissues from 5 patients undergoing surgery for pancreatic cancer and prepared 80-μm slices; tissues were exposed to supramaximal cholecystokinin octapeptide (CCK-8) or ethanol and a low concentration of CCK-8 and analyzed by immunoblot and immunofluorescence analyses. STX2-KO mice and syntaxin 2+/+ C57BL6 mice (controls) were given intraperitoneal injections of supramaximal caerulein (a CCK-8 analogue) or fed ethanol and then given a low dose of caerulein to induce acute pancreatitis, or saline (controls); pancreata were isolated and analyzed by histology and immunohistochemistry. Acini were isolated from mice, incubated with CCK-8, and analyzed by immunofluorescence microscopy or used in immunoprecipitation experiments. Exocytosis was quantified using live-cell exocytosis and Ca2+ imaging analyses and based on formation of exocytotic soluble N-ethyl maleimide sensitive factor attachment protein receptor complexes. Dysregulations in autophagy were identified using markers, electron and immunofluorescence microscopy, and protease activation assays.ResultsHuman pancreatic tissues and dispersed pancreatic acini from control mice exposed to CCK-8 or ethanol plus CCK-8 were depleted of STX2. STX2-KO developed more severe pancreatitis after administration of supramaximal caerulein or a 6-week ethanol diet compared with control. Acini from STX2-KO mice had increased apical exocytosis after exposure to CCK-8, as well as increased basolateral exocytosis, which led to ectopic release of proteases. These increases in apical and basolateral exocytosis required increased formation of fusogenic soluble N-ethyl maleimide sensitive factor attachment protein receptor complexes, mediated by STX3 and STX4. STX2 bound ATG16L1 and prevented it from binding clathrin. Deletion of STX2 from acini increased binding of AT16L1 to clathrin, increasing formation of pre-autophagosomes and inducing autophagy. Induction of autophagy promoted the CCK-8-induced increase in autolysosome formation and the activation of trypsinogen.ConclusionsIn studies of human pancreatic tissues and pancreata from STX2-KO and control mice, we found STX2 to block STX3- and STX4-mediated fusion of zymogen granules with the plasma membrane and exocytosis and prevent binding of ATG16L1 to clathrin, which contributes to induction of autophagy. Exposure of pancreatic tissues to CCK-8 or ethanol depletes acinar cells of STX2, increasing basolateral exocytosis and promoting autophagy induction, leading to activation of trypsinogen.

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Depletion of the adaptor protein NCK increases UV-induced p53 phosphorylation and promotes apoptosis.

Publications

Depletion of the adaptor protein NCK increases UV-induced p53 phosphorylation and promotes apoptosis.

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