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A RasGAP SH3 peptide aptamer inhibits RasGAP-Aurora interaction and induces caspase-independent tumor cell death.

Pamonsinlapatham P, Hadj-Slimane R, Raynaud F, Bickle M, Corneloup C, Barthelaix A, Lepelletier Y, Mercier P, Schapira M, Samson J, Mathieu AL, Hugo N, Moncorgé O, Mikaelian I, Dufour S, Garbay C, Colas P - PLoS ONE (2008)

Bottom Line: However, RasGAP also acts as a positive effector of Ras and exerts an anti-apoptotic activity that is independent of its GAP function and that involves its SH3 (Src homology) domain.It disrupts the interaction between RasGAP and Aurora B kinase.This work identifies the above-mentioned pocket as an interesting therapeutic target to pursue and points its cognate peptide aptamer as a promising guide to discover RasGAP small-molecule drug candidates.

View Article: PubMed Central - PubMed

Affiliation: UFR Biomédicale, Laboratoire de Pharmacochimie Moléculaire et Cellulaire, INSERM U648, Université Paris Descartes, Paris, France.

ABSTRACT
The Ras GTPase-activating protein RasGAP catalyzes the conversion of active GTP-bound Ras into inactive GDP-bound Ras. However, RasGAP also acts as a positive effector of Ras and exerts an anti-apoptotic activity that is independent of its GAP function and that involves its SH3 (Src homology) domain. We used a combinatorial peptide aptamer approach to select a collection of RasGAP SH3 specific ligands. We mapped the peptide aptamer binding sites by performing yeast two-hybrid mating assays against a panel of RasGAP SH3 mutants. We examined the biological activity of a peptide aptamer targeting a pocket delineated by residues D295/7, L313 and W317. This aptamer shows a caspase-independent cytotoxic activity on tumor cell lines. It disrupts the interaction between RasGAP and Aurora B kinase. This work identifies the above-mentioned pocket as an interesting therapeutic target to pursue and points its cognate peptide aptamer as a promising guide to discover RasGAP small-molecule drug candidates.

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RasGAP SH3 peptide aptamer binding specificity.(A) Protein domain mapping of the RasGAP protein. PPP: Proline-rich region; PH: Pleckstrin homology domain; C2: pKC-conserved region 2. (B) Sequence alignment of RasGAP, Nck, and Grb2 SH3 domains. Residues that are conserved between at least two proteins are highlighted in green. (C) Yeast two-hybrid interaction mating assay. LexA fusion proteins (“baits”) were expressed in EGY191α yeast containing a 8 lexAop-lacZ reporter gene. Control and selected peptide aptamers (fused to the B112 activation domain) were expressed in EGY42a yeast. Strains were mated and diploids were grown on X-gal-containing solid medium. 10M is a Cdk2 peptide aptamer and Control is a peptide aptamer randomly picked from the library.
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pone-0002902-g001: RasGAP SH3 peptide aptamer binding specificity.(A) Protein domain mapping of the RasGAP protein. PPP: Proline-rich region; PH: Pleckstrin homology domain; C2: pKC-conserved region 2. (B) Sequence alignment of RasGAP, Nck, and Grb2 SH3 domains. Residues that are conserved between at least two proteins are highlighted in green. (C) Yeast two-hybrid interaction mating assay. LexA fusion proteins (“baits”) were expressed in EGY191α yeast containing a 8 lexAop-lacZ reporter gene. Control and selected peptide aptamers (fused to the B112 activation domain) were expressed in EGY42a yeast. Strains were mated and diploids were grown on X-gal-containing solid medium. 10M is a Cdk2 peptide aptamer and Control is a peptide aptamer randomly picked from the library.

Mentions: Initially, RasGAP has been identified as the main negative regulator of Ras, since its C-terminal GAP domain catalyzes the hydrolysis of GTP-bound Ras to GDP-bound Ras. Subsequently, it has been shown that the N-terminal region of this large protein is essential to trigger downstream signals independently of its GAP activity, and acts as a positive effector of Ras [2]. N-terminal fragments of RasGAP, naturally generated by caspase cleavage, exert either an anti-apoptotic function (fragment N, 1–455) or a pro-apoptotic function (fragments N1, 1–157 and N2, 158–455) [3]. The N-terminal region of RasGAP (1–692) contains a pKC-conserved region 2 (C2) domain, a pleckstrin homology (PH) domain and a SH3 (Src Homology) domain flanked by two SH2 domains (Figure 1A). The SH2 domains mediate interactions with phospho-tyrosine containing proteins such as PDGF-R, EGF-R, v-Src, p62 and p190RhoGAP. The SH3 domain of RasGAP plays an essential role in Ras downstream signaling [2] and influences Rho-mediated cytoskeleton reorganization, independently from Ras [4]. The microinjection of a monoclonal antibody directed against RasGAP SH3 was shown to induce apoptosis in tumor cells but not in non-transformed cells [5]. A few RasGAP SH3-interacting proteins have been discovered so far. First studies have identified a GAP SH3-Binding Protein (G3BP) [6] and an unknown 14 kDa protein [7]. More recently, Aurora kinases have been shown to bind to RasGAP SH3 [8] and these interactions are suspected to play a crucial role in the Ras effector activity of RasGAP in cancer cells.


