Analysis of the interaction of FLAG-TIFA with TRAF6, TRAF2 and c-IAP1 by co-immunoprecipitation v1

ALPK1 (alpha-kinase 1) is an atypical protein kinase that is activated during infection. In particular, ALPK1 is activated by binding of nucleoside-diphosphate-heptoses such as ADP-heptose to its N-terminal domain, enabling ALPK1 to phosphorylate TIFA (TRAF-interacting protein with FHA domain) at Thr9. This induces the interaction of phosphorylated Thr9 with the forkhead-associated domain of another TIFA molecule, leading to the “head-to-tail” polymerisation of TIFA into “TIFAsomes” that recruit TRAF6 (tumour necrosis factor receptor-associated factor 6) and TRAF2, and TRAF2 in turn recruits c-IAP1 (cellular inhibitor of apoptosis protein 1). TRAF6 and c-IAP1 are E3 ligases that generate the Lys63-linked ubiquitin chains required to recruit and activate TAK1 (transforming growth factor β-activated kinase 1), which in turn activates MAPK (mitogen-activated protein kinase) cascades that lead to the activation of JNK (c-Jun N-terminal kinases). One role of JNK is to activate the transcription factor AP-1 (activator protein 1). Another function of TAK1 is to activate the canonical IκB kinase (IKK) complex, which activates the transcription factor NF-κB (nuclear Factor kappa-light-chain-enhancer of activated B cells). In this protocol, FLAG-tagged wildtype and mutant or truncated forms of TIFA are overexpressed in TIFA knockout HEK-Blue cells and their interaction with TRAF6, TRAF2 and c-IAP1 assessed by co-immunoprecipitation.