Elsevier

Cellular Signalling

Volume 18, Issue 12, December 2006, Pages 2223-2229
Cellular Signalling

Identification of a regulatory autophosphorylation site in the serine–threonine kinase RIP2

https://doi.org/10.1016/j.cellsig.2006.05.005Get rights and content

Abstract

Receptor-interacting protein 2 (RIP2) is a serine–threonine kinase that mediates signaling for many receptors of the innate and adaptive immune systems. Toll like receptors (TLR) are an important component of the innate immune response. Stimulation of RIP2-deficient cells with ligands for TLR 2, 3 and 4 results in impaired cytokine production and decreased activation of NF-kB and MAP kinases compared to wild-type cells. Stimulation of TLR 4 with its ligand lipopolysaccaride (LPS) leads to the activation of RIP2 kinase activity and its autophosphorylation. Here we identify serine residue 176 as a site of autophosphorylation using a combination of mass spectrometry and mutational analysis. Mutation of S176 to alanine not only abolishes autophosphorylation of RIP2 but also significantly decreases its catalytic activity. A phospho-specific anti-S176 antibody detects wild-type RIP2 but not kinase-dead RIP2 or the RIP2 S176A mutant. Endogenous RIP2 in THP-1 cells and mouse bone marrow derived macrophages can be detected by the phospho-RIP2 (S176) antibody only after stimulation with LPS suggesting that the antibody recognizes activated RIP2. In summary, our results indicate that S176 is a regulatory autophosphorylation site for RIP2 and that S176 phosphorylation can be used to monitor the activation state of 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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