The amino terminal domain of a novel WD repeat protein from Trypanosoma cruzi contains a non-canonical mitochondrial targeting signal

WD (tryptophan/aspartic acid) repeat proteins perform a wide variety of functions in eukaryotic cells. They are characterised by the presence of a number of conserved repeat motifs that contribute to the beta-propeller structures which are the common feature of this large group of proteins. We report here the properties of the first characterised member of this family in the American trypanosome, Trypanosoma cruzi (TcBPP1). In the CL Brener clone the protein is 482 amino acids long and is predicted to contain four WD repeat motifs, flanked by amino and carboxyl terminal extensions. TcBPP1 is a single copy gene present on a 1.0/1.6 Mb pair of homologous chromosomes in a locus that is syntenic with the corresponding regions of Trypanosoma brucei and Leishmania major chromosomes. Consistent with the proposed hybrid nature of the CL Brener clone, the proteins encoded by the two different alleles share only 97% identity at the amino acid level. To determine subcellular location, we examined transfected parasites for the distribution of green fluorescent protein (GFP) fused with different regions of TcBPP1. These studies demonstrated that a 115 amino acid peptide derived from the amino terminal domain of TcBPP1 is able to target GFP to the mitochondrion. Interestingly this region lacks a typical amino terminal presequence suggesting that mitochondrial import is mediated by an alternative targeting signal.     

A pseudosubstrate peptide inhibits protein kinase C-mediated phosphorylation in permeabilized Rat-1 cells

Activation of protein kinase C (PKC) in Rat-1 fibroblasts leads to rapid phosphorylation of an 80-kDa protein, a major substrate of PKC. Digitonin-permeabilized cells perfectly supported this early response. Introduction of a PKC pseudosubstrate peptide inhibited 80 kDa phosphorylation with an IC50 of 1 microM, while a control peptide had no effect. The results indicate that this semi-intact cell system can be used in combination with the inhibitory pseudosubstrate peptide to study the involvement of PKC in cellular processes.     

A role for protein kinase C-mediated phosphorylation in the mobilization of arachidonic acid in mouse macrophages

Mouse peritoneal macrophages respond to activators of protein kinase C and to zymosan particles and calcium ionophore by rapid enhancement of a phospholipase A pathway and mobilization of arachidonic acid. The pattern of protein phosphorylation induced in these cells by 4 beta-phorbol 12-myristate 13-acetate (PMA), 1,2-dioctanoyl-sn-glycerol, exogenous phospholipase C and by zymosan and ionophore A23187 was found to be virtually identical. The time course of phosphorylation differed among the phosphoprotein bands and in only some of those identified (i.e., those of 45 and 65 kDa) was the phosphorylation sufficiently rapid to be involved in the activation of the phospholipase A pathway. Phosphorylation of lipocortin I or II could not be detected. Down-regulation of kinase C by a 24-h pretreatment with PMA resulted in extensive inhibition of both protein phosphorylation and the mobilization of arachidonic acid in response to PMA or dioctanoylglycerol. The phosphorylation of the 45 kDa protein in response to zymosan and A23187 was also inhibited by pretreatment with PMA, while only arachidonic acid release induced by zymosan was inhibited by this pretreatment. Depletion of intracellular calcium had little effect on kinase C-dependent phosphorylation, although arachidonic acid mobilization is severely inhibited under these conditions. Bacterial lipopolysaccharide and lipid A induced a phosphorylation pattern different from that induced by PMA, and down-regulation of protein kinase C did not affect lipopolysaccharide-induced protein phosphorylation. The results indicate (i) that protein kinase C plays a critical role also in zymosan-induced activation of the phospholipase A pathway mobilizing arachidonic acid; (ii) that such activation requires calcium at some step distal to kinase C-mediated phosphorylation and (iii) that phosphorylation of lipocortins does not explain the kinase C-dependent activation.     

