title: Cytohesin-2 phosphorylation by protein kinase C relieves the constitutive suppression of platelet dense granule secretion by ADP-ribosylation factor 6.
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.
  
  
  OBJECTIVE
  We aimed to validate cytohesin-2 as a PKC substrate in platelets and to determine its role in granule secretion and other platelet responses.
  
  
  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.
authors.affiliation: School of Physiology and Pharmacology, University of Bristol, Bristol, UK.
authors.forename: M T J
authors.initials: MT
authors.lastname: van den Bosch
authors.forename: A W
authors.initials: AW
authors.lastname: Poole
authors.forename: I
authors.initials: I
authors.lastname: Hers
mesh_terms: D000089982:ADP-Ribosylation Factor 6; D000199:Actins / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001792:Blood Platelets / Q000166:cytology / Q000378:metabolism*; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism*; D005434:Flow Cytometry; D020690:GTPase-Activating Proteins / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D008247:Lysosomes / Q000378:metabolism; D013058:Mass Spectrometry; D051379:Mice; D018345:Mice, Knockout; D010766:Phosphorylation; D015539:Platelet Activation; D010974:Platelet Aggregation; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D040901:Proteomics; D022142:Secretory Vesicles / Q000378:metabolism; D001619:beta-N-Acetylhexosaminidases / Q000378:metabolism
chemical_list: C431141:ACTR6 protein, human; D000089982:ADP-Ribosylation Factor 6; D000199:Actins; D002868:Chromosomal Proteins, Non-Histone; D020690:GTPase-Activating Proteins; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; C102474:cytohesin-2; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D011493:Protein Kinase C; D001619:beta-N-Acetylhexosaminidases; C000715348:ARF6 protein, human; C000715727:Arf6 protein, mouse
keywords: ADP-ribosylation factor 6; cytohesin-2; platelets; protein kinase C; secretion