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PubMed2006

Novel protein phosphorylation site identification in spinach stroma membranes by titanium dioxide microcolumns and tandem mass spectrometry.

In this work, spinach stroma membrane, instead of thylakoid, has been investigated for the presence of phosphorylated proteins. We identified seven previously unknown phosphorylation sites by taking advantage of TiO(2) phosphopeptides enrichment coupled to mass spectrometric analysis. Upon illumination at 100 micromol m(-2) s(-1), two novel phosphopeptides belonging to the N-terminal region of Lhcb1 light-harvesting protein were detected: NVSSGS(p)PWYGPDR and T(p)VQSSSPWYGPDR. Moreover, three new threonine residues in CP43 (Thr-6, Thr-8, and Thr-346) and, for the first time, two amino acid residues of the N-terminus of Rieske Fe-S protein of the cytochrome b(6)f complex (Thr-2 and Ser-3) were revealed to be phosphorylated. Since Lhcb1 and CP43 have been reported as mobile proteins, it may be suggested that illumination derived phosphorylation, and consequently the addition of negatively charged groups to the protein, is a necessary condition to induce a significant protein structural change.

Cell Fractionation↗
PubMed2002

Modulation of junctional communication by phosphorylation: protein phosphatases, the missing link in the chain.

Protein phosphorylation has been proposed to control the degree of intercellular gap junctional communication at several steps, from gene expression to protein degradation. In vertebrates, gap junctions are composed of proteins from the "connexin" (Cx) gene family, and the majority of connexins are post-translationally modified by phosphorylation. Alterations in the phosphorylation status of proteins, resulting from the dynamic interplay of protein kinases and protein phosphatases, are thought to be involved in a broad variety of connexin processes (such as the trafficking, assembly/disassembly and degradation, as well as the gating of gap junction channels), but the underlying mechanisms remain poorly understood. Although protein kinases have an established role in this process (see Cruciani and Mikalsen, this issue), less is known about the involvement of protein phosphatases. The present review examines the role played by protein dephosphorylation catalysers in the regulation of gap junctional communication.

Animals↗
PubMed2005

Combining metal oxide affinity chromatography (MOAC) and selective mass spectrometry for robust identification of in vivo protein phosphorylation sites.

BACKGROUND: Protein phosphorylation is accepted as a major regulatory pathway in plants. More than 1000 protein kinases are predicted in the Arabidopsis proteome, however, only a few studies look systematically for in vivo protein phosphorylation sites. Owing to the low stoichiometry and low abundance of phosphorylated proteins, phosphorylation site identification using mass spectrometry imposes difficulties. Moreover, the often observed poor quality of mass spectra derived from phosphopeptides results frequently in uncertain database hits. Thus, several lines of evidence have to be combined for a precise phosphorylation site identification strategy. RESULTS: Here, a strategy is presented that combines enrichment of phosphoproteins using a technique termed metaloxide affinity chromatography (MOAC) and selective ion trap mass spectrometry. The complete approach involves (i) enrichment of proteins with low phosphorylation stoichiometry out of complex mixtures using MOAC, (ii) gel separation and detection of phosphorylation using specific fluorescence staining (confirmation of enrichment), (iii) identification of phosphoprotein candidates out of the SDS-PAGE using liquid chromatography coupled to mass spectrometry, and (iv) identification of phosphorylation sites of these enriched proteins using automatic detection of H3PO4 neutral loss peaks and data-dependent MS3-fragmentation of the corresponding MS2-fragment. The utility of this approach is demonstrated by the identification of phosphorylation sites in Arabidopsis thaliana seed proteins. Regulatory importance of the identified sites is indicated by conservation of the detected sites in gene families such as ribosomal proteins and sterol dehydrogenases. To demonstrate further the wide applicability of MOAC, phosphoproteins were enriched from Chlamydomonas reinhardtii cell cultures. CONCLUSION: A novel phosphoprotein enrichment procedure MOAC was applied to seed proteins of A. thaliana and to proteins extracted from C. reinhardtii. Thus, the method can easily be adapted to suit the sample of interest since it is inexpensive and the components needed are widely available. Reproducibility of the approach was tested by monitoring phosphorylation sites on specific proteins from seeds and C. reinhardtii in duplicate experiments. The whole process is proposed as a strategy adaptable to other plant tissues providing high confidence in the identification of phosphoproteins and their corresponding phosphorylation sites.

