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

BACKGROUND:Protein kinase C is a major regulator of platelet function and secretion. The underlying molecular pathway from to secretion, however, is poorly understood. By a proteomics screen we identified the guanine nucleotide exchange factor cytohesin-2 as a candidate duanyu1531 substrate. OBJECTIVES:We aimed to validate cytohesin-2 as a duanyu1531 substrate in platelets and to determine its role in granule secretion and other platelet responses. METHODS AND RESULTS:Immunoprecipitation was performed with a phosphoserine duanyu1531 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 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 αII b β3 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 duanyu1531 inhibition. CONCLUSIONS:Cytohesin-2 constitutively suppresses platelet dense granule secretion and aggregation by keeping ARF6 in a GTP-bound state. phosphorylation of cytohesin-2 relieves this inhibitory effect, thereby promoting platelet secretion and aggregation. ©2014 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis.

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