AK2 is an AMP-sensing negative regulator of BRAF in tumorigenesis.

The RAS-BRAF signaling is a major pathway of cell proliferation and their mutations are frequently found in human cancers. Adenylate kinase 2 (AK2), which modulates balance of adenine nucleotide pool, has been implicated in cell death and cell proliferation independently of its enzyme activity. Recently, the role of AK2 in tumorigenesis was in part elucidated in some cancer types including lung adenocarcinoma and breast cancer, but the underlying mechanism is not clear. Here, we show that AK2 is a BRAF-suppressor. In in vitro assays and cell model, AK2 interacted with BRAF and inhibited BRAF activity and downstream ERK phosphorylation. Energy-deprived conditions in cell model and the addition of AMP to cell lysates strengthened the AK2-BRAF interaction, suggesting that AK2 is involved in the regulation of BRAF activity in response to cell metabolic state. AMP facilitated the AK2-BRAF complex formation through binding to AK2. In a panel of HCC cell lines, AK2 expression was inversely correlated with ERK/MAPK activation, and AK2-knockdown or -knockout increased BRAF activity and promoted cell proliferation. Tumors from HCC patients showed low-AK2 protein expression and increased ERK activation compared to non-tumor tissues and the downregulation of AK2 was also verified by two microarray datasets (TCGA-LIHC and GSE14520). Moreover, AK2/BRAF interaction was abrogated by RAS activation in in vitro assay and cell model and in a mouse model of HRASG12V-driven HCC, and AK2 ablation promoted tumor growth and BRAF activity. AK2 also bound to BRAF inhibitor-insensitive BRAF mutants and attenuated their activities. These findings indicate that AK2 monitoring cellular AMP levels is indeed a negative regulator of BRAF, linking the metabolic status to tumor growth.

Casein kinase-1γ1 and 3 stimulate tumor necrosis factor-induced necroptosis through RIPK3.

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 (CK1γ) as a candidate necroptosis-promoting factor. Here, we show that the decreased activity or amounts of CK1γ1 and CK1γ3, either by treatment with a chemical inhibitor or knockdown in cells, reduced TNFα-induced necroptosis. Conversely, ectopic expression of CK1γ1 or CK1γ3 exacerbated necroptosis, but not apoptosis. Similar to RIPK1 and RIPK3, CK1γ1 was also cleaved at Asp343 by caspase-8 during apoptosis. CK1γ1 and CK1γ3 formed a protein complex and were recruited to the necrosome harboring RIPK1, RIPK3 and MLKL. In particular, an autophosphorylated form of CK1γ3 at Ser344/345 was detected in the necrosome and was required to mediate the necroptosis. In addition, in vitro assays with purified proteins showed that CK1γ phosphorylated RIPK3, affecting its activity, and in vivo assays showed that the CK1γ-specific inhibitor Gi prevented abrupt death in mice with hypothermia in a model of TNFα-induced systemic inflammatory response syndrome. Collectively, these data suggest that CK1γ1 and CK1γ3 are required for TNFα-induced necroptosis likely by regulating RIPK3.