A RasGAP SH3 peptide aptamer inhibits RasGAP-Aurora interaction and induces caspase-independent tumor cell death.

Pamonsinlapatham P, Hadj-Slimane R, Raynaud F, Bickle M, Corneloup C, Barthelaix A, Lepelletier Y, Mercier P, Schapira M, Samson J, Mathieu AL, Hugo N, Moncorgé O, Mikaelian I, Dufour S, Garbay C, Colas P - PLoS ONE (2008)

RasGAP SH3 peptide aptamer binding specificity.(A) Protein domain mapping of the RasGAP protein. PPP: Proline-rich region; PH: Pleckstrin homology domain; C2: pKC-conserved region 2. (B) Sequence alignment of RasGAP, Nck, and Grb2 SH3 domains. Residues that are conserved between at least two proteins are highlighted in green. (C) Yeast two-hybrid interaction mating assay. LexA fusion proteins (“baits”) were expressed in EGY191α yeast containing a 8 lexAop-lacZ reporter gene. Control and selected peptide aptamers (fused to the B112 activation domain) were expressed in EGY42a yeast. Strains were mated and diploids were grown on X-gal-containing solid medium. 10M is a Cdk2 peptide aptamer and Control is a peptide aptamer randomly picked from the library.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2483412&req=5

pone-0002902-g001: RasGAP SH3 peptide aptamer binding specificity.(A) Protein domain mapping of the RasGAP protein. PPP: Proline-rich region; PH: Pleckstrin homology domain; C2: pKC-conserved region 2. (B) Sequence alignment of RasGAP, Nck, and Grb2 SH3 domains. Residues that are conserved between at least two proteins are highlighted in green. (C) Yeast two-hybrid interaction mating assay. LexA fusion proteins (“baits”) were expressed in EGY191α yeast containing a 8 lexAop-lacZ reporter gene. Control and selected peptide aptamers (fused to the B112 activation domain) were expressed in EGY42a yeast. Strains were mated and diploids were grown on X-gal-containing solid medium. 10M is a Cdk2 peptide aptamer and Control is a peptide aptamer randomly picked from the library.
Mentions: Initially, RasGAP has been identified as the main negative regulator of Ras, since its C-terminal GAP domain catalyzes the hydrolysis of GTP-bound Ras to GDP-bound Ras. Subsequently, it has been shown that the N-terminal region of this large protein is essential to trigger downstream signals independently of its GAP activity, and acts as a positive effector of Ras [2]. N-terminal fragments of RasGAP, naturally generated by caspase cleavage, exert either an anti-apoptotic function (fragment N, 1–455) or a pro-apoptotic function (fragments N1, 1–157 and N2, 158–455) [3]. The N-terminal region of RasGAP (1–692) contains a pKC-conserved region 2 (C2) domain, a pleckstrin homology (PH) domain and a SH3 (Src Homology) domain flanked by two SH2 domains (Figure 1A). The SH2 domains mediate interactions with phospho-tyrosine containing proteins such as PDGF-R, EGF-R, v-Src, p62 and p190RhoGAP. The SH3 domain of RasGAP plays an essential role in Ras downstream signaling [2] and influences Rho-mediated cytoskeleton reorganization, independently from Ras [4]. The microinjection of a monoclonal antibody directed against RasGAP SH3 was shown to induce apoptosis in tumor cells but not in non-transformed cells [5]. A few RasGAP SH3-interacting proteins have been discovered so far. First studies have identified a GAP SH3-Binding Protein (G3BP) [6] and an unknown 14 kDa protein [7]. More recently, Aurora kinases have been shown to bind to RasGAP SH3 [8] and these interactions are suspected to play a crucial role in the Ras effector activity of RasGAP in cancer cells.

Bottom Line: However, RasGAP also acts as a positive effector of Ras and exerts an anti-apoptotic activity that is independent of its GAP function and that involves its SH3 (Src homology) domain.It disrupts the interaction between RasGAP and Aurora B kinase.This work identifies the above-mentioned pocket as an interesting therapeutic target to pursue and points its cognate peptide aptamer as a promising guide to discover RasGAP small-molecule drug candidates.

View Article: PubMed Central - PubMed

Affiliation: UFR Biomédicale, Laboratoire de Pharmacochimie Moléculaire et Cellulaire, INSERM U648, Université Paris Descartes, Paris, France.

ABSTRACT
The Ras GTPase-activating protein RasGAP catalyzes the conversion of active GTP-bound Ras into inactive GDP-bound Ras. However, RasGAP also acts as a positive effector of Ras and exerts an anti-apoptotic activity that is independent of its GAP function and that involves its SH3 (Src homology) domain. We used a combinatorial peptide aptamer approach to select a collection of RasGAP SH3 specific ligands. We mapped the peptide aptamer binding sites by performing yeast two-hybrid mating assays against a panel of RasGAP SH3 mutants. We examined the biological activity of a peptide aptamer targeting a pocket delineated by residues D295/7, L313 and W317. This aptamer shows a caspase-independent cytotoxic activity on tumor cell lines. It disrupts the interaction between RasGAP and Aurora B kinase. This work identifies the above-mentioned pocket as an interesting therapeutic target to pursue and points its cognate peptide aptamer as a promising guide to discover RasGAP small-molecule drug candidates.

Show MeSH
Related in: MedlinePlus