Differential effect of the inhibition of Grb2-SH3 interactions in platelet activation induced by thrombin and by Fc receptor engagement.

INSERM U428, Faculte de Pharmacie, 4 Avenue de l'Observatoire, Paris F-75006, France.
Biochemical Journal (Impact Factor: 4.4). 06/2002; 363(Pt 3):717-25.
Source: PubMed


The adaptor protein Grb2 (growth factor receptor-bound protein 2) is involved in cell proliferation via the Ras signalling pathway. In order to study the role of Grb2 in blood platelet responses, we used a peptide containing two proline-rich sequences derived from Sos (peptidimer), which binds to Grb2-Src homology 3 domain (SH3) with a high affinity, and hence inhibits Grb2-SH3-mediated protein interactions. Platelet aggregation and 5-hydroxytryptamine (serotonin) release measured in the presence of the peptidimer were: (i) significantly decreased when induced by thrombin; and (ii) potentiated when induced by the engagement of the Fc receptor. In thrombin-activated platelets, the Grb2-SH2 domain formed an association with the beta3 subunit of the alphaIIb-beta3 integrin (GPIIb-IIIa), Shc, Syk, Src and SHP1 (SH2-containing phosphotyrosine phosphatase 1), whereas these associations did not occur after the engagement of the receptor for the Fc domain of IgG (FcgammaRIIa) or in resting platelets. Grb2-SH3 domains formed an association with the proline-rich sequences of Sos and Cbl in both resting and activated platelets, since the peptidimer abolished these associations. Inhibition of both fibrinogen binding and platelet aggregation by the peptide RGDS (Arg-Gly-Asp-Ser) had no effect on thrombin-induced Grb2-SH2 domain association with the aforementioned signalling molecules, indicating that these associations occurred during thrombin-induced 'inside-out' signalling. Platelet aggregation induced by direct activation via alphaIIb-beta3 ('outside-in' signalling) was potentiated by the peptidimer. The results show that inhibition of Grb2-SH3 interactions with signal-transduction proteins down-regulates thrombin-induced platelet activation, but also potentiates Fc receptor- and alphaIIb-beta3-mediated platelet activation.

Download full-text


Available from: Wang-Qing Liu
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: This paper describes the design of the highest affinity ligands for Grb2 SH3 domains reported so far. These compounds were designed by combining N-alkyl amino acid incorporation in a proline-rich sequence with subsequent dimerization of the peptoid sequence based on structural data and molecular modeling. Optimization of the linker size is discussed, and the N-alkyl amino acid incorporation into both monomeric halves is reported. Because the affinity for Grb2 of the optimized compounds was too high to be measured using the fluorescent modifications that they induce on the Grb2 emission spectrum, a competition assay was developed. In this test, Grb2 is pulled down from a cellular extract by the initial VPPPVPPRRR peptide bound to Sepharose beads. In the presence of competitors, the test quantifies the amount of Grb2 displaced from the beads. It has enabled us to determine a K(i) value in the 10(-10) M range for the highest affinity Grb2 peptoid analogue dimer.
    Full-text · Article · Jul 2004 · Biochemistry
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Spleen tyrosine kinase (Syk) activation is a key intermediate step in the activation of platelets by the physiologic agonist collagen. We have found that Syk is rapidly ubiquitinated upon activation of platelets by collagen, collagen-related peptide (CRP), and convulxin. The Src family kinase inhibitors prevented Syk phosphorylation and its ubiquitination, indicating that the process is downstream of Src kinases. The ubiquitination of Syk did not cause degradation of the protein as evidenced by the lack of effect of proteasomal and lysosomal inhibitors. We separated ubiquitinated Syk from its nonubiquitinated counterpart and used an in vitro kinase assay to compare their activities. We found that the ubiquitinated Syk appeared to be about 5-fold more active. Using a phosphospecific antibody to Syk (Tyr525/Tyr526) that measures activated Syk, we found that most (60%-75%) of the active Syk is in the ubiquitinated fraction. This result explains the apparent high specific activity of ubiquitinated Syk. In c-Cbl-deficient mice, Syk is not ubiquitinated, implicating c-Cbl as the E3 ligase involved in Syk ubiquitination. Furthermore, Syk is not dephosphorylated in these mice. We propose that c-Cbl plays a regulatory role in glycoprotein VI (GPVI)/Fc receptor gamma (FcRgamma)-chain-dependent platelet activation through its interaction with Syk.
    Full-text · Article · Jun 2005 · Blood
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Macrophage colony-stimulating factor (M-CSF) is a physiological regulator of monocyte-macrophage lineage. Ectopic expression of the M-CSF receptor (M-CSFR, or Fms) in murine myeloid cell line FDC-P1 (FD/Fms cells) results in M-CSF-dependent macrophage differentiation. Previously, we observed that M-CSF induces two temporally distinct phases of mitogen-activated protein kinase (MAPK) phosphorylation. Here we show that levels of phosphorylated MAPK kinase MEK1 follow the same kinetics as MAPK phosphorylation, characterized by an early and transient phase (the first 30 min of M-CSF stimulation) and a late and persistent phase from 4 h of stimulation. The MEK inhibitor U0126 strongly inhibited both phases of MAPK phosphorylation as well as FD/Fms cell differentiation, indicating that MAPK may relay M-CSF differentiation signaling downstream of M-CSFR. Treatment of FD/Fms cells with U0126 during the first hour of M-CSF stimulation reversibly blocked the early phase of MAPK phosphorylation but did not affect differentiation. In contrast, U0126 still inhibited FD/Fms cell differentiation when its addition was delayed by 24 h. This demonstrated that late and persistent MEK activity is specifically required for macrophage differentiation to occur. Furthermore, disrupting Grb2-Sos complexes with a specific blocking peptide did not prevent FD/Fms cells differentiation in response to M-CSF, nor did it abolish MAPK phosphorylation. The role of phosphatidylinositol 3-kinase (PI 3-kinase), another potential regulator of the MAPK pathway, was examined using the specific inhibitor LY294002. This compound could not impede FD/Fms cell commitment to macrophage differentiation and did not significantly affect MAPK phosphorylation in response to M-CSF. Therefore, M-CSF differentiation signaling in myeloid progenitor cells is mediated through persistent MEK activity but it is not strictly dependent upon Grb2-Sos interaction or PI 3-kinase activity.
    Full-text · Article · Dec 2005 · Cellular Signalling
Show more