scholarly journals Bioinformatic and experimental survey of 14-3-3-binding sites

2010 ◽  
Vol 427 (1) ◽  
pp. 69-78 ◽  
Author(s):  
Catherine Johnson ◽  
Sandra Crowther ◽  
Margaret J. Stafford ◽  
David G. Campbell ◽  
Rachel Toth ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinases ◽  
Binding Sites ◽  
Histone Deacetylases ◽  
Cognitive Disorders ◽  
Logic Gates ◽  
Protein Families ◽  
Kinase C ◽  
Dependent Protein ◽  
Physiological Demands

More than 200 phosphorylated 14-3-3-binding sites in the literature were analysed to define 14-3-3 specificities, identify relevant protein kinases, and give insights into how cellular 14-3-3/phosphoprotein networks work. Mode I RXX(pS/pT)XP motifs dominate, although the +2 proline residue occurs in less than half, and LX(R/K)SX(pS/pT)XP is prominent in plant 14-3-3-binding sites. Proline at +1 is rarely reported, and such motifs did not stand up to experimental reanalysis of human Ndel1. Instead, we discovered that 14-3-3 interacts with two residues that are phosphorylated by basophilic kinases and located in the DISC1 (disrupted-in-schizophrenia 1)-interacting region of Ndel1 that is implicated in cognitive disorders. These data conform with the general findings that there are different subtypes of 14-3-3-binding sites that overlap with the specificities of different basophilic AGC (protein kinase A/protein kinase G/protein kinase C family) and CaMK (Ca2+/calmodulin-dependent protein kinase) protein kinases, and a 14-3-3 dimer often engages with two tandem phosphorylated sites, which is a configuration with special signalling, mechanical and evolutionary properties. Thus 14-3-3 dimers can be digital logic gates that integrate more than one input to generate an action, and coincidence detectors when the two binding sites are phosphorylated by different protein kinases. Paired sites are generally located within disordered regions and/or straddle either side of functional domains, indicating how 14-3-3 dimers modulate the conformations and/or interactions of their targets. Finally, 14-3-3 proteins bind to members of several multi-protein families. Two 14-3-3-binding sites are conserved across the class IIa histone deacetylases, whereas other protein families display differential regulation by 14-3-3s. We speculate that 14-3-3 dimers may have contributed to the evolution of such families, tailoring regulatory inputs to different physiological demands.

scholarly journals Prostacyclin inhibits platelet aggregation induced by phorbol ester or Ca2+ ionophore at steps distal to activation of protein kinase C and Ca2+-dependent protein kinases

1989 ◽  
Vol 258 (1) ◽  
pp. 57-65 ◽  
Author(s):  
W Siess ◽  
E G Lapetina
Keyword(s):  
Protein Kinase C ◽  
Platelet Aggregation ◽  
Protein Kinase ◽  
Protein Kinases ◽  
Shape Change ◽  
Ionophore A23187 ◽  
Maximal Effect ◽  
Kinase C ◽  
Dependent Protein ◽  
High Concentrations

Suspensions of aspirin-treated, 32P-prelabelled, washed platelets containing ADP scavengers in the buffer were activated with either phorbol 12,13-dibutyrate (PdBu) or the Ca2+ ionophore A23187. High concentrations of PdBu (greater than or equal to 50 nM) induced platelet aggregation and the protein kinase C (PKC)-dependent phosphorylation of proteins with molecular masses of 20 (myosin light chain), 38 and 47 kDa. No increase in cytosolic Ca2+ was observed. Preincubation of platelets with prostacyclin (PGI2) stimulated the phosphorylation of a 50 kDa protein [EC50 (concn. giving half-maximal effect) 0.6 ng of PGI2/ml] and completely abolished platelet aggregation [ID50 (concn. giving 50% inhibition) 0.5 ng of PGI2/ml] induced by PdBu, but had no effect on phosphorylation of the 20, 38 and 47 kDa proteins elicited by PdBu. The Ca2+ ionophore A23187 induced shape change, aggregation, mobilization of Ca2+, rapid phosphorylation of the 20 and 47 kDa proteins and the formation of phosphatidic acid. Preincubation of platelets with PGI2 (500 ng/ml) inhibited platelet aggregation, but not shape change, Ca2+ mobilization or the phosphorylation of the 20 and 47 kDa proteins induced by Ca2+ ionophore A23187. The results indicate that PGI2, through activation of cyclic AMP-dependent kinases, inhibits platelet aggregation at steps distal to protein phosphorylation evoked by protein kinase C and Ca2+-dependent protein kinases.

