Unanchored K63-linked polyubiquitin chains: a novel second messenger involved in G protein-coupled receptors early signaling events

GPCR rapidly transduced extracellular cues via their ability to activate effector proteins leading to the production of a variety of well-characterized second messenger molecules. We describe here that the E3 ubiquitin ligase tumor necrosis factor receptor associated factor 6 (TRAF6) is required for the rapid and transient production of unanchored lysine (K)63-linked polyubiquitin chains in primary and transformed cells exposed to vasoactive agonists Angiotensin II (Ang II), Lysophosphatidic acid (LPA) and Thrombin (Thr). We further show that unanchored K63-linked polyubiquitin chains accumulate in Transforming Growth Factor beta-activated kinase 1 (TAK1) and IkappaB kinase beta (IKKbeta) immunocomplexes and are required for the T-loop phosphorylation of TAK1 leading to IKKbeta, c- Jun N-terminal kinases (JNK1/2) and p38 mitogen-activated protein kinases (p38) phosphorylation and the induction of transcriptional events. Our results support a novel paradigm in field of GPCR signal transduction, identifying unanchored K63-linked polyubiquitin chains as novel second messenger molecules. Overall design: RNASeq analysis conducted on HaCat cells stimulated with Lysophosphatidic acid (LPA). Production of stable HaCaT expressing FLAG 4x ZnF UBP : Platinum-A (Plat-A) retroviral packaging cell line (Cells BIOLABS) were transfected with the pMRX-ires-puro vector or with the pMRX-ires-puro Flag 4x ZnF UBP vector using Lipofectamine 3000 Transfection Reagent (Invitrogen) according to the manufacturer's instruction. The retroviral pseudoviruses were collected 48 hours after transfection and passed through a 0.45 µm filter. 1 600 000 HaCaT cells were infected with 2 ml of virus containing 10 mg/ml of polybrene for 48 hours and selected for 4 days with 1.5 µg/ml of puromycin, creating stable populations expressing either the pMRX-ires-puro vector or the pMRX-ires-puro Flag 4x ZnF UBP. 2) Stimulations: Serum-starved cells were stimulated with LPA (10 µM) for 15, 30 and 60 min or left untreated. RNA was extracted and submitted to RNASeq analysis