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S369 phosphorylation of RIPK3 by PLK1 during G2/M phases enables its apoptotic function in the ripoptosome independent of necroptosis
Author(s) -
Gupta Kartik,
Liu Bo
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.07417
Subject(s) - necroptosis , ripk1 , plk1 , microbiology and biotechnology , programmed cell death , cleavage (geology) , phosphorylation , kinase , mitosis , fadd , caspase 8 , chemistry , nlrp1 , caspase , biology , apoptosis , biochemistry , cell cycle , paleontology , fracture (geology)
Receptor interacting protein kinase 3 (RIPK3) regulates a form of regulated necrosis called necroptosis. When an altered conformation is induced by via mutations in its kinase site or using chemical inhibitors, RIPK3 associates with an ensemble of proteins called the ripoptosome, containing FADD, RIPK1, Caspase 8 and cFLIP. RIPK3 now becomes decisive in the execution of apoptosis. Paradoxically, in contexts not completely understood, the ripoptosome cleaves RIPK3, highlighting a knowledge gap on how RIPK3 fulfills its role via the ripoptosome which is responsible for its own degradation. Recently, ripoptosome assembly was shown to occur physiologically during mitosis. We found RIPK3 levels to be elevated during the G2/M phases of the cell‐cycle, allowing us to study the mechanism by which RIPK3 resists degradation by the ripoptosome. We now report that polo‐like kinase 1 (PLK1) associates with RIPK3 and phosphorylates it at serine 369 before cells enter mitosis. RIPK3 in this phase has pro‐apoptotic activity but when released from ripoptosome, can elicit necroptosis. Disruption of the PLK1 site via T368A and S369A mutations causes degradation of RIPK3. RIPK3‐degradation can be prevented by the addition of pan‐caspase inhibitor zVAD.fmk, via a cis‐mutation in caspase 8 cleavage site i.e. D333A, but not by PLK1 over‐expression, suggesting that the S369 site regulates the apposing caspase cleavage site (D333) in cis‐manner. Additionally, S369D mutation on RIPK3 prevents its degradation via the ripoptosome whereas TS>>AA version of T368 and S369 is degraded. Together, phosphorylation of RIPK3 at S369 prevents its cleavage in the ripoptosome, thereby retaining its pro‐death activity during G2/M phases. The shift of cell‐death towards RIPK3 may have evolved as a mechanism by which the responsibility of balancing necroptosis vs apoptosis in mitotic cells converges on RIPK3. Support or Funding Information This study was supported by the National Institute of Health R01HL088447 (BL) and R01HL122562 (BL), and American Heart Association 17PRE33670082 (KG).