Activation of Endogenous Protein Kinase C by Halothane in Synaptosomes

1996 ◽  
Vol 84 (3) ◽  
pp. 652-662 ◽  
Author(s):  
Hugh C. Hemmings ◽  
Anna I. B. Adamo
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Protein Kinases ◽  
Synaptic Membranes ◽  
Dependent Protein Kinase ◽  
Kinase C ◽  
Endogenous Protein ◽  
Dependent Protein ◽  
Protein Kinase C Activation ◽  
Synapsin 1

Background Protein kinase C is a signal transducing enzyme that is an important regulator of multiple physiologic processes and a potential molecular target for general anesthetic actions. However, the results of previous studies of the effects of general anesthetics on protein kinase C activation in vitro have been inconsistent. Methods The effects of halothane on endogenous brain protein kinase C activation were analyzed in isolated rat cerebrocortical nerve terminals (synaptosomes) and in synaptic membranes. Protein kinase C activation was monitored by the phosphorylation of MARCKS, a specific endogenous substrate. Results Halothane stimulated basal Ca2+ dependent phosphorylation of MARCKS (Mr = 83,000) in lysed synaptic membranes (2.1-fold; P< 0.01) and in intact synaptosomes (1.4-fold; P< 0.01). The EC50 for stimulation of MARCKS phosphorylation by halothene in synaptic membranes was 1.8 vol%. A selective peptide protein kinase C inhibitor, but not a protein phosphatase inhibitor (okadaic acid) or a peptide inhibitor of Ca2+/calmodulin-dependent protein kinase II, another Ca2+/-dependent signal transducing enzyme, blocked halothane-stimulated MARCKS phosphorylation in synaptic membranes. Halothane did not affect the phosphorylation of synapsin 1, a synaptic vesicle-associated protein substrate for Ca2+/calmodulin-dependent protein kinase II and AMP-dependent protein kinase, in synaptic membranes or intact synaptosomes subjected to KC1-evoked depolarization. However, halothane stimulated synapsin 1 phosphorylation evoked by ionomycin (a Ca2+ ionophore that permeabilizes membranes to Ca2+) in intact synaptosomes. Conclusions Halothane acutely stimulated basal protein kinase C activity in synaptosomes when assayed with endogenous nerve terminal substrates, lipids, and protein kinase C. This effect appeared to be selective for protein kinases C, because two other structurally similar second messenger-regulated protein kinases were not affected. Direct determinations of anesthetic effects on endogenous protein kinase C activation, translocation, and/or down-regulation are necessary to determine the ultimate effect of anesthetics on the protein kinase C signaling pathway in intact cells.

ANP stimulates phospholipase C in cultured RIMCT cells: roles of protein kinases and G protein

1991 ◽  
Vol 260 (4) ◽  
pp. F590-F595 ◽  
Author(s):  
T. Berl ◽  
J. Mansour ◽  
I. Teitelbaum
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Protein Kinases ◽  
G Protein ◽  
Pertussis Toxin ◽  
Cyclic Nucleotide ◽  
Dependent Protein Kinase ◽  
Kinase C ◽  
Dependent Protein ◽  
Stimulation Of

