The role of protein phosphorylation in the regulation of cyclic nucleotide phosphodiesterases

The cyclic nucleotide phosphodiesterases constitute a complex superfamily of enzymes responsible for catalyzing the hydrolysis of cyclic nucleotides. Regulation of cyclic nucleotide phosphodiesterases is one of the two major mechanisms by which intracellular cyclic nucleotide levels are controlled. In many cases the fluctuations in cyclic nucleotide cAMP-specific, calmodulin-stimulated and cGMP-binding phosphodiesterases have been demonstrated to be substrates for protein kinases. Here we review the evidence that hormonally responsive phosphorylation acts to regulate cyclic nucleotide phosphodiesterases. In particular, the cGMP-inhibited phosphodiesterases, which can be phosphorylated by at least two different protein kinases, are activated as a result of phosphorylation. In contrast, phosphorylation of the calmodulin-stimulated phosphodiesterases, which coincides with, a decreased sensitivity to activation by calmodulin, results in decreased phosphodiesterase activity.     

Calpain-mediated proteolysis of microtubule associated proteins MAP1B and MAP2 in developing brain

Microtubule associated proteins MAP1B and MAP2 are important components of the neuronal cytoskeleton. During early development of the brain, MAP1B (340 kDa) is present as two isoforms that differ in their level of phosphorylation, while MAP2 is expressed as a single high molecular weight isoform (MAP2B, 280 kDa) and a low molecular weight form (MAP2C, 70 kDa). In this study we examined and compared the sensitivities of MAP1B and MAP2, obtained from MT preparations and brain homogenates of young rats, to degradation by calcium-activated neutral protease, calpain II. We found that in MAPs prepared from microtubules the two isoforms of MAP1B had comparable sensitivity to calpain-mediated proteolysis. Similarly, the high and low molecular weight forms of MAP2 were equally sensitive to digestion by calpain. However, although both MAPs were very susceptible to calpain-mediated proteolysis, MAP1B was more resistant to degradation by calpain than MAP2. Furthermore, the endogenous degradation of MAPs in neonate brain homogenates was calcium-dependent and inhibited by leupeptin, and the pattern of degradation products for MAP1B and MAP2 was similar to that of calpain-mediated proteolysis. These data suggest that calpain can play a role in the regulation of MAPs levels during brain development, in relation to normal neuronal differentiation and disorders associated with neurodegeneration.     

古菌蛋白激酶的研究进展

磷酸化是蛋白质翻译后修饰(post-translational modification)的主要方式,可由蛋白激酶、磷酸转移酶、磷酸化酶等多种方式催化进行。其中,由蛋白激酶(protein kinases)/磷酸酶(protein phosphatases)介导的可逆的蛋白磷酸化是细胞中信号转导的重要机制,在DNA复制、转录、蛋白质翻译、DNA损伤修复等生命过程中起广泛的调节作用。目前,古菌中蛋白激酶的研究尚属于初期阶段。虽然磷酸化蛋白质组学研究表明,古菌中存在大量的磷酸化蛋白质,但是我们对其具体催化作用的酶及调控机制尚不清楚。本文总结了古菌中已报道的蛋白激酶所参与的生命过程,包括古菌的DNA代谢、细胞代谢、细胞周期和运动机制等四个方面,并对今后的研究提出展望。     

Expression of multiple type 1 phosphoprotein phosphatases in Arabidopsis thaliana

Type 1 phosphoprotein Ser/Thr phosphatases (PP1) are highly conserved enzymes found in all eukaryotes. These enzymes have multiple functions in fungal and animal cells but little is known of their function and regulation in plants. Previous studies in our laboratory indicated that maize and Arabidopsis contain a family of PP1 genes and/or pseudogenes. In this study, we report the isolation of five distinct Arabidopsis cDNA clones (TOPP1, TOPP2, TOPP3, TOPP4 and TOPP5) which encode the catalytic subunit (PP1c) of type 1 protein phosphatases. Genomic Southern blot analyses indicate that these clones are the products of five distinct genes and that an additional 2–3 PP1c genes and/or pseudogenes may be present in the Arabidopsis genome. The derived amino acid sequences of the TOPP clones are very similar to published sequences of PP1c from animals, fungi and plants. Four of the TOPP amino acid sequences show unique structural features not observed in other PP1c sequences from fungi or animals. All of the TOPP genes are expressed in Arabidopsis roots, rosettes and flowers, although TOPP1, TOPP2 and TOPP3 appear to be expressed at higher levels in these tissues than TOPP4 and TOPP5.     

