Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Feb 3;292(5):1691-1704.
doi: 10.1074/jbc.M116.746867. Epub 2016 Nov 30.

The Phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) Binder Rasa3 Regulates Phosphoinositide 3-kinase (PI3K)-dependent Integrin αIIbβ3 Outside-in Signaling

Affiliations
Free PMC article

The Phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) Binder Rasa3 Regulates Phosphoinositide 3-kinase (PI3K)-dependent Integrin αIIbβ3 Outside-in Signaling

Anthony M Battram et al. J Biol Chem. .
Free PMC article

Abstract

The class I PI3K family of lipid kinases plays an important role in integrin αIIbβ3 function, thereby supporting thrombus growth and consolidation. Here, we identify Ras/Rap1GAP Rasa3 (GAP1IP4BP) as a major phosphatidylinositol 3,4,5-trisphosphate-binding protein in human platelets and a key regulator of integrin αIIbβ3 outside-in signaling. We demonstrate that cytosolic Rasa3 translocates to the plasma membrane in a PI3K-dependent manner upon activation of human platelets. Expression of wild-type Rasa3 in integrin αIIbβ3-expressing CHO cells blocked Rap1 activity and integrin αIIbβ3-mediated spreading on fibrinogen. In contrast, Rap1GAP-deficient (P489V) and Ras/Rap1GAP-deficient (R371Q) Rasa3 had no effect. We furthermore show that two Rasa3 mutants (H794L and G125V), which are expressed in different mouse models of thrombocytopenia, lack both Ras and Rap1GAP activity and do not affect integrin αIIbβ3-mediated spreading of CHO cells on fibrinogen. Platelets from thrombocytopenic mice expressing GAP-deficient Rasa3 (H794L) show increased spreading on fibrinogen, which in contrast to wild-type platelets is insensitive to PI3K inhibitors. Together, these results support an important role for Rasa3 in PI3K-dependent integrin αIIbβ3-mediated outside-in signaling and cell spreading.

Keywords: GAP1IP4BP; Ras-related protein 1 (Rap1); Rasa3; cell signaling; integrin; phosphatidylinositide 3-kinase (PI3K); platelet.

Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

FIGURE 1.
FIGURE 1.
Rasa3 binds to PI(3,4,5)P3 and is highly expressed in platelets. A, Rasa3 domain structure. Rasa3 consists of two N-terminal C2 domains (C2A and C2B), a central RasGAP-related domain (RasGAP), and a C-terminal pleckstrin homology (PH)/Btk moiety. B, summary of proteomics data. Rasa3 and Btk were captured on PI(3,4,5)P3-coated (PIP3) beads after incubation with human platelet lysate. The total score of the protein is the sum of all peptide Xcorr values for that protein above the specified score threshold. The area is the mean of the area of the three most intense unique peptides matched to that protein. The coverage values indicate the percentages of the protein sequence covered by the identified peptides. PSM indicates the total number of identified peptide sequences for the protein, and the far-right column shows the number of unique peptide sequences identified for the protein. Rasa3 and Btk were identified in three independent experiments from which the mean values were calculated. C, Western blotting confirmation of Rasa3 and Btk captured on PI(3,4,5)P3-coated beads. Human platelet lysate was incubated with uncoated control beads, PI(3,4,5)P3-coated beads (PIP3 beads), or PI(3,4,5)P3-coated beads following preincubation of the lysate with competing free PI(3,4,5)P3 (PIP3 beads + PIP3). The input material for each sample was blotted for Rasa3, Btk, and α-tubulin as a loading control. The blots are representative of four independent experiments. D, lysates from human platelets (hPlt), mouse platelets (mPlt), and CHO cells were subjected to immunoblotting to analyze the expression of Rasa3 and its substrates Rap1 and Ras. Input material for each cell type was matched by means of protein assay. The blots are representative of three independent experiments.
FIGURE 2.
FIGURE 2.
Sustained Rap1 activation is dependent on PI3K and P2Y12, whereas Ras activation is PI3K-independent. Human washed platelets were incubated with 100 nm wortmannin (Wtm), 1 μm AR-C66096 (ARC), or vehicle control (0.2% DMSO) for 10 min prior to stimulation with 0.2 unit/ml α-thrombin for the indicated time. Rap1-GTP (A and B) or Ras-GTP (C) was extracted from platelets lysates by GST-RalGDS-RBD or GST-Raf1RBD pulldown, respectively. Pulldown samples were immunoblotted for Rap1 or Ras, and total lysate controls were immunoblotted for Rap1, Ras, or pAktS473 and total Akt. The results are expressed as the percentage of the maximum Rap1-GTP (A, n = 4; B, n = 5) or Ras-GTP (C, n = 6) detected using densitometry. The data are expressed as the means ± standard deviation, and statistical analysis is presented as paired Student's t test for each time point to show the effect of wortmannin or AR-C66096 compared with DMSO control (**, p ≤ 0.01; ***, p ≤ 0.001).
FIGURE 3.
FIGURE 3.
Rasa3 translocates to the plasma membrane upon agonist stimulation in a PI3K/P2Y12-dependent manner and colocalizes with integrin αIIbβ3. A, human washed platelets were incubated with 100 nm wortmannin (Wtm), 1 μm AR-C66096 (ARC), or vehicle control (0.2% DMSO) for 10 min prior to stimulation with 0.2 unit/ml α-thrombin (αT) for 5 min. Cytosolic and membrane fractions were separated by ultracentrifugation and immunoblotted alongside a platelet whole cell lysate (WCL) for Rasa3, Rap1, ERK (cytosolic control), FcRγ (membrane control), and talin. The blots are representative of three independent experiments. B, quantification of Rasa3 levels in cytosol and membrane fractions from platelet fractionation experiments (n = 3). The data are expressed as means ± standard deviation, and statistical analysis shows the effect of thrombin + vehicle control, thrombin + wortmannin, or thrombin + AR-C66096 compared with basal control (*, p ≤ 0.05). C and D, platelets stimulated with 0.2 unit/ml α-thrombin for 5 min or untreated platelets were fixed in 4% paraformaldehyde and spun onto glass coverslips. Adhered platelets were permeabilized and stained with antibodies against integrin αIIbβ3, Rasa3, and Rap1. Localization was identified using Alexa Fluor 568 (integrin αIIbβ3, magenta), Alexa Fluor 488 (Rasa3, green), and Alexa Fluor 350 (Rap1, blue) secondary antibodies. Images were captured using a spinning disk confocal module (PerkinElmer UltraVIEW ERS 6FE confocal microscope) at 100× magnification. C, representative extended focus images of three separate experiments. Scale bars, 0.5 μm (left panel, single cell images) and 2 μm (right panel, multiple cell images). D, analysis of immunofluorescence data to determine colocalization between Rasa3 and Rap1, Rasa3 and integrin αIIbβ3, and integrin αIIbβ3 and Rap1 (left panel), and submembrane localization (right panel) was performed using Volocity software, with submembrane defined as 0.5 μm from the outermost point of the cell. The results are expressed as means ± standard deviation (n = 3; *, p ≤ 0.05).
FIGURE 4.
FIGURE 4.
