Visit our Expo - Redox and Inflammation signaling 2012

Session III : Protein kinase cascades as therapeutic targets Poster III, 30
Design and functional activity of phosphopeptides with potential immunomodulating
capacity, based on the sequence of Grb2 associated binder 1 (Gab1)
Akos Kertesz1, Balazs Takacs1, Gyorgyi Varadi2, Gabor K. Toth2, Gabriella Sarmay1
1Department of Immunology, Lorand Eotvos University, Budapest 1117, Hungary, email:
sarmayg@cerberus.elte.hu and 2Department of Medical Chemistry, University of
Szeged, Szeged 6720, Hungary, e-mail: tgabor@mdche.szote.u-szeged.hu
Grb2-associated binder 1 (Gab1) is a scaffolding/adaptor protein involved in the signal
transduction pathways of growth factors, cytokines, and antigen receptors. Gab adaptor
proteins have several tyrosine residues which are phosphorylated upon ligand mediated
tyrosine kinase activation and bind signaling molecules with SH2 domains, such as SHP-2
tyrosine phosphatase, and phosphatidyl inositol 3-kinase (PI 3-K). We hypothesized that
phosphopeptides corresponding to motifs of Gab1 may interfere with the corresponding
signaling molecules in B cells, thus modulating the cell activation. Our main goal is to design
cell membrane permeable phosphopeptides based on the sequence of Gab1 that may regulate
the B-cell response.
Phosphopeptides representing Gab1’s tyrosine phosphorylated motifs were synthesized and
tested. Using peptide pull down assay, we identified SHP-2, Lyn and PLCg binding to the
same motif, 621GD-LD633. Furthermore, the 621GD-LD633 phosphopeptide bound and
powerfully activated SHP-2, while the PI3-K binding 442EL-PN453 and 467IQ-GP478
peptides less efficiently activated the phosphatase. In order to test the role of the 621-633
motif on B-cell signaling, the phosphopeptide was coupled to a membrane permeable
transporting peptide, octanoyl-R8 (OR8). Confocal laser scanning microscopy analysis of
cells exposed to the BODIPY dye labeled peptide showed that the peptide is taken up by B
cells and it is localized mostly in lysosome. Next, B cells were treated with membrane
permeable phosphopeptide, OR8(GD-LD) then the alteration of the intracellular free [Ca2+]
and tyrosine phosphorylation of intracellular proteins were tested. OR8(GD-LD) transiently
raised the level of [Ca 2+] in B-cells, and selectively modified the phosphorylation of certain
intracellular proteins.
Conclusion: cell membrane permeable phosphopeptides designed based on the sequence of
Gab adaptor protein may selectively modulate intracellular signaling pathways thus may be a
source of further drug development.
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