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

M T J van den Bosch, A W Poole, I Hers

Research output: Contribution to journalArticle (Academic Journal)

11 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)726-35
Number of pages10
JournalJournal of Thrombosis and Haemostasis
Volume12
Issue number5
DOIs
Publication statusPublished - May 2014

Bibliographical note

©2014 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis.

Fingerprint Dive into the research topics of 'Cytohesin-2 phosphorylation by protein kinase C relieves the constitutive suppression of platelet dense granule secretion by ADP-ribosylation factor 6'. Together they form a unique fingerprint.

Cite this