Identification of 14-3-3 proteins in human platelets: effects of synthetic peptides on protein kinase C activation

Abstract

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.

Protein Kinase C μ Is Negatively Regulated by 14-3-3 Signal Transduction Proteins

“…14-3-3 binding to several signal transducers (7-10) including PKC isotypes (18,41,42) has been reported, but controversial data exist as to the functional role of these interactions (7,8,41). Of relevance to the findings reported here, 14-3-3 has been described to inhibit PKC regulated interleukin-2 expression in T cells by preventing its translocation to the membrane (18).…”

Expression of 14-3-3 ζ and interaction with protein kinase C in the rat retina in early diabetes

“…Many investigators have reported that 14-3-3s act as potential regulators of PKC, which is controlled by the interactions between them [10][11][12][13]. However, the effects of 14-3-3 on PKC have been controversial, showing conflicting results of inhibition or activation [5,9,13], and the biological significance of this event remains unclear.…”

“…Moreover, this isotype is present in significant amounts in the retina, as well as in the brain, and is necessary for light adaptation processes or differentiation in photoreceptors [7,8]. It is well established that 14-3-3 proteins function as a protein kinase C (PKC) regulator [9][10][11][12][13]. In vitro, 14-3-3 ζ has been reported to be an endogenous PKC inhibitor [5,14], but the role of 14-3-3 ζ in the diabetic retina remains unclear.…”

14-3-3ζ Mediates Integrin-induced Activation of Cdc42 and Rac

“…Platelets contain high levels of the , ␤, and ␥ isoforms and lower levels of the ⑀ and isoforms of 14-3-3 (2). The only isoform that has been shown to interact with GP Ib␣ is 14-3-3.…”

“…Cas , BAD, and phosphatidylinositol 3-kinase (1)(2)(3)(4)(5), proteins such as Cdc25 and Wee1 that are involved in cell cycle control (6,7), and proteins such as FKHRLl and DAF-16 (8,9) that are involved in regulation of transcription.…”