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

Abstract

Summary: Background: 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. Objectives: 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. Conclusions: 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. ©2014 The Authors.