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Identifier

000388618

Title

Structural study of the interaction between ETS2 Repressor Factor (ERF) and kinase Erk2

Alternative Title

Δομική μελέτη της αλληλεπίδρασης του παράγοντα ERF (ETS2 repressor factor) με την κίναση Erk2

Author

Βογιατζή, Αγγελική Χ.

Thesis advisor

Μαυροθαλασσίτης, Γεώργιος

Abstract

ERF (ETS2 Repressor Factor) is a eukaryotic transcriptional repressor exerting tumor suppressor activity and is shown to be implicated in mouse embryonic development as well as in human disease in the case of ERF-related craniosynostosis. The function of ERF in cells is mainly regulated by phosphorylation by ERKs (Extracellular signal- Regulated Kinases), an event that takes place inside nucleus upon mitogenic stimuli, resulting in the rapid export of ERF towards the cytoplasm and the subsequent activation of genes previously suppressed by this factor. Although the association of ERF with Erk2 has been shown to be mediated by two FXF motifs lying in the EID (ERK interaction domain) of ERF, additional regions of this domain seem to participate in intermolecular contacts that confer overall stability to this complex. Therefore, the aim of this study was the purification of the two partners and the subsequent crystallization of the complex they form in order to gain an insight into all those structural elements that determine this particular interaction. His-tagged rat Erk2, produced in its double- phosphorylated activated form due to its co-expression with the constitutively active mutant of the upstream kinase MEK1, was initially isolated from the bacterial lysate by immobilized metal- affinity chromatography (IMAC) performed on Ni⁺²- NTA resin. The separation of the double- phosphorylated activated form of the protein from the non- phosphorylated inactive one was accomplished by anion exchange chromatography performed on Mono Q HR5/5 column. GST-tagged human ERF- EID was isolated in the first step of the purification procedure by glutathione S- transferase (GST) - affinity chromatography. Since GST-ERF (EID) was extremely sensitive to proteolysis and GST protein itself has a dimeric tertiary structure, the formation of heterodimers between the full-length protein and the proteolytic fragments of its polypeptide chain could not be prevented. Nevertheless we were successful in the isolation of a population consisting solely of full-length GST-ERF homodimers by gel filtration chromatography performed on Sephacryl S-200 HR16/60 column. Finally, in order to prevent the dimerization event, we performed the replacement of the GST-tag by a 6xHis-tag followed by protein purification by IMAC on Ni⁺²-NTA column as well as cation exchange chromatography on Mono S HR5/5. We anticipate that this work upon completion will not only shed light on the intricate interaction existing between ERF and kinase Erk2, but it will also pave the way towards the identification of a selective inhibitor of this particular association, with potential therapeutic implication in cases in which an increase in the nuclear ERF levels is required, as is the case of ERF-related craniosynostosis.

Language

English

Subject

FXF motif

FXF μοτίβο

GST-ERF

Gel filtration

HIS-TAGGED ERK2

Ion exchange chromatography

NI-NTA affinity

Phosphorylation

Protein purification