Phosphorylation of cytosolic proteins by casein kinases in human erythrocytes. Response to ionic strength and to 2,3-bisphosphoglycerate

The endogenous phosphorylation of human erythrocyte cytosolic proteins is markedly increased when the crude cytosol, prior to incubation in the presence of [y-³²P] ATP, is submitted to DEAE-cellulose chromatography. Some proteins, including 22 and 23 kDa proteins, are preferentially phosphorylated by cytosolic casein kinase CS, whereas other proteins, including 42 kDa protein, are preferentially phosphorylated by casein kinase CTS. The CS-catalyzed phosphorylation is strongly inhibited by physiological ionic strength (150 mM KCl or NaCl) and by physiological levels (3 mM) of 2,3-bisphosphoglycerate, while CTS-catalyzed phosphorylation is unaffected. The very poor endogenous phosphorylation of these proteins in the crude cytosol may be due to the presence of other cytosolic inhibitors which are removed by DEAE-cellulose chromatography.     

Identification of Tau and MAP2 as novel substrates of Rho-kinase and myosin phosphatase

Rho-kinase and myosin phosphatase are implicated in the phosphorylation-state of myosin light chain downstream of Rho, which is thought to induce smooth muscle contraction and stress fibre formation in non-muscle cells. Here, we found that microtubule-associated proteins, Tau and MAP2, interacted with the myosin-binding subunit (MBS) of myosin phosphatase, and were the possible substrates of both Rho-kinase and myosin phosphatase. We determined the phosphorylation sites of Tau (Thr245, Thr377, Ser409) and MAP2 (Ser1796) by Rho-kinase. We also found that Rho-kinase phosphorylated Tau at Ser262 to some extent. Phosphorylation by Rho-kinase decreased the activity of Tau to promote microtubule assembly in vitro. Substitutions of Ala for Ser/Thr at the phosphorylation sites of Tau (Tau-AAA) did not affect the activity to promote microtubule assembly, while substitutions of Asp for Ser/Thr (Tau-DDD), which are expected to mimic the phosphorylation-state of Tau, slightly reduced the activity. When Tau, or mutated forms of Tau, were expressed in PC12 cells, followed by treatment with cytochalasin D, they promoted extension of the cell process in a cytochalasin-dependent manner. However, Tau-DDD showed the weaker activity in this capacity than wild-type Tau or Tau-AAA. These results suggest that the phosphorylation-state of these residues of Tau affects its activity both in vitro and in vivo. Thus, it is likely that the Rho-kinase/MBS pathway regulates not only the actin-myosin system but also microtubule dynamics.     

Cloning and expression of a new human polypeptide which regulates protein phosphorylation in Escherichia coli

Summary A 1,820bp full-length clone encoding for a new human protein was isolated from a gt11 placental cDNA library using anti-human hexokinase antibodies. The cDNA complete sequence includes a 12 by 5 noncoding region, a single open reading frame encoding a protein of 55 KDa (HP-10) and a 177 by non-coding with two putative polyadenylation signals upstream of 3poly(A)tail. The deduced amino acid sequence reveals a sequence of 492 amino acids that contains a stretch of 7 glutamic acid from position 169 and one potential glycosylation site at position 274. Although antibodies against hexokinase recognize the fusion protein and antibodies against the fusion protein recognize hexokinase, HP-10 is not human hexokinase, by a number of criteria including the alignment of determined amino acid sequences.In searching for a possible functional role of HP-10 its cDNA was inserted into a procaryotic vector which allows the expression of the non-fused protein. Bacteria expressing the HP-10 encoded protein were isolated and found to have a dramatic increase in endogenous phosphorylated proteins. Since HP-10 does not have a protein kinase activity per se it should be considered a new regulatory phosphorylation protein which is active in E. coli Abbreviations HK Hexokinase (EC 2.7.1.1)     