Rasa3 suppresses Rap1 and Ras activation in integrin αIIbβ3-expressing CHO cells. A, CHO cells that were allowed to adhere to glass-bottomed dishes coated with 0.1 mg/ml poly-l-lysine were transfected with GFP alone (GFP) or GFP-conjugated wild-type Rasa3 (WT Rasa3). 16 h after transfection, cell medium was replaced with imaging medium, and the cells were imaged on a spinning disk confocal microscope at 63× magnification. The images are representative of three independent experiments. Scale bar, 20 μm. B–E, CHO cells were transfected with GFP alone or GFP-conjugated WT Rasa3, Rasa3-ΔC2, Rasa3 (R371Q), or Rasa3 (P489V). CHO cells were unstimulated or stimulated with 50 μm SFLLRN for 5 min. Rap1-GTP or Ras-GTP was extracted from platelets lysates by GST-RalGDS-RBD or GST-Raf1RBD pulldown, respectively. Pulldown samples were blotted for Rap1 or Ras, and total lysate controls were immunoblotted for Rap1 or Ras, pAktS473, GFP, talin, and α-tubulin (loading control). B and C, representative blots from at least four independent experiments. D and E, quantification of Rap1-GTP (D, n = 4–9) or Ras-GTP (E, n = 4–7) bands by densitometry, expressed as means ± standard deviation of the percentage of the stimulated GFP control detected. The values are compared with the basal or stimulated GFP control to test for significance (***, p ≤ 0.001).
FIGURE 5.
FIGURE 5.
Integrin αIIbβ3-mediated spreading of CHO cells is inhibited by Rasa3 Rap1GAP activity. A and B, CHO cells were allowed to adhere to 100 μg/ml fibrinogen, in the absence or presence of 10 μg/ml abciximab, at 37 °C. Adherent cells were fixed and stained with CruzFluor 594-phalloidin (red) and DAPI (blue). Images were acquired using a Leica AF6000 wide field microscope at 40× magnification. A, representative images of spread CHO cells in the absence (−abcix) or presence (+abcix) of 10 μg/ml abciximab. Scale bar, 32 μm. B, cell area was analyzed by measuring the phalloidin staining per cell using ImageJ software. The results are expressed as means ± standard deviation (n = 4; ***, p ≤ 0.001). C and D, CHO cells were transfected with GFP alone or GFP-conjugated WT Rasa3, Rasa3-ΔC2, Rasa3 (R371Q), or Rasa3 (P489V) and then allowed to adhere to 100 μg/ml fibrinogen at 37 °C. Adherent cells were fixed and stained with CruzFluor 594-phalloidin (red) and DAPI (blue). Prior to the spreading assay, CHO cells were transfected with GFP alone or GFP-conjugated WT Rasa3, Rasa3-ΔC2, Rasa3 (R371Q), or Rasa3 (P489V). GFP (green) expression indicates transfected cells. The images were acquired using a Leica AF6000 wide field microscope at 40× magnification. C, representative images of spread CHO cells transfected with GFP, WT Rasa3, Rasa3-ΔC2, Rasa3 (R371Q), or Rasa3 (P489V). Scale bar, 32 μm. D, cell area was analyzed by measuring the phalloidin staining per cell using ImageJ software. The results are expressed as means ± standard deviation compared with GFP control (n = 3–7; ***, p ≤ 0.001).
FIGURE 6.
FIGURE 6.
Rasa3 hlb and scat forms have deficient RasGAP activity and reduced Rap1GAP activity upon stimulation. A–D, CHO cells were transfected with GFP alone or GFP-conjugated WT Rasa3, Rasa3 (H794L), or Rasa3 (G125V), and Rap1-GTP (A) or Ras-GTP (D) activation assays were carried out as described for Fig. 4 (B–E). A and D, representative blots from at least four independent experiments. B and C, quantification of blots, expressed as means ± standard deviation of the percentage of the stimulated GFP control (B, n = 4–6; C, n = 4–6) detected. The values are compared with the basal or stimulated GFP control to test for significance (*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001). E and F, 25 nm recombinant Rasa3 (WT, R371Q, H794L, or G125V) was incubated with [γ-32P]GTP-loaded 1 μm Rap1b or H-Ras for 10 min at 25 °C, and GAP activity was measured as described under “Experimental Procedures” (n = 3; ***, p ≤ 0.001). G and H, CHO cells were transfected with GFP alone or GFP-conjugated WT Rasa3, Rasa3 (H794L), or Rasa3 (G125V) and then allowed to adhere to 100 μg/ml fibrinogen at 37 °C. Adherent cells were fixed and stained with CruzFluor 594-phalloidin (red) and DAPI (blue). GFP (green) expression indicates transfected cells. Images were acquired using a Leica AF6000 wide field microscope at 40× magnification. G, cell area was analyzed as described for Fig. 5D. The results are expressed as means ± standard deviation compared with GFP control (n = 4–5; ***, p ≤ 0.001). H, representative images of spread CHO cells transfected with GFP, WT Rasa3, Rasa3 (H794L), or Rasa3 (G125V). Scale bar, 32 μm.
FIGURE 7.
FIGURE 7.
Spreading of Rasa3H794L/H794L mouse platelets on fibrinogen. Platelets from CalDAG-GEF1+/− Rasa3H794L/H794L mice were incubated with 100 nm wortmannin or vehicle control for 10 min before being allowed to spread on 100 μg/ml fibrinogen for 60 min at 37 °C. After fixation, platelets were stained with Alexa Fluor 594-phalloidin. A, representative images of spread platelets from wild-type, CalDAG-GEF1+/− Rasa3+/H794L, and CalDAG-GEF1+/− Rasa3H794L/H794L mice. Scale bar, 10 μm. B, quantification of platelet spreading by measuring the extent of phalloidin staining per platelet using ImageJ software (n = 3).
FIGURE 8.
FIGURE 8.
Role of Rasa3 and PI3K in platelet integrin αIIbβ3 outside-in signaling. A, fibrinogen binding to activated integrin αIIbβ3 initiates outside-in signaling, including the activation of PI3K. PI3K reduces Rasa3 Rap1GAP activity, thus allowing CalDAG-GEFI-mediated Rap1 activation to occur uninhibited. Activated GTP-bound Rap1 then promotes further signaling processes that lead to cell spreading. B, Rap1GAP-inactive forms of Rasa3 (shown in “red”), such as Rasa3 (H794L), are unable to mediate Rap1 inactivation, leading to enhanced cell spreading. C, inhibition of PI3K by wortmannin releases the Rap1GAP activity of Rasa3, thus enabling the conversion of Rap1 into its inactive GDP-bound state. As a result, cell spreading is severely reduced. Note that the role of PI3K in platelet spreading is upstream of Rasa3, and therefore cells containing intrinsically Rap1GAP-inactive forms of Rasa3 are insensitive to PI3K inhibition.

