Innate immunity kinase TAK1 phosphorylates Rab1 on a hotspot for posttranslational modifications by host and pathogen

Abstract

TGF-β activated kinase 1 (TAK1) is a critical signaling hub responsible for translating antigen binding signals to immune receptors for the activation of the AP-1 and NF-κB master transcriptional programs. Despite its importance, known substrates of TAK1 are limited to kinases of the MAPK and IKK families and include no direct effectors of biochemical processes. Here, we identify over 200 substrates of TAK1 using a chemical genetic kinase strategy. We validate phosphorylation of the dynamic switch II region of GTPase Rab1, a mediator of endoplasmic reticulum to Golgi vesicular transport, at T75 to be regulated by TAK1 in vivo. TAK1 preferentially phosphorylates the inactive (GDP-bound) state of Rab1. Phosphorylation of Rab1 disrupts interaction with GDP dissociation inhibitor 1 (GDI1), but not guanine exchange factor (GEF) or GTPaseactivating protein (GAP) enzymes, and is exclusive to membranelocalized Rab1, suggesting phosphorylation may stimulate Rab1 membrane association. Furthermore, we found phosphorylation of Rab1 at T75 to be essential for Rab1 function. Previous studies established that the pathogen Legionella pneumophila is capable of hijacking Rab1 function through posttranslational modifications of the switch II region. Here, we present evidence that Rab1 is regulated by the host in a similar fashion, and that the innate immunity kinase TAK1 and Legionella effectors compete to regulate Rab1 by switch II modifications during infection.

PINK1-dependent phosphorylation of Serine111 within the SF3 motif of Rab GTPases impairs effector interactions and LRRK2 mediated phosphorylation at Threonine72

“…Our analysis also revealed that a number of other kinases can regulate Rab phosphorylation including MST3 and TAK1 that are Thr72 directed kinases and MSK1, ZAP70, PAK2, MAP4K5, MAPKAPK3, JNK, PBK, TAK1, and NEK6 whose sites of Rab phosphorylation are unknown. TAK1 has previously been shown to phosphorylate Thr72 in Rab1 and this is important for immunity against Legionella infection [29]. Our studies suggest that the mechanism by which LRRK2 phosphorylates Rab Thr72 is distinct from MST3 and TAK1, and in future it will be important to undertake NMR studies to address this.…”

PPM1H phosphatase counteracts LRRK2 signaling by selectively dephosphorylating Rab proteins

“…The PPM1H related, PPM1M enzyme ( Figure 5B) also dephosphorylated Rab10 when overexpressed in cells, to a lower extent than PPM1H ( Figure 5A). In corresponding biochemical studies, we also found that PPM1M displayed weak activity towards phosphorylated Rab10 ( [16], including Rab7A phosphorylated at Ser72 by TBK1 [51] or LRRK1 [52] and Rab1 that is phosphorylated at Thr75 by TAK1 [53]. It will be interesting to probe whether PPM1M, PPM1J or indeed PPM1H might dephosphorylate other Rab proteins.…”

cTag-PAPERCLIP Reveals Alternative Polyadenylation Promotes Cell-Type Specific Protein Diversity and Shifts Araf Isoforms with Microglia Activation

“…We also demonstrate that the Araf APA isoform switch is a common response for microglia/macrophage to multiple TLR ligands. Our results suggest that a detailed dissection of the molecular mechanism responsible for the Araf APA isoform switch will be of interest; we note that inhibition of TAK1, a MAP3K kinase controlling major inflammatory response pathways in microglia and macrophage (Goldmann et al, 2013; Levin et al, 2016), does not prevent the downregulation of Araf(S) in RAW264.7 cells (Fig. S7), suggesting that the p38, JNK and NF-kappaB pathways are not major contributors to the Araf isoform switch in this cell-type.…”