Translocation of pleckstrin requires its phosphorylation and newly formed ligands.
Pleckstrin is the major substrate of protein kinase C (PKC) in platelets. We sought to determine whether pleckstrin phosphorylation is sufficient to target the soluble protein to binding sites. Permeabilization of platelets by streptolysin O (SLO) was used to separate bound and soluble pleckstrin. Platelets were incubated with phorbol 12-myristate 13-acetate (PMA) and/or guanosine 5'-[gamma-thio]triphosphate (GTP[S]) in the presence of [gamma-(32)P]ATP and SLO. PMA stimulated pleckstrin phosphorylation, but this pleckstrin diffused from permeabilized platelets. Addition of GTP[S] with PMA caused up to 40-50% of pleckstrin to be retained within platelets and enhanced secretion of platelet 5-hydroxytryptamine. PKC alpha pseudosubstrate peptide inhibited pleckstrin phosphorylation, the binding of pleckstrin and secretion. After extraction of permeabilized platelets containing bound pleckstrin with Triton X-100, the protein was solubilized. Thus, phosphorylated pleckstrin was retained in platelets only after activation of GTP-binding proteins that stimulate the formation of membrane-bound pleckstrin ligands. Translocation of pleckstrin may facilitate the associated secretion.
Sloan
Denis C
DC
Department of Biochemistry, McMaster University, Hamilton, ON, Canada L8N 3Z5.
Wang
Ping
P
Bao
Xiankun
X
Haslam
Richard J
RJ
D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D001798:Blood Proteins / Q000378:metabolism*; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000502:physiology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006801:Humans; D008024:Ligands; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D021381:Protein Transport; D011994:Recombinant Proteins / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D001798:Blood Proteins; D008024:Ligands; D010750:Phosphoproteins; D011994:Recombinant Proteins; C039077:platelet protein P47; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D013755:Tetradecanoylphorbol Acetate