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Publication - Professor Ingeborg Hers

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

    Citation

    van den Bosch, MTJ, Poole, AW & Hers, I, 2014, ‘Cytohesin-2 phosphorylation by protein kinase C relieves the constitutive suppression of platelet dense granule secretion by ADP-ribosylation factor 6’. Journal of Thrombosis and Haemostasis, vol 12., pp. 726-35

    Abstract

    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 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 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.

    Full details in the University publications repository