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Quantitative proteomics reveals novel interaction partners of Rac1 in pancreatic ?-cells: Evidence for increased interaction with Rac1 under hyperglycemic conditions.

ABSTRACT: Rac1, a small G protein, regulates physiological insulin secretion from the pancreatic ?-cell. Interestingly, Rac1 has also been implicated in the onset of metabolic dysfunction of the ?-cell under the duress of hyperglycemia (HG). This study is aimed at the identification of interaction partners of Rac1 in ?-cells under basal and HG conditions. Using co-immunoprecipitation and UPLC-ESI-MS/MS, we identified 324 Rac1 interaction partners in INS-1832/13?cells, which represent the largest Rac1 interactome to date. Furthermore, we identified 27 interaction partners that exhibited increased association with Rac1 in ?-cells exposed to HG. Western blotting (INS-1832/13?cells, rat islets and human islets) and co-immunoprecipitation (INS-1832/13?cells) further validated the identity of these Rac1 interaction partners including regulators of GPCR-G protein-effector coupling in the islet. These data form the basis for future investigations on contributory roles of these Rac1-specific signaling pathways in islet ?-cell function in health and diabetes.

SUBMITTER: Damacharla D

PROVIDER: S-EPMC6686664 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Quantitative proteomics reveals novel interaction partners of Rac1 in pancreatic β-cells: Evidence for increased interaction with Rac1 under hyperglycemic conditions.

Damacharla Divyasri D Thamilselvan Vijayalakshmi V Zhang Xiangmin X Mestareehi Aktham A Yi Zhengping Z Kowluru Anjaneyulu A

Molecular and cellular endocrinology 20190613

Rac1, a small G protein, regulates physiological insulin secretion from the pancreatic β-cell. Interestingly, Rac1 has also been implicated in the onset of metabolic dysfunction of the β-cell under the duress of hyperglycemia (HG). This study is aimed at the identification of interaction partners of Rac1 in β-cells under basal and HG conditions. Using co-immunoprecipitation and UPLC-ESI-MS/MS, we identified 324 Rac1 interaction partners in INS-1832/13 cells, which represent the largest Rac1 inte ...[more]

PMID: 31202817

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Project description:Background/aimsWe recently reported increased phosphorylation (at S536) of the p65 subunit of NFκB (Rel A) in pancreatic beta (INS-1 832/13) cells following exposure to hyperglycemic (HG) conditions. We also demonstrated that HG-induced S536 phosphorylation of p65 is downstream to the regulatory effects of CARD9 since deletion of CARD9 expression significantly attenuated HG-induced S536 phosphorylation of p65 in beta cells. The overall objective of the current investigation is to identify putative mechanisms underlying HG-induced phosphorylation of p65 in islet beta cells following exposure to HG conditions.MethodsINS-1 832/13 cells were incubated in low glucose (LG; 2.5 mM) or high glucose (HG; 20 mM) containing media for 24 hours in the absence or presence of small molecule inhibitors of G protein prenylation and activation. Non-nuclear and nuclear fractions were isolated from INS-1 832/13 cells using a commercially available (NE-PER) kit. Degree of S536 phosphorylation of the p65 subunit was quantified by western blotting and densitometry.ResultsHG-induced p65 phosphorylation was significantly attenuated by inhibitors of protein prenylation (e.g., simvastatin and L-788,123). Pharmacological inhibition of Tiam1-Rac1 (e.g., NSC23766) and Vav2-Rac1 (e.g., Ehop-016) signaling pathways exerted minimal effects on HG-induced p65 phosphorylation. However, EHT-1864, a small molecule compound, which binds to Rac1 thereby preventing GDP/GTP exchange, markedly suppressed HG-induced p65 phosphorylation, suggesting that Rac1 activation is requisite for HG-mediated p65 phosphorylation. Lastly, EHT-1864 significantly inhibited nuclear association of STAT3, but not total p65, in INS-1 832/13 cells exposed to HG conditions.ConclusionActivation of Rac1, a step downstream to HG-induced activation of CARD9, might represent a requisite signaling step in the cascade of events leading to HG-induced S536 phosphorylation of p65 and nuclear association of STAT3 in pancreatic beta cells. Data from these investigations further affirm the role(s) of Rac1 as a mediator of metabolic stress- induced dysfunction of the islet beta cell.

Dataset Information

Quantitative proteomics reveals novel interaction partners of Rac1 in pancreatic ?-cells: Evidence for increased interaction with Rac1 under hyperglycemic conditions.

Publications

Quantitative proteomics reveals novel interaction partners of Rac1 in pancreatic β-cells: Evidence for increased interaction with Rac1 under hyperglycemic conditions.

Similar Datasets

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