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14-3-3 Mediated regulation of the tumor suppressor protein, RASSF1A

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Abstract

Death receptor-dependent apoptosis is an important mechanism of growth control. It has been demonstrated that Ras association domain family protein 1A (RASSF1A) is a tumor suppressor protein involved in death receptor-dependent apoptosis. However, it is unclear how RASSF1A-mediated cell death is initiated. We have now detailed 14-3-3 dependent regulation of RASSF1A-mediated cell death. We demonstrate that basal association of RASSF1A with 14-3-3 was lost following stimulation with tumor necrosis factor alpha (TNFα) or TNFα related apoptosis inducing ligand (TRAIL). Subsequent to the loss of 14-3-3 association, RASSF1A associated with modulator of apoptosis (MOAP-1) followed by death receptor association with either TNFα receptor 1 (TNF-R1) or TRAIL receptor 1 (TRAIL-R1). 14-3-3 association required basal phosphorylation by the serine/threonine kinase, glycogen synthase kinase 3β (GSK-3β), on serine 175, 178, and 179. Mutation of these critical serines resulted in the loss of 14-3-3 association and earlier recruitment of RASSF1A to MOAP-1, TNF-R1, and TRAIL-R1. Furthermore, stable cells containing a triple serine mutant of RASSF1A [serine (S) 175 to alanine (A) [S175A], S178A, and S179A] resulted in increased basal cell death, enhanced Annexin V staining and enhanced cleavage of poly (ADP-ribose) polymerase (PARP) following TNFα stimulation when compared to stable cells containing wild type RASSF1A. RASSF1A-mediated cell death is, therefore, tightly controlled by 14-3-3 association.

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Acknowledgments

We would like to thank Christina Onyskiw for her excellent technical assistance with our experiments. We would like to thank Dr. Victor Yu and NaiYang Fu for the MOAP-1 expression construct; Dr. Gerd Pfeifer for the GST expression constructs for RASSF1A, 1C, and RASSF5; Dr. Yutaka Hata for human Myc-tagged RASSF6B; Dr. Andrew Chalmers for human HA-tagged RASSF7; and Dr. Michael Yaffe for the HA-14-3-3 expression constructs. Grant support for this study was provided by the University of Alberta, Faculty of Medicine and Department of Pediatrics (N031000425) (SB); by the Women and Children’s Health Research Institute (WCO14) (SB, HA-G, JDO), Canadian Institutes of Health Research (MOP-79494) (CO), Alberta Heritage Foundation for Medical Research (G220170170) (SB), and Canadian Foundation for Innovation/Alberta Small Equipment Grants Program (13118); and by the Office of the Provost and VP Academic Award (SLC, RCC, DP and RXW).

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Authors and Affiliations

  1. Department of Pediatrics, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, T6G 2N8, Canada

    Haya Abu Ghazaleh, Renfred S. Chow, Sheryl L. Choo, Diana Pham, Jamie D. Olesen, Russell X. Wong, Christina Onyskiw & Shairaz Baksh

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  1. Haya Abu Ghazaleh
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Correspondence to Shairaz Baksh.

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Supplementary material 1 (DOC 39 kb)

10495_2009_451_MOESM2_ESM.pdf

Supplemental Fig. S1 Specificity of RASSF1A/14-3-3 associations. a and b, HA (a) or GST (b) tagged RASSF proteins were ectopically expressed in U2OS cells. Associated RASSF isoforms were recovered by 14-3-3 immunoprecipitation (IP) followed by immunoblotting (IB) with the indicated antibodies. (PDF 119 kb)

10495_2009_451_MOESM3_ESM.pdf

Supplemental Fig. S2 Loss of RASSF1A self association with 14-3-3 binding site mutants. Green fluorescent protein (GFP) tagged RASSF1A and HA tagged RASSF1A WT or serine mutants were ectopically expressed in U2OS cells. Associated GFP-RASSF1A was recovered by immunoprecipitation with an anti-HA antibody followed by immunoblotting with the indicated antibodies. (PDF 14 kb)

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Ghazaleh, H.A., Chow, R.S., Choo, S.L. et al. 14-3-3 Mediated regulation of the tumor suppressor protein, RASSF1A. Apoptosis 15, 117–127 (2010). https://doi.org/10.1007/s10495-009-0451-6

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