Mitochondrial targeting of intact CYP2B1 and CYP2E1 and N-terminal truncated CYP1A1 proteins in Saccharomyces cerevisiae--role of protein kinase A in the mitochondrial targeting of CYP2E1

Previously we showed that intact rat cytochrome P450 2E1, cytochrome P450 2B1 and truncated cytochrome P450 1A1 are targeted to mitochondria in rat tissues and COS cells. However, some reports suggest that truncated cytochrome P450 2E1 is targeted to mitochondria. In this study, we used a heterologous yeast system to ascertain the conservation of targeting mechanisms and the nature of mitochondria-targeted proteins. Mitochondrial integrity and purity were established using electron microscopy, and treatment with digitonin and protease. Full-length cytochrome P450 2E1 and cytochrome P450 2B1 were targeted both to microsomes and mitochondria, whereas truncated cytochrome P450 1A1 (+ 5 and + 33/cytochrome P450 1A1) were targeted to mitochondria. Inability to target intact cytochrome P450 1A1 was probably due to lack of cytosolic endoprotease activity in yeast cells. Mitochondrial targeting of cytochrome P450 2E1 was severely impaired in protein kinase A-deficient cells. Similarly, a phosphorylation site mutant cytochrome P450 2E1 (Ser129A) was poorly targeted to the mitochondria, thus confirming the importance of protein kinase A-mediated protein phosphorylation in mitochondrial targeting. Mitochondria-targeted proteins were localized in the matrix compartment peripherally associated with the inner membrane and their ethoxyresorufin O-dealkylation, erythromycin N-demethylase, benzoxyresorufin O-dealkylation and nitrosodimethylamine N-demethylase activities were fully supported by yeast mitochondrial ferredoxin and ferredoxin reductase.     

Role of troponin I phosphorylation in protein kinase C-mediated enhanced contractile performance of rat myocytes

Our goal was to define the role of phosphorylated cardiac troponin-I in the adult myocyte contractile performance response to activated protein kinase C. In agreement with earlier work, endothelin enhanced both adult rat myocyte contractile performance and cardiac troponin-I phosphorylation. Protein kinase C participated in both responses. The role of cardiac troponin-I phosphorylation in the contractile function response to protein kinase C was further investigated using gene transfer into myocytes of troponin-I isoforms/mutants lacking one or more phosphorylation sites previously identified in purified cardiac troponin-I. Sarcomeric replacement with slow skeletal troponin-I-abrogated protein kinase C-mediated troponin-I phosphorylation. In functional studies, endothelin slowed relaxation in myocytes expressing slow skeletal troponin-I, while the relaxation rate increased in myocytes expressing cardiac troponin-I. Based on these results, acceleration of myocyte relaxation during protein kinase C activation largely depended on cardiac troponin-I phosphorylation. Experiments with troponin-I isoform chimeras provided evidence that phosphorylation sites in the amino portion of cardiac troponin I-mediated the protein kinase C acceleration of relaxation. The cardiac troponin-I Thr-144 phosphorylation site identified in earlier biochemical studies was not significantly phosphorylated during the acute contractile response. Thus, amino-terminal protein kinase C-dependent phosphorylation sites in cardiac troponin-I are likely responsible for the accelerated relaxation observed in adult myocytes.     

Altered levels and protein kinase C-mediated phosphorylation of substrates in normal and transformed mouse lung epithelial cells.

Protein phosphorylation and protein kinase C (PKC) levels were analyzed in intact cultures of spontaneously transformed, chemically transformed, and untransformed mouse pulmonary epithelial cell lines. It was found that although the transformed cell lines contained about 80% less protein kinase C, measured as total enzyme activity or binding of [3H]phorbol ester, phosphorylation events after phorbol ester treatment could still be easily detected. A commonly described Mr 80-kDa protein kinase C substrate (p80, 80 K, MARKS) was identified using 2D-PAGE, following phosphorylation in intact cells, and found to have reduced availability for phosphorylation in the transformed cell lines C4SE9, C1SA5 and NULB5 in comparison to the untransformed C4E10 and C1C10. Available levels of p80 were further analyzed in heat-denatured extracts from all cell lines using partially purified bovine brain PKC and correlated well with changes seen in intact cells. It was also noted that all transformed cell lines contained large amounts of a family of phosphoproteins of Mr 55-65 kDa, that could not be detected in the untransformed cell lines and whose phosphorylation state was increased by protein kinase C activation. This protein was found to be located in the nucleus. Hence, spontaneously and chemically transformed mouse pulmonary epithelial cells exhibit reduced levels of PKC, along with an altered pattern of PKC-mediated phosphorylation.     

