title: Unambiguous characterization of site-specific phosphorylation of leucine-rich repeat Fli-I-interacting protein 2 (LRRFIP2) in Toll-like receptor 4 (TLR4)-mediated signaling.
abstract: In the TLR4 signaling pathways, we previously characterized a signal regulator, LRRFIP2, that modulates the time course-dependent changes in NF-κB activity through its dynamic interaction with the TLR adaptor protein, MyD88. However, little is known about the driving force behind the LPS-inducible dynamics between LRRFIP2 and MyD88. We have therefore designed a multiplex label-free quantitative proteomics method to investigate dynamic changes of LRRFIP2 phosphorylation upon LPS stimulation. Given our observation that LRRFIP2 binds to MyD88 through its serine-rich domain in which most of serine residues have the propensity to be phosphorylated, we used collision-activated dissociation- and electron transfer dissociation-based methods in a complementary manner to unambiguously localize phosphorylation sites in the peptides constituting the serine-rich domain. Among 23 phosphorylation sites identified and first quantified by the label-free approach and then verified by the AACT/SILAC (amino acid-coded tagging/stable isotope labeling in cell culture)-based quantitation method, phosphorylation at serine 202 showed a significant LPS-induced dynamic change during the full-course cellular response to LPS stimulation. The substitution of serine 202 with nonphosphorylated residues by site-directed mutagenesis resulted in a weakened LRRFIP2-MyD88 interaction and a concurrently reduced activity in downstream NF-κB. Taking these results together, phosphorylation at serine 202 was found to regulate the dynamics of the LRRFIP2-MyD88 interaction, which in turn modulated the strength and duration of TLR4 signaling. Strategically, we have demonstrated the importance of precise identification of the biologically relevant phosphorylation site(s) using comprehensive mass spectrometry-based quantitative proteomics approaches in guiding downstream biological characterization experiments, which could otherwise be both time- and cost-consuming for a large number of phosphorylation possibilities.
authors.affiliation: Program in Molecular Biology and Biotechnology, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
authors.forename: Harsha P
authors.initials: HP
authors.lastname: Gunawardena
authors.forename: Yi
authors.initials: Y
authors.lastname: Huang
authors.forename: Roma
authors.initials: R
authors.lastname: Kenjale
authors.forename: Haiyang
authors.initials: H
authors.lastname: Wang
authors.forename: Ling
authors.initials: L
authors.lastname: Xie
authors.forename: Xian
authors.initials: X
authors.lastname: Chen
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology; D051379:Mice; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D053594:Myeloid Differentiation Factor 88 / Q000235:genetics / Q000378:metabolism; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D011485:Protein Binding / Q000187:drug effects / Q000235:genetics; D017434:Protein Structure, Tertiary; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D051197:Toll-Like Receptor 4 / Q000819:agonists / Q000235:genetics / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; C120550:LRRFIP2 protein, human; D008070:Lipopolysaccharides; C507779:MYD88 protein, human; C507780:Myd88 protein, mouse; D053594:Myeloid Differentiation Factor 88; D016328:NF-kappa B; C495345:TLR4 protein, human; C493487:Tlr4 protein, mouse; D051197:Toll-Like Receptor 4; C440499:lipopolysaccharide, Escherichia coli O111 B4