CZ.1.07/2.3.00/ Employment of Best Young Scientists for International Cooperation Empowerment 2 nd Postdoc Presentation Day Name of the postdoc: Jan Ryneš

Molecular interactions of the scaffold protein Axin Mentor: Dr. Lukáš Trantírek CEITEC, Centre for Structural Biology Research group Biomolecular NMR Spectroscopy Start date of postdoc (in the project):

Axin is a scaffold protein involved in multiple signaling pathways 3 Binding regions for Axin partners RGS DIX unstructured hAxin1

Goal of the project: manipulate activity of the Wnt, JNK, p53, and TGF pathways via specific inhibition of interactions between Axin and its partners 4

5 Identification of binding sites for Axin partners using peptide microarray screen Printed Axin peptide library Recombinant protein (CK1e) Detection antibody overlapping peptides, covering the protein print in spots on a glass slide RGSDIX hAxin1 Binding sites for CK1e:

Deletion mutants of Axin used in CoIP assays 6 DIX RGSDIX RGS DIX RGSDIX RGS wt Δ1 Δ2 Δ3 Δ1+2 Δ1+3 Δ2+3 Δ1+2+3

Single binding epitope is sufficient for the interaction of Axin with CK1e; deletion of all sites abolish the interaction 7 IP: α-Myc WB: α-CK1e Input: α-CK1e Input: α-Myc wt Δ1 Δ2 Δ3 Δ1+2Δ1+3 Δ2+3 Δ1+2+3 Ctrl Myc-hAxin1

Synthetic peptides mimicking Axin binding-site can be used to inhibit the interaction in vitro 8 RGSDIXHisGST CK1e Axin based peptide inhibitor pulldown: GST WB: α-CK1e Input: α-His peptide - + RGSDIXHisGST CK1e X

Future directions 9 -use chemically stabilized synthetic peptides for testing the inhibition in cells -functional studies (effect of Axin binding on catalytic activity of CK1e, effect of blocking single interactions on the activity of different pathways using luciferase reporter system) -apply the approach for other Axin – binding partners, e.g. p53, c-Myc, Pin1, MEKK1…

Expected publication of the results: papers in peeer reviewed journals (e.g. JBC, Biochemistry, EMBO J) - review on Axin interactions - presentation on a cell signaling conference (2014 / 2015) 10