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Cytohesin-2 phosphorylation by protein kinase C relieves the constitutive suppression of platelet dense granule secretion by ADP-ribosylation factor 6.

van den Bosch MT, Poole AW, Hers I - J. Thromb. Haemost. (2014)

Bottom Line: By western blotting we showed that different agonists induced cytohesin-2 phosphorylation by PKC.Flow cytometry data indicate that α-granule release and integrin αII b β3 activation were not affected by cytohesin-2 inhibition.As shown by western blotting, ARF6 interacted with cytohesin-2 and was present in an active GTP-bound form under basal conditions.

View Article: PubMed Central - PubMed

Affiliation: School of Physiology and Pharmacology, University of Bristol, Bristol, UK.

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Conventional PKC isoforms mediate the phosphorylation of cytohesin-2. Washed human platelets (4 × 108 mL−1) were treated for 15 min with 0.2% DMSO vehicle (Veh), the control BIM compound BIM V (10 μm), the broad-spectrum inhibitors PKC inhibitor BIM IX (2 μm), BIM I (5 μm) and Go 6983 (10 μm), or the PKCα/β selective inhibitor ruboxistaurin (10 μm) (A). Alternatively, mouse wild-type (WT) and PKCα knockout (PKCα−/−) mouse platelets (2 × 108 mL−1) were used (B). Platelets were stimulated with 0.2 U mL−1 α-thrombin (αT) in the presence of 10 μm ADP and lysed at the indicated time-points. Clarified whole cell lysates and immunoprecipitations using the pSer PKC substrate antibody were immunoblotted for cytohesin-2 (CYH2). The arrows (➤) indicate the 47 kDa CYH2 band and the asterisk (*) indicates a non-specific band. Results are representative of at least three independent experiments.
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fig02: Conventional PKC isoforms mediate the phosphorylation of cytohesin-2. Washed human platelets (4 × 108 mL−1) were treated for 15 min with 0.2% DMSO vehicle (Veh), the control BIM compound BIM V (10 μm), the broad-spectrum inhibitors PKC inhibitor BIM IX (2 μm), BIM I (5 μm) and Go 6983 (10 μm), or the PKCα/β selective inhibitor ruboxistaurin (10 μm) (A). Alternatively, mouse wild-type (WT) and PKCα knockout (PKCα−/−) mouse platelets (2 × 108 mL−1) were used (B). Platelets were stimulated with 0.2 U mL−1 α-thrombin (αT) in the presence of 10 μm ADP and lysed at the indicated time-points. Clarified whole cell lysates and immunoprecipitations using the pSer PKC substrate antibody were immunoblotted for cytohesin-2 (CYH2). The arrows (➤) indicate the 47 kDa CYH2 band and the asterisk (*) indicates a non-specific band. Results are representative of at least three independent experiments.

Mentions: In addition to BIM IX, other pharmacological inhibitors of PKC were used to assess phosphorylation of cytohesin-2. The broad-spectrum PKC inhibitors BIM I and Go 6983, as well as the PKCα/β selective inhibitor ruboxistaurin 24, blocked phosphorylation of cytohesin-2 upon stimulation (Fig.2A). These results suggest that cytohesin-2 phosphorylation is mediated through the conventional PKC isoform PKCα/β. Moreover, the inactive analogue (BIM V) did not affect cytohesin-2 phosphorylation, thereby serving as a control for non-specific effects. In addition, to further explore which PKC isoform is responsible for the phosphorylation of cytohesin-2, we performed similar experiments using PKCα knockout (PKCα−/−) mouse platelets (Fig.2B). Cytohesin-2 phosphorylation was, however, not affected in PKCα−/− platelets, but was blocked by ruboxistaurin, suggesting that PKCβ may be principally responsible for the phosphorylation of cytohesin-2.


Cytohesin-2 phosphorylation by protein kinase C relieves the constitutive suppression of platelet dense granule secretion by ADP-ribosylation factor 6.

van den Bosch MT, Poole AW, Hers I - J. Thromb. Haemost. (2014)

Conventional PKC isoforms mediate the phosphorylation of cytohesin-2. Washed human platelets (4 × 108 mL−1) were treated for 15 min with 0.2% DMSO vehicle (Veh), the control BIM compound BIM V (10 μm), the broad-spectrum inhibitors PKC inhibitor BIM IX (2 μm), BIM I (5 μm) and Go 6983 (10 μm), or the PKCα/β selective inhibitor ruboxistaurin (10 μm) (A). Alternatively, mouse wild-type (WT) and PKCα knockout (PKCα−/−) mouse platelets (2 × 108 mL−1) were used (B). Platelets were stimulated with 0.2 U mL−1 α-thrombin (αT) in the presence of 10 μm ADP and lysed at the indicated time-points. Clarified whole cell lysates and immunoprecipitations using the pSer PKC substrate antibody were immunoblotted for cytohesin-2 (CYH2). The arrows (➤) indicate the 47 kDa CYH2 band and the asterisk (*) indicates a non-specific band. Results are representative of at least three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4238808&req=5

fig02: Conventional PKC isoforms mediate the phosphorylation of cytohesin-2. Washed human platelets (4 × 108 mL−1) were treated for 15 min with 0.2% DMSO vehicle (Veh), the control BIM compound BIM V (10 μm), the broad-spectrum inhibitors PKC inhibitor BIM IX (2 μm), BIM I (5 μm) and Go 6983 (10 μm), or the PKCα/β selective inhibitor ruboxistaurin (10 μm) (A). Alternatively, mouse wild-type (WT) and PKCα knockout (PKCα−/−) mouse platelets (2 × 108 mL−1) were used (B). Platelets were stimulated with 0.2 U mL−1 α-thrombin (αT) in the presence of 10 μm ADP and lysed at the indicated time-points. Clarified whole cell lysates and immunoprecipitations using the pSer PKC substrate antibody were immunoblotted for cytohesin-2 (CYH2). The arrows (➤) indicate the 47 kDa CYH2 band and the asterisk (*) indicates a non-specific band. Results are representative of at least three independent experiments.
Mentions: In addition to BIM IX, other pharmacological inhibitors of PKC were used to assess phosphorylation of cytohesin-2. The broad-spectrum PKC inhibitors BIM I and Go 6983, as well as the PKCα/β selective inhibitor ruboxistaurin 24, blocked phosphorylation of cytohesin-2 upon stimulation (Fig.2A). These results suggest that cytohesin-2 phosphorylation is mediated through the conventional PKC isoform PKCα/β. Moreover, the inactive analogue (BIM V) did not affect cytohesin-2 phosphorylation, thereby serving as a control for non-specific effects. In addition, to further explore which PKC isoform is responsible for the phosphorylation of cytohesin-2, we performed similar experiments using PKCα knockout (PKCα−/−) mouse platelets (Fig.2B). Cytohesin-2 phosphorylation was, however, not affected in PKCα−/− platelets, but was blocked by ruboxistaurin, suggesting that PKCβ may be principally responsible for the phosphorylation of cytohesin-2.

Bottom Line: By western blotting we showed that different agonists induced cytohesin-2 phosphorylation by PKC.Flow cytometry data indicate that α-granule release and integrin αII b β3 activation were not affected by cytohesin-2 inhibition.As shown by western blotting, ARF6 interacted with cytohesin-2 and was present in an active GTP-bound form under basal conditions.

View Article: PubMed Central - PubMed

Affiliation: School of Physiology and Pharmacology, University of Bristol, Bristol, UK.

Show MeSH
Related in: MedlinePlus