植物蛋白磷酸化的检测方法

蛋白磷酸化是一种重要的蛋白质翻译后修饰方式,几乎参与植物所有生命过程的调节。蛋白磷酸化过程主要指在蛋白激酶的催化作用下,将三磷酸腺苷(ATP)上的γ位磷酸基团转移到底物蛋白特定氨基酸残基上的过程。底物蛋白上被磷酸化的常见氨基酸有丝氨酸、苏氨酸及酪氨酸,磷酸基团与氨基酸中的羟基通过酯键连接。该文详细描述了几种常用的蛋白质体外及体内磷酸化的检测方法及注意事项。     

Phosphorylation of vertebrate nonmuscle and smooth muscle myosin heavy chains and light chains

In this article we review the various amino acids present in vertebrate nonmuscle and smooth muscle myosin that can undergo phosphorylation. The sites for phosphorylation in the 20 kD myosin light chain include serine-19 and threonine-18 which are substrates for myosin light chain kinase and serine-1 and/or-2 and threonine-9 which are substrates for protein kinase C. The sites in vertebrate smooth muscle and nonmuscle myosin heavy chains that can be phosphorylated by protein kinase C and casein kinase II are also summarized.Original data indicating that treatment of human T-lymphocytes (Jurkat cell line) with phorbol 12-myristate 13-acetate results in phosphorylation of both the 20 kD myosin light chain as well as the 200 kD myosin heavy chain is presented. We identified the amino acids phosphorylated in the human T-lymphocytes myosin light chains as serine-1 or serine-2 and in the myosin heavy chains as serine-1917 by 1-dimensional isoelectric focusing of tryptic phosphopeptides. Untreated T-lymphocytes contain phosphate in the serine-19 residue of teh myosin light chain and in a residue tentatively identified as serine-1944 in the myosin heavy chain.Abbreviations MLC myosin light chain - MHC myosin heavy chain - Tris tris(hydroxymethyl)aminomethane - EGTA [ethylenebis(oxyethylenenitrilo)]tetraacetic acid - EDTA ethylenediaminetetraacetate - TPCK N-tosyl-L-phenylalanine chloromethyl ketone - PMA phorbol 12-myristate 13-acetate     

The replication band of Euplotes is enriched with phosphoproteins

Summary— Employing several antibodies to phosphorylated protein epitopes, we demonstrate by immunostaining that the macronuclear replication band (RB) of the ciliated protozoan Euplotes eurystomus contains a high concentration of phosphoproteins. Enrichment is principally within the rear zone of the RB, the region of DNA synthesis and chromatin assembly. By immunoblot analysis, the various antibodies reacted with a diversity of macronuclear phosphoproteins, one of which was phosphorylated histone Hl. This diversity of phosphoproteins was also supported by examination of the macronuclear matrix generated by high NaCl extraction. Available evidence clearly indicates that the ultrastructural wave of chromatin modulation accompanying DNA replication is spatially correlated with a wave of localized nuclear protein phosphorylation.     

Protein phosphorylation in rat liver mitochondria

Summary Incubation of rat liver mitochondria in the presence of either [³²P] Pi or ₃₂ ^y -P] ATP resulted in a phosphorylation of four proteins with Mr 50, 47, 44 and 36 kDa, respectively. The endogenous phosphorylation of these proteins in the presence of [³²P] Pi was markedly influenced by the osmolarity of the incubation medium and differentially affected by various effectors of mitochondrial functions, such as Ca²⁺, oligomycin, FCCP, arsenite and dichloroacetate. In particular, the 36 kDa protein, unlike the other proteins, appears to be phosphorylated also by direct incorporation of [³²P], independently of respiratory chain-linked ATP synthesis. The four proteins, located in the mitoplasts, seem to be phosphorylated by diiferent protein kinases, as suggested by the observation that the endogenous phosphorylation of 36 kDa protein resulted selectively increased by addition of exogenous protein kinases, such as casein kinases S and TS. A tentative identification of these phosphorylatable protein is discussed.     