Role of angiotensin II type 1A receptor phosphorylation, phospholipase D, and extracellular calcium in isoform-specific protein kinase C membrane translocation responses

The angiotensin II type 1A receptor (AT(1A)R) plays an important role in cardiovascular function and as such represents a primary target for therapeutic intervention. The AT(1A)R is coupled via G(q) to the activation of phospholipase C, the hydrolysis of phosphoinositides, release of calcium from intracellular stores, and the activation of protein kinase C (PKC). We show here that PKCbetaI and PKCbetaII exhibit different membrane translocation patterns in response to AT(1A)R agonist activation. Whereas PKCbetaII translocation to the membrane is transient, PKCbetaI displays additional translocation responses: persistent membrane localization and oscillations between the membrane and cytosol following agonist removal. The initial translocation of PKCbetaI requires the release of calcium from intracellular stores and the activation of phospholipase C, but persistent membrane localization is dependent upon extracellular calcium influx. The mutation of any of the three PKC phosphorylation consensus sites (Ser-331, Ser-338, and Ser-348) localized within the AT(1A)R C-tail significantly increases the probability that persistent increases in diacylglycerol levels and PKCbetaI translocation responses will be observed. The persistent increase in AT(1A)R-mediated diacylglycerol formation is mediated by the activation of phospholipase D. Although the persistent PKCbetaI membrane translocation response is absolutely dependent upon the PKC activity-dependent recruitment of an extracellular calcium current, it does not require the activation of phospholipase D. Taken together, we show that the patterning of AT(1A)R second messenger response patterns is regulated by heterologous desensitization and PKC isoform substrate specificity.     

Homologous desensitisation of the mouse leukotriene B4 receptor involves protein kinase C-mediated phosphorylation of serine 127.

Murine leukotriene B(4) (LTB(4)) receptor (mBLT1) cDNA was identified by searching the EST database using human LTB(4) receptor as the query sequence. Expression of functional mBLT1 after injection of in vitro transcribed cRNA into Xenopus laevis oocytes was demonstrated as LTB(4)-evoked, Ca(2+)-activated Cl(-) currents recorded by two-electrode voltage clamp. From mBLT1-expressing oocytes, a dose-dependent relationship between the Ca(2+)-activated Cl(-) current and LTB(4) concentration was demonstrated with an apparent EC(50) of 6.7 nM. Following LTB(4) stimulation of mBLT1, we observed two transient, spatially distinct Ca(2+)-activated, inwardly directed Cl(-) currents in the oocytes: a fast peak current requiring relatively high LTB(4) concentrations, and a slowly progressing Cl(-) current. Nucleotides, PGE(2), 12R-hydroxy-5, 8, 14-cis-10-trans-eicosatetraenoic acid, and LTD(4) did not activate mBLT1. U75302, specifically targeting BLT1, significantly reduced LTB(4)-evoked Cl(-) currents. Repetitive LTB(4) administration desensitized the LTB(4)-evoked currents. Activation of protein kinase C (PKC) by PMA addition completely eliminated the LTB(4)-evoked currents, whereas down-regulation of PKC by prolonged PMA exposure (20 h) impaired mBLT1 desensitisation. In addition, Ser-127-Ala substitution of the PKC consensus phosphorylation site on the second intracellular loop prevented the mBLT1 desensitisation. These data indicate that PKC-mediated phosphorylation at Ser-127 leads to mBLT1 desensitisation.     