We examined the possibility that, in addition to stimulation of guanylate cyclase (GC), atrial natriuretic peptide (ANP) also activates phospholipase C (PLC) in cultured rat inner medullary collecting tubule (RIMCT) cells. ANP (10(-12)M) causes marked release of inositol trisphosphate (IP3) at a concentration that does not stimulate GC. Concentrations of ANP that stimulate GC (greater than or equal to 10(-10) M) result in attenuated IP3 release. Similarly, exogenous dibutyryl guanosine 3',5'-cyclic monophosphate (10(-6) M) markedly inhibits the response to 10(-10) M ANP. Inhibition of cyclic nucleotide-dependent protein kinase by H 8, but not inhibition of protein kinase C by H 7, restores the response to 10(-8) and 10(-6) M ANP. Therefore, activation of cyclic nucleotide-dependent protein kinase inhibits ANP-stimulated PLC activity. Activation of protein kinase C by phorbol 12-myristate-13-acetate (PMA) decreases ANP-stimulated IP3 production. Pretreatment with H 7, but not H 8, prevents inhibition by PMA. To explore a potential role for G proteins, we examined the effect of guanine nucleotide analogues on ANP-stimulated IP3 production in saponin-permeabilized cells. ANP-stimulated IP3 production is enhanced by GTP gamma S and is inhibited by GDP beta S. Similarly, preincubation with pertussis toxin prevents ANP-stimulated IP3 release. We conclude that ANP stimulates PLC in RIMCT cells via a pertussis toxin-sensitive G protein. Stimulation of PLC is inhibited on activation of either cyclic nucleotide or Ca2+-phospholipid dependent protein kinases.

scholarly journals Isolation and identification of a novel cellular protein that potently activates Ca2+/phospholipid-dependent protein kinase (protein kinase C)

1991 ◽  
Vol 279 (3) ◽  
pp. 695-698 ◽  
Author(s):  
S A Goueli
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Binding Sites ◽  
Reaction Medium ◽  
Cellular Protein ◽  
Isolation And Identification ◽  
Dependent Protein Kinase ◽  
Heat Stable ◽  
Kinase C ◽  
Dependent Protein

A novel heat-stable cellular protein that significantly stimulated Ca2+/phospholipid-dependent protein kinase (protein kinase C, PKC) was identified. It is ubiquitous and has a molecular mass of over 150 kDa, and was shown to be specific for PKC. It activates PKC in the absence and presence of phospholipids; however, its maximal stimulatory potential was achieved when optimal amounts of phospholipids were also present in the reaction medium. Thus, in comparison with classical cofactors such as phospholipid, where activation of PKC mainly involves interaction with the regulatory domain of PKC, the mechanism of activation of PKC by the activator appears to involve several binding sites.

scholarly journals Dystrophin is phosphorylated by endogenous protein kinases

1993 ◽  
Vol 293 (1) ◽  
pp. 243-247 ◽  
Author(s):  
M Luise ◽  
C Presotto ◽  
L Senter ◽  
R Betto ◽  
S Ceoldo ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Protein Kinases ◽  
Cyclic Gmp ◽  
Casein Kinase ◽  
Cytoskeletal Proteins ◽  
Kinase C ◽  
Endogenous Protein ◽  
Dependent Protein

Dystrophin, the protein coded by the gene missing in Duchenne muscular dystrophy, is assumed to be a component of the membrane cytoskeleton of skeletal muscle. Like other cytoskeletal proteins in different cell types, dystrophin bound to sarcolemma membranes was found to be phosphorylated by endogenous protein kinases. The phosphorylation of dystrophin was activated by cyclic AMP, cyclic GMP, calcium and calmodulin, and was inhibited by cyclic AMP-dependent protein kinase peptide inhibitor, mastoparan and heparin. These results suggest that membrane-bound dystrophin is a substrate of endogenous cyclic AMP- and cyclic GMP-dependent protein kinases, calcium/calmodulin-dependent kinase and casein kinase II. The possibility that dystrophin could be phosphorylated by protein kinase C is suggested by the inhibition of phosphorylation by staurosporin. On the other hand dystrophin seems not to be a substrate for protein tyrosine kinases, as shown by the lack of reaction of phosphorylated dystrophin with a monoclonal antiphosphotyrosine antibody. Sequence analysis indicates that dystrophin contains seven potential phosphorylation sites for cyclic AMP- and cyclic GMP-dependent protein kinases (all localized in the central rod domain of the molecule) as well as several sites for protein kinase C and casein kinase II. Interestingly, potential sites of phosphorylation by protein kinase C and casein kinase II are located in the proximity of the actin-binding site. These results suggest, by analogy with what has been demonstrated in the case of other cytoskeletal proteins, that the phosphorylation of dystrophin by endogenous protein kinases may modulate both self assembly and interaction of dystrophin with other cytoskeletal proteins in vivo.

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