Chemical cross-linking of pleckstrin in human platelets: evidence for oligomerization of the protein and its dissociation by protein kinase C.
The major substrate of protein kinase C(PKC) in platelets is the 40 kDa protein, pleckstrin. Addition of the homobifunctional reagent, bis(sulphosuccinimidyl)suberate (BS3), to platelet lysate, cytosol fraction or to electropermeabilized platelets resulted in cross-linking of pleckstrin to give higher-molecular-mass complexes of 68 kDa, 90 kDa and 100-120 kDa respectively, which were visualized by immunoblotting with an anti-pleckstrin antibody. Higher levels of cross-linking were observed in permeabilized platelets than in platelet lysates. The yields of the cross-linked complexes were much reduced after dilution of platelet lysate or lysis of electropermeabilized platelets and, in the case of the 90 kDa and 100-120 kDa species, after activation of PKC by phorbol 12-myristate 13-acetate. Similar experiments with purified pleckstrin indicated that the 90 kDa and 100-120 kDa species consist, at least in part, of pleckstrin dimers and higher oligomers. After incubation of purified pleckstrin (0.45 mg/ml) for 1 h with 2 mM BS3, about 25% of the protein was present in cross-linked species. The results indicate that pleckstrin undergoes a reversible self-association that can be prevented by phosphorylation of the protein, and also interacts with an unidentified platelet protein of about 28 kDa.
McDermott
A M
AM
Department of Biochemistry, McMaster University, Hamilton, Ontario, Canada.
Haslam
R J
RJ
D001792:Blood Platelets / Q000737:chemistry / Q000378:metabolism*; D001798:Blood Proteins / Q000737:chemistry / Q000378:metabolism*; D002463:Cell Membrane Permeability; D003432:Cross-Linking Reagents; D004789:Enzyme Activation; D006801:Humans; D010750:Phosphoproteins; D011487:Protein Conformation; D011493:Protein Kinase C / Q000187:drug effects / Q000378:metabolism*; D013347:Subcellular Fractions / Q000737:chemistry / Q000378:metabolism; D013388:Succinimides; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D001798:Blood Proteins; D003432:Cross-Linking Reagents; D010750:Phosphoproteins; D013388:Succinimides; C039077:platelet protein P47; C035760:bis(sulfosuccinimidyl)suberate; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate