title: Lysine residues of IGF-I are substrates for transglutaminases and modulate downstream IGF-I signalling.
abstract: Numerous studies have reported associations between IGF-I and other extra cellular matrix (ECM) proteins, including fibronectin (FN), integrins, IGF-binding proteins (IGFBPs) and through IGFBPs, with vitronectin (VN). Nevertheless, the precise nature and mechanisms of these interactions are still being characterised. In this paper, we discuss transglutaminases (TGases) as a constituent of the ECM and provide evidence for the first time that IGF-I is a lysine (K)-donor substrate to TGases. When IGF-I was incubated with an alpha-2 plasmin inhibitor-derived Q peptide in the presence of tissue transglutaminase (TG2), an IGF-I:Q peptide cross-linked species was detected using Western immunoblotting and confirmed by mass spectrometry. Similar findings were observed in the presence of Factor XIIIa (FXIIIa) TGase. To identify the precise location of this K-donor TGase site/s on IGF-I, all the three IGF-I K-sites, individually and collectively (K27, K65 and K68), were substituted to arginine (R) using site-directed mutagenesis. Incubation of these K→R IGF-I analogues with Q peptide in the presence of TG2 or FXIIIa resulted in the absence of cross-linking in IGF-I analogues bearing arginine substitution at site 68. This established that K68 within the IGF-I D-domain was the principal K-donor site to TGases. We further annotated the functional significance of these K→R IGF-I analogues on IGF-I mediated actions. IGF-I analogues with K→R substitution within the D-domain at K65 and K68 hindered migration of MCF-7 breast carcinoma cells and correspondingly reduced PI3-K/AKT activation. Therefore, this study also provides first insights into a possible functional role of the previously uncharacterised IGF-I D-domain.
authors.affiliation: Queensland University of Technology, Faculty of Health, Institute of Health and Biomedical Innovation, Kelvin Grove, Brisbane, QLD 4059, Australia. Electronic address: manaswini.sivaramakrishnan@uqconnect.edu.au.
authors.forename: Manaswini
authors.initials: M
authors.lastname: Sivaramakrishnan
authors.affiliation: Queensland University of Technology, Faculty of Health, Institute of Health and Biomedical Innovation, Kelvin Grove, Brisbane, QLD 4059, Australia.
authors.forename: Tristan I
authors.initials: TI
authors.lastname: Croll
authors.affiliation: Queensland University of Technology, Faculty of Health, Institute of Health and Biomedical Innovation, Kelvin Grove, Brisbane, QLD 4059, Australia.
authors.forename: Rajesh
authors.initials: R
authors.lastname: Gupta
authors.affiliation: Queensland University of Technology, Faculty of Health, Institute of Health and Biomedical Innovation, Kelvin Grove, Brisbane, QLD 4059, Australia.
authors.forename: Dario
authors.initials: D
authors.lastname: Stupar
authors.affiliation: Queensland University of Technology, Faculty of Health, Institute of Health and Biomedical Innovation, Kelvin Grove, Brisbane, QLD 4059, Australia.
authors.forename: Derek R
authors.initials: DR
authors.lastname: Van Lonkhuyzen
authors.affiliation: Queensland University of Technology, Faculty of Health, Institute of Health and Biomedical Innovation, Kelvin Grove, Brisbane, QLD 4059, Australia.
authors.forename: Zee
authors.initials: Z
authors.lastname: Upton
authors.affiliation: Queensland University of Technology, Faculty of Health, Institute of Health and Biomedical Innovation, Kelvin Grove, Brisbane, QLD 4059, Australia.
authors.forename: Gary K
authors.initials: GK
authors.lastname: Shooter
mesh_terms: D000595:Amino Acid Sequence; D002465:Cell Movement / Q000187:drug effects; D003432:Cross-Linking Reagents / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D026122:Factor XIIIa / Q000378:metabolism; D006801:Humans; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000378:metabolism; D007334:Insulin-Like Growth Factor I / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008239:Lysine / Q000378:metabolism*; D061986:MCF-7 Cells; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D017526:Receptor, IGF Type 1 / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013329:Structure-Activity Relationship; D013379:Substrate Specificity / Q000187:drug effects; D020349:Surface Plasmon Resonance; D011503:Transglutaminases / Q000378:metabolism*; D019096:Vitronectin / Q000494:pharmacology
chemical_list: D003432:Cross-Linking Reagents; D018972:Insulin-Like Growth Factor Binding Protein 3; D010455:Peptides; D011993:Recombinant Fusion Proteins; D019096:Vitronectin; D007334:Insulin-Like Growth Factor I; D026122:Factor XIIIa; D011503:Transglutaminases; D017526:Receptor, IGF Type 1; D051057:Proto-Oncogene Proteins c-akt; D008239:Lysine
keywords: D-domain of IGF-I; ECM; FN; FXIIIa; IGF binding proteins; IGF-I; IGFBPs; Insulin-like growth factor-1; Migration; RTK; SPR; TG2; TGases; Transglutaminase; VN; Viability; extra-cellular matrix; factor thirteen transglutaminase; fibronectin; receptor tyrosine kinases; surface plasmon resonance; tissue transglutaminase; transglutaminases; vitronectin