Casein kinase-1 gamma 1 and 3 stimulate tumor necrosis factor-induced necroptosis through RIPK3

Abstract

Upon necroptosis activation, receptor interacting serine/threonine kinase (RIPK)1 and RIPK3 form a necrosome complex with pseudokinase mixed lineage kinase-like (MLKL). Although protein phosphorylation is a key event for RIPK1 and RIPK3 activation in response to a necroptosis signal, relatively little is known about other factors that might regulate the activity of these kinases or necrosome formation. Through a gain-of-function screen with 546 kinases and 127 phosphatases, we identified casein kinase 1 gamma (CK1 gamma) as a candidate necroptosis-promoting factor. Here, we show that the decreased activity or amounts of CK1 gamma 1 and CK1 gamma 3, either by treatment with a chemical inhibitor or knockdown in cells, reduced TNF alpha-induced necroptosis. Conversely, ectopic expression of CK1 gamma 1 or CK1 gamma 3 exacerbated necroptosis, but not apoptosis. Similar to RIPK1 and RIPK3, CK1 gamma 1 was also cleaved at Asp(343) by caspase-8 during apoptosis. CK1 gamma 1 and CK1 gamma 3 formed a protein complex and were recruited to the necrosome harboring RIPK1, RIPK3 and MLKL. In particular, an autophosphorylated form of CK1 gamma 3 at Ser(344)(/345) was detected in the necrosome and was required to mediate the necroptosis. In addition, in vitro assays with purified proteins showed that CK1 gamma phosphorylated RIPK3, affecting its activity, and in vivo assays showed that the CK1 gamma-specific inhibitor Gi prevented abrupt death in mice with hypothermia in a model of TNF alpha-induced systemic inflammatory response syndrome. Collectively, these data suggest that CK1 gamma 1 and CK1 gamma 3 are required for TNF alpha-induced necroptosis likely by regulating RIPK3.