Manuscript handled by: C. Gachet
Cytohesin-2 phosphorylation by protein kinase C relieves the constitutive suppression of platelet dense granule secretion by ADP-ribosylation factor 6
Article first published online: 22 MAY 2014
©2014 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.
Journal of Thrombosis and Haemostasis
Volume 12, Issue 5, pages 726–735, May 2014
How to Cite
Cytohesin-2 phosphorylation by protein kinase C relieves the constitutive suppression of platelet dense granule secretion by ADP-ribosylation factor 6. J Thromb Haemost 2014; 12: 726–35., , .
Final decision: P. H. Reitsma, 19 February 2014
- Issue published online: 22 MAY 2014
- Article first published online: 22 MAY 2014
- Accepted manuscript online: 3 MAR 2014 04:06AM EST
- Manuscript Accepted: 19 FEB 2014
- Manuscript Received: 17 OCT 2013
- British Heart Foundation. Grant Numbers: FS/11/62/28934, RG/10/006/28299
- ADP-ribosylation factor 6;
- protein kinase C;
Protein kinase C (PKC) is a major regulator of platelet function and secretion. The underlying molecular pathway from PKC to secretion, however, is poorly understood. By a proteomics screen we identified the guanine nucleotide exchange factor cytohesin-2 as a candidate PKC substrate.
We aimed to validate cytohesin-2 as a PKC substrate in platelets and to determine its role in granule secretion and other platelet responses.
Methods and results
Immunoprecipitation was performed with a phosphoserine PKC substrate antibody followed by mass spectrometry, leading to the identification of cytohesin-2. By western blotting we showed that different agonists induced cytohesin-2 phosphorylation by PKC. Protein function was investigated using a pharmacological approach. The cytohesin inhibitor SecinH3 significantly enhanced platelet dense granule secretion and aggregation, as measured by lumi-aggregometry. Flow cytometry data indicate that α-granule release and integrin αIIbβ3 activation were not affected by cytohesin-2 inhibition. Lysosome secretion was assessed by a colorimetric assay and was also unchanged. As shown by western blotting, ARF6 interacted with cytohesin-2 and was present in an active GTP-bound form under basal conditions. Upon platelet stimulation, this interaction was largely lost and ARF6 activation decreased, both of which could be rescued by PKC inhibition.
Cytohesin-2 constitutively suppresses platelet dense granule secretion and aggregation by keeping ARF6 in a GTP-bound state. PKC-mediated phosphorylation of cytohesin-2 relieves this inhibitory effect, thereby promoting platelet secretion and aggregation.