Dephosphorylation of the thylakoid membrane light-harvesting complex-II by a stromal protein phosphatase

Light-harvesting complex-II (LHC-II) phosphatase activity has generally been examined in the intact thylakoid membrane. A recent report of peptide-phosphatase activity associated with the chloroplast stromal fraction (Hammer, M.F. et al. (1995) Photosynth Res 44: 107–115) has led to the question of whether this activity is capable of dephosphorylating membrane-bound LHC-II. To this end, heat-treated thylakoid membranes were examined as a potential LHC-II phosphatase substrate. Following incubation of the thylakoid membrane at 60°C for 15 min, the endogenous protein phosphatase and kinase activities were almost eliminated. Heat-inactivated phosphomembranes exhibited minimal dephosphorylation of the light harvesting complex-II. Peptide-phosphatase activities isolated from the thylakoid and stromal fraction were able to dephosphorylate LHC-II in heat-inactivated phosphomembranes. The stromal phosphatase showed highest activity against LHC-II at pH 9. Dephosphorylation of the LHC-II by the stromal enzyme was not inhibited by molybdate, vanadate or tungstate ions, but was partially inhibited by EDTA and a synthetic phosphopeptide mimicking the LHC-II phosphorylation site. Thus, the previously identified stromal phosphatase does appear capable of dephosphorylating authentic LHC-II in vivo.Abbreviations CPP chymotryptic phosphopeptides - LHC-II light-harvesting complex of Photosystem II - MP protein phosphatase fractionated from the thylakoid membrane - P_2Thr synthetic phosphopeptide MRK-SAT(p)TKKVW - SP protein phosphatase fractionated from the stromal compartment     

Phosphorylation of ankyrin down-regulates its cooperative interaction with spectrin and protein 3

Ankyrin mediates the primary attachment between beta spectrin and protein 3. Ankyrin and spectrin interact in a positively cooperative fashion such that ankyrin binding increases the extent of spectrin tetramer and oligomer formation (Giorgi and Morrow: submitted, 1988). This cooperative interaction is enhanced by the cytoplasmic domain of protein 3, which is prepared as a 45-41-kDa fragment generated by chymotryptic digestion of erythrocyte membranes. Using sensitive isotope-ratio methods and nondenaturing PAGE, we now demonstrate directly (1) the enhanced affinity of ankyrin for spectrin oligomers compared to spectrin dimers; (2) a selective stimulation of the affinity of ankyrin for spectrin oligomer by the 43-kDa cytoplasmic domain of protein 3; and (3) a selective reduction in the affinity of ankyrin for spectrin tetramer and oligomer after its phosphorylation by the erythrocyte cAMP-independent membrane kinase. The phosphorylation of ankyrin does not affect its binding to spectrin dimer. Ankyrin also enhances the rate of interconversion between dimer-tetramer-oligomer by 2-3-fold at 30 degrees C, and in the presence of the 43-kDa fragment, ankyrin stimulates the rate of oligomer interconversions by nearly 40-fold at this temperature. These results demonstrate a long-range cooperative interaction between an integral membrane protein and the peripheral cytoskeleton and indicate that this linkage may be regulated by covalent protein phosphorylation. Such interactions may be of general importance in nonerythroid cells.     

