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 PKC 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 β, γ and ζ 14-3-3 isoforms. In addition, low levels of the ε and η 14-3-3 isoforms were detected. In washed, saponin-permeabilized platelets incubated with [γ-32P]ATP, thrombin- and phorbol 12-myristate 13-acetate (PMA)-induced phosphorylation of several proteins (66, 45, and 20 kDa) 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.
Identification of 14-3-3 proteins in human platelets: effects of synthetic peptides on protein kinase C activation
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Caroline P. D. WHEELER-JONES, Michele P. LEARMONTH, Harry MARTIN, Alastair AITKEN; Identification of 14-3-3 proteins in human platelets: effects of synthetic peptides on protein kinase C activation. Biochem J 1 April 1996; 315 (1): 41–47. doi: https://doi.org/10.1042/bj3150041
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