Identification of 14-3-3 proteins in human platelets: effects of synthetic peptides on protein kinase C activation.
The 14-3-3 proteins inhibit protein kinase C (PKC) activity in vitro and contain conserved sequences that resemble the pseudosubstrate domain of PKC and the C-terminus of the annexins. In the present study we have identified the isoforms of 14-3-3 in human platelets and used synthetic peptides derived from the regions with similarity to PKC and annexins to examine the potential role of 14-3-3 in regulating platelet activity. Immunoblotting studies with isoform-specific antisera raised against the acetylated peptides corresponding to the N-termini of 14-3-3 showed that these cells express high levels of the beta, gamma and zeta 14-3-3 isoforms. In addition, low levels of the epsilon and eta 14-3-3 isoforms were detected. In washed, saponin-permeabilized platelets incubated with [gamma-32P]ATP, thrombin- and phorbol 12-myristate 13-acetate (PMA)-induced phosphorylation of several proteins (66, 45, and 20kDa) was inhibited by preincubation with AS peptide (KNVVGARRSSWRVISSIEQK) based on the pseudosubstrate-like region of the 14-3-3 family. A control peptide of similar size had no effect on PKC-mediated phosphorylation. PMA caused a rapid translocation of PKC activity from the cytosol to the particulate fraction of saponin-permeabilized platelets that was unaffected by either the AS peptide or a peptide derived from the annexin-like 14-3-3 domain (MKGDYYRYLAEVATGDD). These results suggest that isoforms of the 14-3-3 family may play an important physiological role as inhibitors of PKC activity in human platelets but are unlikely to be involved in controlling association of PKC with the membrane.
Wheeler-Jones
C P
CP
Vascular Biology Research Centre, Biomedical Sciences Division, King's College London, U.K.
Learmonth
M P
MP
Martin
H
H
Aitken
A
A
D048948:14-3-3 Proteins; D000595:Amino Acid Sequence; D000818:Animals; D001792:Blood Platelets / Q000737:chemistry / Q000187:drug effects* / Q000201:enzymology*; D002463:Cell Membrane Permeability / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D007536:Isomerism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000494:pharmacology*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000097:blood* / Q000187:drug effects; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology*; D012503:Saponins / Q000494:pharmacology; D012756:Sheep; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014446:Tyrosine 3-Monooxygenase
D048948:14-3-3 Proteins; D010446:Peptide Fragments; D011506:Proteins; D012503:Saponins; D014446:Tyrosine 3-Monooxygenase; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate