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.
Casein kinases I (CKI) are serine/threonine protein kinases widely expressed in a range of eukaryotes including yeast, mammals and plants. They have been shown to play a role in diverse physiological events including membrane trafficking. CKIa is associated with synaptic vesicles and phosphorylates some synaptic vesicle associated proteins including SV2. In this report, we show that syntaxin-1A is phosphorylated in vitro by CKI on Thr21. Casein kinase II (CKII) has been shown previously to phosphorylate syntaxin-1A in vitro and we have identified Ser14 as the CKII phosphorylation site, which is known to be phosphorylated in vivo. As syntaxin-1A plays a key role in the regulation of neurotransmitter release by forming part of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex, we propose that CKI may play a role in synaptic vesicle exocytosis.Keywords: CKI; CKII; syntaxin-1A; trafficking.Casein kinase I (CKI) belongs to a family of serine/ threonine protein kinases with seven isoforms identified in mammals (CKI a, b, d, e, c1, c2, and c3; reviewed in [1]). The kinase domain is highly conserved between members of the CKI family but unique N-and C-terminal tails characterize each isoform. In yeast, the functions of CKI have been much more extensively studied compared to their mammalian counterparts. Recently, many reports have linked yeast CKIs to cytokinesis and vesicle trafficking especially in endocytosis [2][3][4][5][6][7][8][9]. Mammalian CKIs appear to have similar functions and also have been involved in DNA repair, circadian rhythms, and wnt signaling. Like their yeast counterparts, CKIcs have been implicated in cytokinesis and in membrane trafficking [10]. CKIa interacts with and phosphorylates the clathrin adapter that is involved in endocytosis. CKIa has been found to colocalize in neurones with synaptic vesicle markers and to phosphorylate some synaptic vesicle associated proteins including SV2 [12]. More importantly, the phosphorylation of SV2 by CKI modulates its ability to interact with synaptotagmin [13]. SV2 plays a role in neurotransmitter release suggesting a role for CKI in this biological process. We have recently identified centaurin-a 1 , a protein shown to associate with presynaptic vesicular structures [14], as a novel CKI partner [15].In this report, we have identified syntaxin-1A as a novel substrate for CKI, which further supports a role for CKI in membrane trafficking. Indeed, the involvement of syntaxin-1A in neurotransmitter release is well documented (reviewed in [16,17]). Regulated neurotransmitter secretion is the key step in synaptic transmission and is the basis of intercellular communication in the nervous system. Synaptic vesicle exocytosis is regulated by Ca 2+ and by a large number of proteins (reviewed in [17,18]). Syntaxin-1A is associated with the presynaptic membrane and associates with the plasma membrane protein SNAP-25 and the synaptic vesicle protein synaptobrevin to form a ÔSNARE complexÕ. Assembly of this complex is necessary ...
The 14-3-3 family are homo- and heterodimeric proteins whose biological role has been unclear for some time, although they are now gaining acceptance as a novel type of 'adaptor' protein that modulates interactions between components of signal transduction pathways, rather than by direct activation or inhibition. It is becoming apparent that phosphorylation of the binding partner and possibly also the 14-3-3 proteins may regulate these interactions. 14-3-3 isoforms interact with a novel phosphoserine (Sp) motif on many proteins, RSX1,2SpXP. The two isoforms that interact with Raf-1 are phosphorylated in vivo on Ser185 in a consensus sequence motif for proline-directed kinases. The crystal structure of 14-3-3 indicates that this phosphorylation could regulate interaction of 14-3-3 with its target proteins. We have now identified a number of additional phosphorylation sites on distinct mammalian and yeast isoforms.
An antithrombin (AT) variant Ala382 to Thr (AT-TRI) was identified by mass spectrometric techniques. The variant behaved as a substrate rather than a thrombin inhibitor, but, contrary to previously described P12 AT variants, AT-TRI, expressed as a heterozygous dominant trait, caused severe thromboembolic tendency beginning in their teens in affected members of an English family. In addition, it underwent the S-to-R conformational state transition as evidenced by an increased resistance to thermal denaturation on active centre cleavage, but did not react with a monoclonal antibody, 4C9, directed against a neoepitope that is present on complexed and cleaved normal AT. Antithrombin-TRI, in plasma, was also associated with an abnormal high molecular weight (M(r)) 194,000) component composed of non-covalently-linked antithrombin molecules. This component (D194) showed low affinity for heparin and was devoid of antithrombin progressive activity. D194, isolated by ammonium sulphate precipitation and three chromatographic steps (heparin Sepharose, ion exchange and immunoaffinity), migrated as a single band of M(r) 60,000 on SDS-PAGE under both reducing and non-reducing conditions and was recognized by monospecific anti-human antithrombin antibodies, but did not immunoreact with antibodies raised against a number of proteins including albumin and thrombin. The above data and the fact that the 15 N-terminal amino acids of this M(r) 60,000 band were identical to that of normal antithrombin indicated that the inactive D194 component was composed of aggregated antithrombin molecules, possibly antithrombin trimers. In conclusion, early adulthood severe thromboembolic tendency, failure to expose the 4C9 epitope, and presence of aggregated AT molecules in the plasma are characteristic features of AT-TRI not previously described in other ALA-382 THR mutations.
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