The Fic Domain: Regulation of Cell Signaling by Adenylylation

Abstract

Summary We show that the secreted antigen, IbpA, of the respiratory pathogen Histophilus somni induces cytotoxicity in mammalian cells via its Fic domains. Fic domains are defined by a core HPFxxGNGR motif and are conserved from bacteria to humans. We demonstrate that the Fic domains of IbpA catalyze a unique reversible adenylylation event that uses ATP to add an adenosine monophosphate (AMP) moiety to a conserved tyrosine residue in the switch I region of Rho GTPases. This modification requires the conserved histidine of the Fic core motif and renders Rho GTPases inactive. We further demonstrate that the only human protein containing a Fic domain, HYPE (Huntingtin yeast-interacting protein E), also adenylylates Rho GTPases in vitro. Thus, Fic domain containing proteins are a new class of enzymes that mediate bacterial pathogenesis as well as a previously unrecognized eukaryotic post-translational modification that may regulate key signaling events.

A New Chemical Handle for Protein AMPylation at the Host–Pathogen Interface

“…The performance of N 6 pATP was also verified against a background of mammalian cell lysate. In accordance with previous reports, [9][10][11] in-gel fluorescence analysis of HeLa lysates treated with the probe and the abovementioned recombinant AMP transferases revealed labeling of a distinct band in the molecular weight range of small GTPases. Finally, in a preliminary experiment the authors applied their probe to mass spectrometry-based identification of an AMPylated substrate within a complex protein mixture.…”

“…[6][7][8] . Key bacterial AMPylators characterized to date include VopS, a cytotoxin of Vibrio parahaemolyticus, that AMPylates Rho family GTPases at a conserved threonine residue; [9] IbpA secreted by Histophilus somni, that AMPylates the same target protein but modifies a tyrosine residue instead of a threonine; [10] and finally DrrA (also known as SidM), a Legionella pneumophila effector that AMPylates Rab1 GTPases also at a tyrosine. [11] Both VopS and IbpA mediate AMPylation using a so-called Fido catalytic motif (canonical sequence: [12] and target the host cell cytoskeleton, whilst DrrA utilizes an adenylyl transferase domain (canonical sequence: Gx11DxD) [11] and targets host cell protein trafficking.…”

“…Bacterial Fido domain proteins are involved in cell division, whilst HYPE, a human Fido motif protein, may regulate interactions of small GTPases with their binding partners. [10] Furthermore, metabolic labeling experiments using 32 P-α-ATP show numerous radioactive protein bands in human cell lysates, suggesting the presence of additional AMPylating enzymes. [9] AMPylation is rapidly emerging as a fundamental mechanism serving to regulate protein-protein interactions and cell signaling in normal cells and during the invasion of mammalian cells by bacteria.…”

“…[15] In turn, AMPylated substrate proteins have been identified by the combination of radioactive isotope labeling and targeted mass spectrometric analysis both on the MS and MS/MS level. [9][10][11]16] Two additional developments that complement the above-mentioned approaches include the elucidation of mass spectrometric fragmentation characteristics of AMPylation based on the analysis of synthetic peptides modified at serine, threonine, and tyrosine residues, [17] and the generation of a polyclonal antibody specific towards threonineAMPylation. [18] In a very recent report, a potentially more general, chemistry-based methodology for labeling AMP transferase substrates has been proposed by Hang, Orth and coworkers.…”

Intrinsic regulation of FIC-domain AMP-transferases by oligomerization and automodification

“…FIC domains encode enzymatic activities that modulate target protein function by diverse posttranslational modifications (1,2). The vast majority of known Fic proteins are AMP-transferases that use ATP to catalyze the transfer of an AMP moiety onto a target hydroxyl side chain (3,4). This reaction is akin to the situation in protein kinases, which catalyze γ-phosphate transfer onto target side chains.…”