Subcellular Localisation of 14-3-3 Isoforms in Rat Brain Using Specific Antibodies

Abstract: The 14-3-3 protein family, which is present at particularly high concentrations in mammalian brain, is known to be involved in various cellular functions, including protein kinase C regulation and exocytosis. Despite the fact that most of the 14-3-3 proteins are cytosolic, a small but significant proportion of 14-3-3 in brain is tightly and selectively associated with some membranes. Using a panel of isoform-specific antisera we find that the ε, η, γ, β, and ζ isoforms are all present in purified synaptic membranes but absent from mitochondrial and myelin membranes. In addition, the η, ε, and γ isoforms but not the β and ζ isoforms are associated with isolated synaptic junctions. When different populations of synaptosomes were fractionated by a nonequilibrium Percoll gradient procedure, the ε and γ isoforms were present and the β and ζ isoforms were absent from the membranes of synaptosomes sedimenting in the more dense parts of the gradient. The finding that these proteins are associated with different populations of synaptic membranes suggests that they are selectively expressed in different classes of neurones and raises the possibility that some or all of them may influence neurotransmission by regulating exocytosis and/or phosphorylation.     

Human 14-3-3 Protein: Radioimmunoassay, Tissue Distribution, and Cerebrospinal Fluid Levels in Patients with Neurological Disorders

Abstract: An antiserum to human 14-3-3 protein has been produced in rabbits. The protein was a poor antigen and attempts to improve immunogenicity were unsuccessful. A radioimmunoassay was developed using the antiserum, ¹²⁵I- 14-3-3-2, and unlabelled 14-3-3-2 as standards. The assay had a sensitivity limit of 2.5 ng.m1⁻¹. The minor component of human 14-3-3 protein (14-3-3-1 protein) cross-reacted to approximately 10% in the assay. Human tissues were surveyed for 14-3-3 protein by two-dimensional electrophoresis and by radioimmunoassay. Two-dimensional electrophoresis showed a 14-3-3 protein complex in brain, intestine, and testis, but not in other tissues. Radioimmunoassay showed that although brain had the highest concentration of 14-3-3 (13.3 μg. mg⁻¹ soluble protein), immunoreactivity was present in all tissues, with the concentration in intestine and testis approaching 50% of the brain level. Lower levels (less than 1.0 μg. mg⁻¹ soluble protein) were seen in liver, kidney, skeletal muscle, and erythrocytes. The immunoreactivity present in tissues other than brain showed the same molecular weight and charge characteristics as authentic 14-3-3 protein. The radioimmunoassay also detected 14-3-3 protein in serum (50 ng.m1⁻¹) and in CSF (5-130 ng.ml⁻¹). The immunoreactivity present in CSF appeared to be intact 14-3-3 protein. CSF 14-3-3 levels were measured in 82 patients with various neurological disorders. Measurements of this protein did not appear sufficiently discriminating to be o f diagnostic value.     

A Cdc2-related protein kinase hPFTAIRE1 from human brain interacting with 14-3-3 proteins

hPFTAIRE1 （PFTK1）, a Cdc2-related protein kinase, is highly expressed in human brain. It exhibits cytoplasmic distribution in Hela cells, although it contains two nuclear localization signals （NLSs） in its N-terminus. To search for its substrates and regulatory components, we screened a two-hybrid library by using the full-length hPFTAIRE1 as a bait. Four 14-3-3 isoforms （β,ε,η,τ） were identified interacting with the hPFTAIRE1. We found a putative 14-3-3 binding consensus motif（RHSSPSS） in the hPFTAIRE 1, which overlapped with its second NLS. Deletion of the RHSSPSS motif or substitution of Ser^119 gwithAla in the conserved binding motif abolished the specific interaction between the hPFTAIRE 1 and the 14-3 -3 proteins. The mutant S 120A hPFTAIRE1 also showed a weak interaction to the 14-3-3 proteins. The results suggested that the Ser^119 is crucial for the interaction between hPFTAIREI and the 14-3-3 proteins. All the hPFTAIRE1 mutants distributed in cytoplasm of Hela cells and human neuroblastoma cells （SH-SY5Y） when fused to the C-terminus of a green fluorescent protein （GFP）, indicating that binding with the 14-3-3 proteins does not contribute to the subcellular localization of the hPFTAIRE1, although the binding may be involved in its signaling regulation.     

Activation of Protein Kinase C by Purified Bovine Brain 14-3-3: Comparison with Tyrosine Hydroxylase Activation

Abstract: In the course of the purification of 14-3-3 protein (14-3-3) we found that 14-3-3 isolated from bovine forebrain activates protein kinase C (PKC), rather than the previously reported protein kinase C inhibitory activity (KCIP). We have characterized the 14-3-3 activation of PKC. The physical properties of purified PKC activator are the same as those previously reported for 14-3-3 and KCIP; i.e., (1) it is composed of subunits of molecular weight 32,000, 30,000, and 29,000; (2) it is homogeneous with respect to molecular weight, as judged by native gradient-gel electrophoresis, with a molecular weight of 53,000; and (3) it is composed of at least six isoforms when analyzed by reverse-phase HPLC. The concentration dependence of PKC activation by 14-3-3 is in the same range as that shown previously for KCIP inhibition of PKC, and as that required for 14-3-3 activation of tyrosine hydroxylase; a maximal stimulation of two- to three-fold occurs at 40–100 µg/ml. 14-3-3's activation of PKC is sensitive to α-chymotrypsin digestion but is not heat labile. Activation is specific to PKC; at least two other protein kinases, cyclic AMP- and calcium/calmodulin-dependent protein kinases, are not activated. The activation of PKC by 14-3-3 is independent of phosphatidylserine and calcium and, as such, is an alternative mechanism for the activation of PKC that obviates its translocation to membranes.     

