A Sos‐derived peptidimer blocks the Ras signaling pathway by binding both Grb2 SH3 domains and displays antiproliferative activity

Abstract

With the aim of interrupting the growth factor-stimulated Ras signaling pathway at the level of the Grb2-Sos interaction, a peptidimer, made of two identical proline-rich sequences from Sos linked by a lysine spacer, was designed using structural data from Grb2 and a proline-rich peptide complexed with its SH3 domains. The peptidimer affinity for Grb2 is 40 nM whereas that of the monomer is 16 microM, supporting the dual recognition of both Grb2 SH3 domains by the dimer. At 50 nM, the peptidimer blocks selectively Grb2-Sos complexation in ER 22 (CCL 39 fibroblasts overexpressing epidermal growth factor receptor) cellular extracts. The peptidimer specifically recognizes Grb2 and does not interact with PI3K or Nck, two SH3 domain-containing adaptors. The peptidimer was modified to enter cells by coupling to a fragment of Antennapedia homeodomain. At 10 microM, the conjugate inhibits the Grb2-Sos interaction (100%) and MAP kinase (ERK1 and ERK2) phosphorylation (60%) without modifying cellular growth of ER 22 cells. At the same concentration, the conjugate also inhibits both MAP kinase activation induced by nerve growth factor or epidermal growth factor in PC12 cells, and differentiation triggered by nerve growth factor. Finally, when tested for its antiproliferative activity, the conjugate was an efficient inhibitor of the colony formation of transformed NIH3T3/HER2 cells grown in soft agar, with an IC50 of around 1 microM. Thus, the designed peptidimers appear to be interesting leads to investigate signaling and intracellular processes and for designing selective inhibitors of tumorigenic Ras-dependent processes.

The C-terminal SH3 domain of the adapter protein Grb2 binds with high affinity to sequences in Gab1 and SLP-76 which lack the SH3-typical P-x-x-P core motif

“…While small compounds with impressive SH3 domain blocking activity are still missing, several laboratories, including our own, have successfully introduced SH3 domain binding sequences into living cells to modulate signal transduction events. In the case of Grb2, a recent report showed that a peptide which can interact with both SH3 domains of Grb2 has superior biological activity compared to a peptide targeting just one of the domains (Cussac et al 1999). This study used a binding motif for the Grb2 SH3(C) domain which is derived from the Grb2 SH3(N) domain binding sequence of the SoS protein and has a signi®cantly lower anity for Grb2 SH3(C) than the new binding peptides described here (see Table 3 for anities).…”

“…In prior studies it was found that a murine SoSderived peptide which binds strongly to the Grb2 SH3(N) domain can also bind to the Grb2 SH3(C) domain Cussac et al, 1999). This peptide is known to form a typical type II polyproline helix and contains the P-x-x-P-x-R motif typically found in good Grb2 SH3(N) binding motifs (indicated by ®lled arrow heads below the peptide).…”

GRB2 Links Signaling to Actin Assembly by Enhancing Interaction of Neural Wiskott-Aldrich Syndrome Protein (N-WASp) with Actin-related Protein (ARP2/3) Complex

“…Isolated N-terminal and Cterminal SH3 domains of Grb2 were obtained by solid phase peptide synthesis as described (50,51). The Sos-derived peptide dimer (VPP-PVPPRRR-Aha-K-Aha-RRRPPVPPPV) was synthesized as described (52). The SH2 domain binding phosphotyrosine m-arylphospho-Tyr-␣-methylphospho-Tyr-Asn-NH 2 tripeptide (pYpYN) was prepared as described (53).…”

“…3A), which binds both SH3 domains (Fig. 3B) and blocks the Ras signaling pathway (52). The pYpYN phosphorylated tripeptide, which mimics the targets of the SH2 domain of Grb2, caused a weak inhibition in the absence as well as in the presence of Grb2.…”

Crk family adaptors–signalling complex formation and biological roles

“…These peptides bind with higher a nities than the corresponding peptides completely made up of naturally occurring amino acids. In vivo studies with these compounds are still missing, but it is already clear from the current literature that Grb2 and Crk/CRKL SH3 domain blocker peptides can be carried into cells with peptidevector systems, thereby inhibiting adaptor protein signalling (Cussac et al, 1999;Kardinal et al, 2000Kardinal et al, , 2001Sakkab et al, 2001).…”