Identification of a human Akt3 (protein kinase B gamma) which contains the regulatory serine phosphorylation site

The family of protein kinases called Akt, protein kinase B (PKB), or related to A and C kinase (RAC) have been implicated in numerous biological processes including adipocyte and muscle differentiation, glycogen synthesis, glucose uptake, apoptosis and cellular proliferation. There are 3 known isoforms of this enzyme in mammalian cells (1/alpha, 2/beta and 3/gamma). Akt1 and 2 contain a key regulatory serine phosphorylation site in the carboxy-terminal region of the protein. However, the reported sequence of the rat Akt3 protein differed significantly from this in that it lacked 25 amino acids in the C-terminal region, including this key regulatory serine phosphorylation site (Biochem. Biophys. Res. Commun. 216, 526-534). In the present studies we show that the deduced sequence of human Akt3 contains this serine and that it is phosphorylated in response to insulin. These results indicate that human Akt3 is regulated similarly to Akt1 and Akt2.     

The NPK1 mitogen-activated protein kinase kinase kinase contains a functional nuclear localization signal at the binding site for the NACK1 kinesin-like protein

The tobacco mitogen-activated protein kinase kinase kinase NPK1 localizes to the equatorial region of phragmoplasts by interacting with kinesin-like protein NACK1. This leads to activation of NPK1 kinase at late M phase, which is necessary for cell plate formation. Until now, its localization during interphase has not been reported. We investigated the subcellular localization of NPK1 in tobacco-cultured BY-2 cells at interphase using indirect immunofluorescence microscopy and fusion to green fluorescent protein (GFP). Fluorescence of anti-NPK1 antibodies and GFP-fused NPK1 were detected only in the nuclei of BY-2 cells at interphase. Examination of the amino acid sequence of NPK1 showed that at the carboxyl-terminal region in the regulatory domain, which contains the binding site of NACK1, NPK1 contained a cluster of basic amino acids that resemble a bipartite nuclear localization signal (NLS). Amino acid substitution mutations in the critical residues in putative NLS caused a marked reduction in nuclear localization of NPK1 in BY-2 cells, indicating that this sequence is functional in tobacco BY-2 cells. We also found that the 64-amino acid sequence at the carboxyl terminus that contains NLS sequence is essential for interaction with NACK1, and that mutations in the NLS sequence prevented NPK1 from interacting with NACK1. Thus, the amino acid sequence at the carboxyl-terminal region of NPK1 has dual functions for nuclear localization during interphase and binding NACK1 in M phase.     

Role of protein phosphorylation in neuronal signal transduction

Protein phosphorylation is involved in the regulation of a wide variety of physiological processes in the nervous system. Studies in which purified protein kinases or kinase inhibitors have been microinjected into defined cells while a specific response is monitored have demonstrated that protein phosphorylation is both necessary and sufficient to mediate responses of excitable cells to extracellular signals. The precise molecular mechanisms involved in neuronal signal transduction processes can be further elucidated by identification and characterization of the substrate proteins for the various protein kinases. The roles of three such substrate proteins in signal transduction are described in this article: 1) synapsin I, whose phosphorylation increases neurotransmitter release and thereby modulates synaptic transmission presynaptically; 2) the nicotinic acetylcholine receptor, whose phosphorylation increases its rate of desensitization and thereby modulates synaptic transmission postsynaptically; and 3) DARPP-32, whose phosphorylation converts it to a protein phosphatase inhibitor and which thereby may mediate interactions between dopamine and other neurotransmitter systems. The characterization of the large number of additional phosphoproteins that have been found in the nervous system should elucidate many additional molecular mechanisms involved in signal transduction in neurons.     

Dynamics of protein kinase C-mediated phosphorylation of the complement C5a receptor on serine 334

