AKAP13, a RhoA GTPase-specific Guanine Exchange Factor, Is a Novel Regulator of TLR2 Signaling

Oren Shibolet,Cosmas Giallourakis,Ian Rosenberg,Tobias Mueller,Ramnik J Xavier,Daniel K Podolsky

doi:10.1074/jbc.m704426200

Oren Shibolet, Cosmas Giallourakis + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m704426200

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Abstract

Members of the guanine exchange factor (GEF) family of scaffold proteins are involved in the integration of signal flow downstream of many receptors in adaptive immunity. However, the full complement of GEFs that function downstream of Toll-like receptors (TLRs) requires further identification and functional understanding. By systematically integrating expression profiles from immune and epithelial cells with functional studies, we demonstrate that protein kinase A anchoring protein 13 (AKAP13), a scaffold protein with GEF activity, is an activator of NF-kappaB downstream of TLR2 signaling. Stimulation of the human macrophage cell line THP-1 and epithelial cells with a TLR2 ligand caused a significant up-regulation in AKAP13 mRNA, corresponding to an increase in protein expression. Analysis of TLR2 reporter cell lines deficient in AKAP13 expression revealed significantly reduced NF-kappaB activation and reduced secretion of interleukin-8 and MCP-1 in response to specific ligand stimulation. Furthermore, NF-kappaB activation was partially inhibited by a GEF-deficient AKAP13 mutant. AKAP13 was also involved in phosphorylation of JNK but not of extracellular signal-regulated kinase ERK1 and -2 following ligand stimulation. Together, our results suggest that AKAP13 plays a role in TLR2-mediated NF-kappaB activation and suggest that GEF-containing scaffold proteins may confer specificity to innate immune responses downstream of TLRs.

Highlights

Toll-like receptors (TLRs) can selectively activate the MyD88-dependent pathway (TLR1, -2, -5, -6, -7, -8, and -9), MyD88-independent pathway (TLR3), or both (TLR4) [2] and are localized on intracellular organelles and/or the cell membrane [3, 4]
We show that AKAP13, a scaffold protein that is a member of the A-kinase anchoring protein family and has guanine exchange factor (GEF) activity, is induced by TLR2 ligands and mediates nuclear factor ␬B (NF-␬B) activation via TLR2
Regulator—A combined computational and experimental approach outlined in Fig. 1 was developed in order to identify GEFs in the human genome that might act as modulators of TLR signaling

Summary

Introduction

TLRs can selectively activate the MyD88-dependent pathway (TLR1, -2, -5, -6, -7, -8, and -9), MyD88-independent pathway (TLR3), or both (TLR4) [2] and are localized on intracellular organelles and/or the cell membrane [3, 4]. Similar results were obtained when AKAP13 expression was assessed following TLR2 ligand stimulation in HEK293 cells stably transfected with TLR2 (HEK-TLR2) (data not shown). By integrating protein domain analysis and gene expression profiles on a genome-wide scale for RhoGEFs, we hypothesized that AKAP13 may be involved in TLR2 signaling, and we focused on elucidating its potential function.

Results

Conclusion