PEGylation of lysine residues reduces the pro-migratory activity of IGF-I

Sivaramakrishnan, Manaswini, Kashyap, Abhishek S., Amrein, Beat, Saenger, Stafani, Meier, Sonja, Staudenmaier, Christian , Upton, Zee, & Metzger, Friedrich (2013) PEGylation of lysine residues reduces the pro-migratory activity of IGF-I. Biochimica et Biophysica Acta (BBA) - General Subjects, 1830(10), pp. 4734-4742.

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Abstract

Background:

The insulin-like growth factor (IGF) system is composed of ligands and receptors which regulate cell proliferation, survival, differentiation and migration. Some functions are regulated via intracellular signaling cascades, others by involvement of the extracellular milieu, including binding proteins and other extracellular matrix proteins. However, understanding of their functions and the exact nature of these interactions remains incomplete.

Methods:

IGF-I was PEGylated at its lysine sites - K27, K65 and K68. Binding of PEG-IGF-I to the IGFBPs was analyzed using BIAcore and its ability to activate the IGF-IR was assessed using IGF-IR phosphorylation assay. Furthermore, functional consequences of PEGylating the lysine residues of IGF-I was investigated using cell viability and cell migration assays. In addition, particular downstream signaling pathways regularly implicated in these mechanisms were also dissected using phospho-AKT and phospho-ERK1/2 assays.

Results:

In this study, IGF-I specifically PEGylated at lysine 27 (PEG-K27), 65 (PEG-K65) or 68 (PEG-K68) were employed. Receptor phosphorylation was only reduced by 2-fold with PEG-K65 and PEG-K68 over all the time points tested, and as observed in two cell types, 3T3 fibroblasts and MCF-7 breast cancer cells. PEGylation at K27 resulted in a much larger effect, with more than 10-fold lower activation for 3T3 fibroblasts and a ~3 fold reduced IGF-IR activation for MCF-7 breast cancer cells over 15 minutes. In addition, all PEG-IGF-I variants demonstrated a ten-fold reduction in the association rate to IGF binding proteins (IGFBPs). Functionally, all PEG variants completely lost their ability to induce cell migration in the presence of IGFBP-3/vitronectin (VN) complexes as compared to IGF-I; in contrast, cell viability was fully preserved. Further investigations into the downstream signaling pathways revealed that the PI3-K/AKT pathway was preferentially affected upon treatment with the PEG-IGF-I variants compared to the MAPK/ERK pathway.

Conclusion:

PEGylation of IGF-I has an impact on cell migration but not cell viability.

General significance:

PEG-IGF-I may differentially modulate IGF-I mediated functions that are dependent on its interaction with its receptor as well as key extracellular proteins such as VN and IGFBPs.

Impact and interest:

2 citations in Scopus
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3 citations in Web of Science®

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ID Code: 62652
Item Type: Journal Article
Refereed: Yes
Keywords: Insulin-like growth factor, IGF, PEG, migration, viability
DOI: 10.1016/j.bbagen.2013.06.012
ISSN: 0006-3002
Subjects: Australian and New Zealand Standard Research Classification > BIOLOGICAL SCIENCES (060000) > BIOCHEMISTRY AND CELL BIOLOGY (060100)
Divisions: Current > Schools > School of Biomedical Sciences
Current > QUT Faculties and Divisions > Faculty of Health
Current > Institutes > Institute of Health and Biomedical Innovation
Copyright Owner: Copyright 2013 Elsevier
Copyright Statement: This is the author’s version of a work that was accepted for publication in Biochimica et Biophysica Acta (BBA) - General Subjects. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Biochimica et Biophysica Acta (BBA) - General Subjects, [in press, (2013)] DOI: 10.1016/j.bbagen.2013.06.012
Deposited On: 18 Sep 2013 22:52
Last Modified: 03 Nov 2014 19:02

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