Upon agonist binding, the C5a anaphylatoxin receptor (C5aR) is rapidly phosphorylated on phosphorylation sites that are located within the C-terminal domain of the receptor. Previous studies suggested that C5aR phosphorylation proceeds in a hierarchical manner with serine 334 presenting a highly accessible priming site that controls subsequent phosphorylation at other positions. To better understand the dynamics of Ser-334 phosphorylation, we generated site-specific monoclonal antibodies that specifically react with phosphoserine 334. In differentiated U937 cells, which endogenously express C5aR, stimulation with low C5a concentrations resulted in a very rapid (t((1/2)) approximately 20 s), albeit transient, receptor phosphorylation. Whole cell phosphorylation assays with specific inhibitors as well as in vitro phosphorylation assays with recombinant enzymes and peptide substrates revealed that phosphorylation of Ser-334 is regulated by protein kinase C-beta and a calyculin A-sensitive protein phosphatase. Surprisingly, at high concentrations (>10 nM) of C5a, the protein kinase C-mediated phosphorylation of Ser-334 was essentially blocked. This could be attributed to the even faster (t((1/2)) < 5 s) binding of beta-arrestin to the receptor. Analysis of C5aR Ser/Ala mutants that possess a single intact serine residue either at position 334 or at neighboring positions 327, 332, or 338 revealed functional redundancy of C-terminal phosphorylation sites since all 4 serine residues could individually support C5aR internalization and desensitization. This study is among the first to analyze in a detailed manner, using a non-mutational approach, modifications of a defined phosphorylation site in a G protein-coupled receptor and to correlate these findings with functional parameters of receptor deactivation.     

Calcium-calmodulin-dependent protein kinase II and protein kinase C-mediated phosphorylation and activation of D-myo-inositol 1,4, 5-trisphosphate 3-kinase B in astrocytes.

D-myo-Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) 3-kinase catalyzes the production of D-myo-inositol 1,3,4,5-tetrakisphosphate from the second messenger Ins (1,4,5)P3. Transient and okadaic acid-sensitive activation of Ins(1,4,5)P3 3-kinase by 8-10-fold is observed in homogenates prepared from rat cortical astrocytes after incubation with either carbachol or UTP. 12-O-Tetradecanoylphorbol-13-acetate provokes the activation of Ins(1,4,5)P3 3-kinase by 2-fold in both cell systems. The kinase was purified by calmodulin-Sepharose from the two cell systems. Enzyme activity corresponding to the silver-stained 88-kDa protein could be regenerated after SDS-polyacrylamide gel electrophoresis. Antibodies to two distinct peptides chosen in the primary structure of human Ins(1,4,5)P3 3-kinase B recognized the astrocytic native isoform. In [32P]orthophosphate-preincubated cells, a major phosphorylated 88-kDa enzyme could be purified and identified in cells in response to receptor activation or 12-O-tetradecanoylphorbol-13-acetate treatment. Calmodulin kinase II inhibitors (i.e. KN-93 and KN-62) and a protein kinase C inhibitor (i.e. calphostin C) prevented the phosphorylation of the 88-kDa isoenzyme. In addition to enzyme activation, a redistribution of Ins(1,4,5)P3 3-kinase from soluble to particulate fraction of astrocytes was observed. In vitro phosphorylation of the purified enzyme by calmodulin kinase II and protein kinase C added together resulted in a maximal 60-70-fold activation.     

信号识别颗粒(SRP)在膜蛋白定位中的作用

依赖信号识别颗粒(signal recognition particle,SRP)的共翻译转运是所有生命体中的一个保守途径,它将新生肽链的翻译与转运耦联在一起。超过30%的新合成的多肽链被SRP转运到正确位置。最近的研究表明,大肠杆菌中SRP抑制子可以规避SRP的需求。当SRP缺失时,翻译控制在介导膜蛋白定位方面起着关键作用。本综述总结了SRP底物如何在存在或缺失SRP的情况下转运到适当的位置以及翻译速率降低如何补偿SRP的缺失。我们还讨论了不同蛋白质对SRP的依赖程度。这一回顾将为进一步研究SRP功能及膜蛋白定位提供新思路。     

Identification and characterization of a mitochondrial targeting signal in rat cytochrome P450 2E1 (CYP2E1)