14-3-3 binding to the IGF-1 receptor is mediated by serine autophosphorylation

The phosphoserine-binding 14-3-3 proteins have been implicated in playing a role in mitogenic and apoptotic signaling pathways. Binding of 14-3-3 proteins to phosphoserine residues in the C-terminus of the insulin-like growth factor-1 receptor (IGF-1R) has been described to occur in a variety of cell systems, but the kinase responsible for this serine phosphorylation has not been identified yet. Here we present evidence that the isolated dimeric insulin-like growth factor-1 receptor kinase domain (IGFKD) contains a dual specific (i.e. tyrosine/serine) kinase activity that mediates autophosphorylation of C-terminal serine residues in the enzyme. From the total phosphate incorporation of approximately 4 mol per mol kinase subunit, 1 mol accounts for serine phosphate. However, tyrosine autophosphorylation proceeds more rapidly than autophosphorylation of serine residues (t(1/2) approximately 1 min vs. t(1/2) approximately 5 min). Moreover, dot-blot and far-Western analyses reveal that serine autophosphorylation of IGFKD is sufficient to promote binding of 14-3-3 proteins in vitro. The proof that dual kinase activity of IGFKD is necessary and sufficient for 14-3-3 binding was obtained with an inactive kinase mutant that was phosphorylated on serine residues in a stoichiometric reaction with the catalytically active enzyme. Thus, the IGF-1R itself might be responsible for the serine autophosphorylation which leads to recognition of 14-3-3 proteins in vivo.     

Phosphorylation and 14-3-3 binding of Arabidopsis trehalose-phosphate synthase 5 in response to 2-deoxyglucose

Trehalose-6-phosphate is a 'sugar signal' that regulates plant metabolism and development. The Arabidopsis genome encodes trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphatase (TPP) enzymes. It also encodes class II proteins (TPS isoforms 5-11) that contain both TPS-like and TPP-like domains, although whether these have enzymatic activity is unknown. In this paper, we show that TPS5, 6 and 7 are phosphoproteins that bind to 14-3-3 proteins, by using 14-3-3 affinity chromatography, 14-3-3 overlay assays, and by co-immunoprecipitating TPS5 and 14-3-3 isoforms from cell extracts. GST-TPS5 bound to 14-3-3s after in vitro phosphorylation at Ser22 and Thr49 by either mammalian AMP-activated protein kinase (AMPK) or partially purified plant Snf1-related protein kinase 1 (SnRK1s). Dephosphorylation of TPS5, or mutation of either Ser22 or Thr49, abolished binding to 14-3-3s. Ser22 and Thr49 are both conserved in TPS5, 7, 9 and 10. When GST-TPS5 was expressed in human HEK293 cells, Thr49 was phosphorylated in response to 2-deoxyglucose or phenformin, stimuli that activate the AMPK via the upstream kinase LKB1. 2-deoxyglucose stimulated Thr49 phosphorylation of endogenous TPS5 in Arabidopsis cells, whereas phenformin did not. Moreover, extractable SnRK1 activity was increased in Arabidopsis cells in response to 2-deoxyglucose. The plant kinase was inactivated by dephosphorylation and reactivated by phosphorylation with human LKB1, indicating that elements of the SnRK1/AMPK pathway are conserved in Arabidopsis and human cells. We hypothesize that coordinated phosphorylation and 14-3-3 binding of nitrate reductase (NR), 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (F2KP) and class II TPS isoforms mediate responses to signals that activate SnRK1.     

Post-translationally modified 14-3-3 isoforms and inhibition of protein kinase C

This report compares the ability of individual members of the 14-3-3 protein family to inhibit particular protein kinase C (PKC) isoforms. We also show that two of these 14-3-3 isoforms ( and ) specific to mammalian and avian brain arein vivo post-translationally modified forms of and respectively. The presence of this modification enhances the activity of 14-3-3 as an inhibitor of protein kinase C nearly two fold.A method for analysing isoforms of 14-3-3 on acid-urea gels is also described. This permits the complete separation of all major isoforms and their unequivocal identification by a range of isoform specific antisera. The activity of recombinant 14-3-3 and isoforms renatured by a novel method after separation by reverse phase HPLC are compared. The effects of diacylglycerol and the phorbol ester, PMA (phorbol 12-myristate 13 acetate) on the inhibition suggest that one of the sites of interaction of 14-3-3 may be the cysteine-rich (C1) domain in PKC.     

Regulation of Dyrk1A kinase activity by 14-3-3

Dual-specificity tyrosine(Y) regulated kinase 1A (DYRK1A) is a serine/threonine protein kinase implicated in mental retardation resulting from Down syndrome. In this study, we carried out yeast two-hybrid screening to find proteins regulating DYRK1A kinase activity. We identified 14-3-3 as a Dyrk1A interacting protein, which is consistent with the previous finding of the interaction between the yeast orthologues Yak1p and Bmh1/2p. We showed the interaction between Dyrk1A and 14-3-3 in vitro and in vivo. The binding required the N-terminus of Dyrk1A and was independent of the Dyrk1A phosphorylation status. Functionally, 14-3-3 binding increased Dyrk1A kinase activity in a dose dependent manner in vitro. In vivo, a small peptide inhibiting 14-3-3 binding, sc138, decreased Dyrk1A kinase activity in COS7. In summary, these results suggest that DYRK1A kinase activity could be regulated by the interaction of 14-3-3.