Identification of a regulatory autophosphorylation site in the serine–threonine kinase RIP2
Introduction
RIP2 was originally described as a serine/threonine kinase implicated in the activation of NF-kB and apoptosis [8], [16], [22]. Subsequent studies with RIP2-deficient mice have identified RIP2 as an important mediator of signaling in the immune system as many signaling pathways of the innate as well as the adaptive immune response are impaired in RIP2-deficient animals [2], [12], [21]. Stimulation of RIP2-deficient macrophages and fibroblast with ligands for TLR 2, 3, and 4 results in decreased cytokine production and activation of NF-kB and MAPK signaling. RIP2-deficient mice are resistant to LPS-induced septic shock [2], [12]. RIP2 deficiency also results in impaired signaling through the T cell receptor and the receptors for interleukin-18 and -12 [2], [12], [21].
RIP2 contains an N-terminal kinase domain followed by an intermediate region and a C-terminal caspase recruitment domain (CARD). Overexpression of RIP2 activates NF-kB and Jun N-terminal kinase and induces apoptosis in mammalian cell lines [8], [16], [22]. This activity is independent of the kinase activity as a kinase-dead mutant of RIP2 exerts the same effect [16], [22]. RIP2 also functions as adaptor molecule and interacts with numerous signaling molecules including TRAFs, IRAK and NEMO as well as toll like receptors [9], [14], [16], [17], [22]. The CARD domain mediates RIP2 association with other CARD domain containing molecules such as NOD1 and NOD2 [1], [19], [22]. NOD1 and NOD2 are cytoplasmic pathogen-recognition receptors that sense intracellular peptidoglycans [3], [10]. Mutations in NOD2 are associated with a subset of Crohn's disease [7], [18]. RIP2 appears to mediate the signaling triggered by stimulation of NODS, leading to activation of NF-kB [12].
Although RIP2 has been implicated in many inflammatory pathways little is known about the molecular mechanism of its function. Stimulation with LPS leads to the transient association of RIP2 with TLR4 as well as TRAF6 and IRAK [14]. It also results in activation of RIP2 kinase and autophosphorylation [14]. However, no endogenous substrates or upstream activating kinase(s) of RIP2 have been identified for the TLR pathway. RIP2 has been proposed to function primarily as a scaffolding protein rather than a kinase [17]. This is supported by our previous observations that RIP2 kinase activity is not required for the LPS signaling. Nevertheless, LPS stimulation leads to increased kinase activity and autophosphorylation [14]. In order to better understand the role of autophoshorylation for its function we set out to identify and characterize the autophosphorylation site(s) of RIP2.
Section snippets
Reagents
Myc-tagged human RIP2 vector, TRAF6 and IRAK1 expression vectors were described previously [14]. A kinase-dead mutant of RIP2 (RIP2 KD) was generated by mutating aspartic acid 146 to alanine [22] and cloned into the expression vector pTracer (Invitrogen, Carlsbad, CA). RIP2ΔCARD (residues 1–435) lacks the CARD domain and is expressed in the vector pCI (Promega, Madison, WI) with an N-terminal myc-tag [1]. In the RIP2ΔCARD S176A construct S176 was mutated to alanine. All constructs were verified
Purification of recombinant RIP2 protein
We have previously shown that LPS stimulation of THP-1 cells leads to an increase in RIP2 kinase activity as measured by autophosphorylation [14]. For many kinases autophoshorylation has been shown to play an important regulatory role for their function [11]. In order to identify the sites(s) of autophosphorylation of RIP2 we performed mass spectrometry analysis on recombinant protein subjected to in vitro autophosphorylation. The kinase domain of RIP2 (RIP2 1–311) was cloned into a
Discussion
Protein kinases are important players in many cellular processes and their activity needs to be tightly regulated. Regulation is achieved by a variety of mechanisms that include phosphorylation and control by additional regulatory domains or subunits [11]. Many kinases are activated by phosphorylation in a particular region in the center of the kinase domain, the so-called activation loop. The activation loop is defined as the region spanning the conserved sequence DFG of subdomain VII and APE
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2021, Clinical ImmunologyCitation Excerpt :In this newer work, the initial findings that NOD1/2 is involved in cell death pathways could not be confirmed. [5,67–69] RIP2 signaling depends on its N-terminal kinase domain, containing serine/threonine and tyrosine kinase activities [23,24], but also on its C-terminal CARD domain mediating CARD-CARD assembly with activated NOD1/2 [20,21] leading to [10,16,24,70–72] NF-κB activation and the release of pro-inflammatory cytokines [73,74]. The mechanism by which RIP2 regulates autophagy remains subject of controversial discussion [75,76].