Cytochrome P450 2E1 (CYP2E1) lacking the hydrophobic NH(2)-terminal hydrophobic transmembrane domain is specifically targeted to mitochondria, where it is processed to a soluble and catalytically active form (Delta2E1) with a mass of about 40 kDa. Small amounts of Delta2E1 were also observed in mitochondria isolated from rat liver, indicating that this form of CYP2E1 is also present in vivo. In the present study the mitochondrial targeting signal was identified and characterized by the use of several NH(2)-terminally truncated and mutated forms of CYP2E1 that were expressed in the mouse H2.35 hepatoma cell line. Two potential mitochondrial targeting sequences were identified in the NH(2) terminus of CYP2E1. Deletion of the first potential mitochondrial targeting sequence located between amino acids 50 and 65, as in Delta(2-64)2E1, still resulted in mitochondrial targeting and processing, but when, in addition to the first, the second potential mitochondrial targeting sequence located between amino acids 74 and 95 was also deleted, as in Delta(2-95)2E1, the mitochondrial targeting was abolished. Mutation of the four positively charged Arg and Lys residues present in this sequence to neutral Ala residues resulted in the abrogation of mitochondrial targeting. Deletion of a hydrophobic stretch of amino acids between residues 76 and 83 also abolished mitochondrial targeting and import. Once imported in the mitochondria, these constructs were further processed to the mature protein Delta2E1. It is concluded that mitochondrial targeting of CYP2E1 is mediated through a sequence located between residues 74 and 95 and that positively charged residues as well as a hydrophobic stretch present in the beginning of this sequence are essential for this process.     

Chronic ethanol exposure increases levels of protein kinase C delta and epsilon and protein kinase C-mediated phosphorylation in cultured neural cells.

Exposure to ethanol for several days increases the number and function of dihydropyridine-sensitive Ca2+ channels in excitable tissues. In the neural cell line PC12, this process is blocked by inhibitors of protein kinase C (PKC), suggesting that PKC mediates ethanol-induced increases in Ca2+ channels. We report that treatment with 25-200 mM ethanol for 2-8 days increased PKC activity in PC12 cells and NG108-15 neuroblastoma-glioma cells. Detailed studies in PC12 cells showed that ethanol also increased phorbol ester binding and immunoreactivity to PKC delta and PKC epsilon. These changes were associated with increased PKC-mediated phosphorylation. Ethanol did not activate the enzyme directly, nor did ethanol increase levels of diacylglycerol. Ethanol-induced increases in PKC levels may promote up-regulation of Ca2+ channels, and may also regulate the expression and function of other proteins involved in cellular adaptation to ethanol.     

The focal adhesion targeting domain of focal adhesion kinase contains a hinge region that modulates tyrosine 926 phosphorylation

The focal adhesion targeting (FAT) domain of focal adhesion kinase (FAK) is critical for recruitment of FAK to focal adhesions and contains tyrosine 926, which, when phosphorylated, binds the SH2 domain of Grb2. Structural studies have shown that the FAT domain is a four-helix bundle that exists as a monomer and a dimer due to domain swapping of helix 1. Here, we report the NMR solution structure of the avian FAT domain, which is similar in overall structure to the X-ray crystal structures of monomeric forms of the FAT domain, except that loop 1 is longer and less structured in solution. Residues in this region undergo temperature-dependent exchange broadening and sample aberrant phi and psi angles, which suggests that this region samples multiple conformations. We have also identified a mutant that dimerizes approximately 8 fold more than WT FAT domain and exhibits increased phosphorylation of tyrosine 926 both in vitro and in vivo.     

Role of dynamin, Src, and Ras in the protein kinase C-mediated activation of ERK by gonadotropin-releasing hormone

G-protein-coupled receptors are a large group of integral membranal receptors, which in response to ligand binding initiate diverse downstream signaling. Here we studied the gonadotropin-releasing hormone (GnRH) receptor, which uses Gq for its downstream signaling. We show that extracellular signal-regulated kinase (ERK) activation is fully dependent on protein kinase C (PKC), but only partially dependent on Src, dynamin, and Ras. Receptor tyrosine kinases, FAK, Gbetagamma, and beta-arrestin, which were implicated in some G-protein-coupled receptor signaling to MAPK cascades, do not play a role in the GnRH to ERK pathway. Our results suggest that the activation of ERK by GnRH involves two distinct signaling pathways, which converge at the level of Raf-1. The main pathway involves a direct activation of Raf-1 by PKC, and this step is partially dependent on a second pathway consisting of Ras activation, which occurs in a dynamin-dependent manner, downstream of Src.