Functional characteristics of the maize RNA-binding protein MA16

The maize RNA-binding protein MA16 is a non-ribosomal nucleolar protein widely distributed in different maize tissues. We have previously shown that the MA16 protein binds preferentially to guanosine-and uridine-rich sequences. As a step towards the identification of specific targets with which MA16 interacts within the cell, we investigated the RNA-binding affinities and several other aspects of the protein by using binding assays and immunochemistry. The MA16 protein showed a wide spectrum of RNA-binding activities with lower affinities to several RNAs that was salt and heparin-sensitive indicative of electrostatic interactions, and higher affinities to particular RNAs including rRNA and translatable mRNA sequences. Among the RNAs found associated with MA16 protein was that encoding MA16 itself. This observation raises the possibility that MA16 gene expression could be self-regulated. Immunoprecipitation studies showed that in vivo MA16 was phosphorylated and that MA16 interacts with RNAs through complex association with several proteins. These results suggest that both phosphorylation and interaction with other proteins may be involved in determining RNA-binding specificities of MA16 in the cell.     

Muscarinic-cholinoceptor mediated attenuation of phospholamban phosphorylation induced by inhibition of phosphodiesterase in ventricular cardiomyocytes: Evidence against a cAMP- dependent effect

In intact guinea pig ventricles, acetylcholine (ACH) has been shown to attenuate the positive inotropic effects of isobutylmethylxanthine (IBMX), a phosphodiesterase inhibitor, by reducing protein phosphorylation without altering cAMP levels. In the present study, we tested the hypothesis that the cAMP-independent inhibitory action of ACH is also evident in isolated cardiomyocytes. cAMP-dependent protein kinase (PKA) activity ratio (-cAMP/+cAMP) and phosphorylation of phospholamban (PLB) were determined in unlabeled and ³²P-labeled guinea pig ventricular cardiomyocytes, respectively. IBMX increased PKA activity ratio and phosphorylation of PLB in a dose-dependent manner. When cardiomyocytes were incubated simultaneously with IBMX (0-1 mM) and ACH (2 M), ACH attenuated PLB phosphorylation stimulated by low concentration (10-100 M) but not by high concentrations (> 200 M) of IBMX. EC₅₀ value for IBMX-induced phosphorylation of PLB was 32 ± 6 M and increased nearly 3-fold after addition of ACH while PKA activity ratio remained unchanged. The rank order of cyclic nucleotide derivatives to phosphorylate PLB was 8 bromo-cAMP > dibutyryl cAMP > 8 bromo-cGMP > dibutyryl cGMP. ACH reduced phosphorylation of PLB stimulated by 8 bromo-cAMP. We conclude that in isolated cardiomyocytes (1) ACH inhibits phosphorylation of PLB stimulated by either IBMX or 8 bromo-cAMP and (2) ACH does not lower IBMX-stimulated PKA activity ratio. These effects of ACH on PLB phosphorylation cannot be explained by a reduction in IBMX-stimulated cAMP levels but may involve the activation of protein phosphatases.     

利用一种新的网上工具Scansite进行蛋白质磷酸化预测

Scansite分析软件是近两年建立的一种新的利用因特网,基于蛋白质分子中较短的模序进行蛋白质磷酸化和蛋白质蛋白质相互作用预测的工具。这里综述了Scansite的使用方法、功能介绍及与其他磷酸化分析软件的比较,并展望了Scansite在进行磷酸化预测中面临的问题和应用前景。     

Molecular cloning of a novel phosphorylation-dependent inhibitory protein of protein phosphatase-1 (CPI17) in smooth muscle: its specific localization in smooth muscle

The cDNA encoding a phosphorylation-dependent inhibitory protein of protein phosphatase-1 (PP1) was isolated from a porcine aorta library. The coding region represented the complete amino acid sequence of this protein comprised of a novel 147-residue polypeptide, which we termed CPI17, a 17-kDa PKC-potentiated inhibitory protein of PP1. As well as the native CPI17 from porcine aorta, the recombinant protein completely suppressed the PP1 activity (IC₅₀=0.18 nM) by the stoichiometric thiophosphorylation. The CPI17 mRNA is expressed in smooth muscle tissues such as aorta and bladder, whereas little expression was observed in heart, skeletal muscle, and non-muscle tissues. These results suggest a specific regulatory mechanism of the PP1 activity through CPI17 in smooth muscle.