Cytohesin-2 phosphorylation by protein kinase C relieves the constitutive suppression of platelet dense granule secretion by ADP-ribosylation factor 6.
Bottom Line: By western blotting we showed that different agonists induced cytohesin-2 phosphorylation by PKC.Flow cytometry data indicate that α-granule release and integrin αII b β3 activation were not affected by cytohesin-2 inhibition.As shown by western blotting, ARF6 interacted with cytohesin-2 and was present in an active GTP-bound form under basal conditions.
Affiliation: School of Physiology and Pharmacology, University of Bristol, Bristol, UK.Show MeSH
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Mentions: In addition to BIM IX, other pharmacological inhibitors of PKC were used to assess phosphorylation of cytohesin-2. The broad-spectrum PKC inhibitors BIM I and Go 6983, as well as the PKCα/β selective inhibitor ruboxistaurin 24, blocked phosphorylation of cytohesin-2 upon stimulation (Fig.2A). These results suggest that cytohesin-2 phosphorylation is mediated through the conventional PKC isoform PKCα/β. Moreover, the inactive analogue (BIM V) did not affect cytohesin-2 phosphorylation, thereby serving as a control for non-specific effects. In addition, to further explore which PKC isoform is responsible for the phosphorylation of cytohesin-2, we performed similar experiments using PKCα knockout (PKCα−/−) mouse platelets (Fig.2B). Cytohesin-2 phosphorylation was, however, not affected in PKCα−/− platelets, but was blocked by ruboxistaurin, suggesting that PKCβ may be principally responsible for the phosphorylation of cytohesin-2.
Affiliation: School of Physiology and Pharmacology, University of Bristol, Bristol, UK.