Similar articles

See all similar articles

Cited by 9 articles

See all "Cited by" articles

References

    1. Kucera G. L., and Rittenhouse S. E. (1990) Human platelets form 3-phosphorylated phosphoinositides in response to α-thrombin, U46619, or GTPγS. J. Biol. Chem. 265, 5345–5348 - PubMed
    1. Jackson S. P., Schoenwaelder S. M., Yuan Y., Rabinowitz I., Salem H. H., and Mitchell C. A. (1994) Adhesion receptor activation of phosphatidylinositol 3-kinase: von Willebrand factor stimulates the cytoskeletal association and activation of phosphatidylinositol 3-kinase and pp60c-src in human platelets. J. Biol. Chem. 269, 27093–27099 - PubMed
    1. Heraud J. M., Racaud-Sultan C., Gironcel D., Albigès-Rizo C., Giacomini T., Roques S., Martel V., Breton-Douillon M., Perret B., and Chap H. (1998) Lipid products of phosphoinositide 3-kinase and phosphatidylinositol 4′,5′-bisphosphate are both required for ADP-dependent platelet spreading. J. Biol. Chem. 273, 17817–17823 - PubMed
    1. Canobbio I., Stefanini L., Cipolla L., Ciraolo E., Gruppi C., Balduini C., Hirsch E., and Torti M. (2009) Genetic evidence for a predominant role of PI3Kβ catalytic activity in ITAM- and integrin-mediated signaling in platelets. Blood 114, 2193–2196 - PubMed
    1. Martin V., Guillermet-Guibert J., Chicanne G., Cabou C., Jandrot-Perrus M., Plantavid M., Vanhaesebroeck B., Payrastre B., and Gratacap M.-P. (2010) Deletion of the p110β isoform of phosphoinositide 3-kinase in platelets reveals its central role in Akt activation and thrombus formation in vitro and in vivo. Blood 115, 2008–2013 - PubMed

Publication types

MeSH terms

Substances

Feedback