Journal Article↗
PubMed1988

cAMP-dependent protein phosphorylation of membrane proteins in the parotid gland, platelets and liver. Comparison of a 22-kDa phosphoprotein from rat parotid microsomes (protein III) with phosphoproteins of similar molecular size from platelet and liver membranes.

Stimulation of secretion in exocrine secretory glands leads to the phosphorylation of a 22-kDa membrane protein (protein III) whose function is still unknown [Jahn et al. (1980) Eur. J. Biochem. 112, 345-352; Jahn & Söling (1980) Proc. Natl Acad. Sci. USA 78, 6903-6906]. This report describes the comparison of this protein with phosphorylated membrane proteins of similar molecular mass in platelets and liver. Incubation of platelets with agents which raise the intracellular cAMP concentration results in the phosphorylation of a 22-kDa protein which is also phosphorylated in membrane preparations by endogenous kinases or by exogenous cAMP-dependent protein kinase. It is shown that this protein is distinct from protein III although both proteins have the same molecular mass and are substrates of cAMP-dependent protein kinase. In contrast to platelets, protein III could be demonstrated in liver microsomes. This indicates that the function of protein III is not exclusively linked to the stimulus-secretion coupling in exocrine cells.

Animals↗
PubMed1998

Effects of inositol hexasulphate, a casein kinase inhibitor, on dentine phosphorylated proteins in organ culture of mouse tooth germs.

To study the effects of impaired protein phosphorylation on dentine formation and mineralization, inositol hexasulphate, an intracellular type I and type II casein kinase inhibitor, was used in an in vitro organotypic culture system. Mandibular first molar tooth germs were dissected from 18-day-old mouse embryos and cultured for 11 days with and without inositol hexasulphate at different concentrations. At 0.04-0.08 mM inhibitor, cellular alterations were not detected. Dentine displayed the characteristic purple-blue colour when Stains all, a specific stain for extracellular phosphoproteins, was used. At 0.1 mM, dentine failed to stain and mineralization did not occur, as seen from the von Kossa method. The presence of numerous lysosome-like vesicles inside cells indicated that the experiment was at the limits of cytotoxicity; higher concentrations induced severe cellular alterations. Therefore, quantitative radioautography was carried out on germs treated or not with the inhibitor at 0.1 mM. [33P]-phosphate incorporation showed that grain density in inhibited germs compared with that in control germs was about double in odontoblasts and half in the predentine/dentine compartment. In the presence of inositol hexasulphate the incorporation of [3H]serine into odontoblast cell bodies was unchanged between 2 and 24 h while in predentine/dentine, grain density was higher between 1 and 4 h, and reduced at 24 h. Both with [33P]phosphate and [3H]serine, labelling was seen throughout the porous dentine formed in vitro and not as a band located at the predentine/dentine junction, as is the case in vivo. With [3H]proline, in the presence of the inhibitor, a small reduction of grain density occurred in cell bodies, no significant difference was seen between 1 and 4 h in predentine/dentine, and more silver grains were present after 24 h both in cells and in the matrix. The radioautographic data support the view that the inhibitor interacts mostly with post-transductional phosphorylation and does not alter significantly other cell synthetic pathways and functions. Finally, the experiments presented here confirm that phophorylated proteins have a key role in dentine mineralization.

Analysis of Variance↗
PubMed2001

Protein phosphorylation in mitochondria from human placenta.

The aim of this study was to investigate whether mitochondria from human placenta contain phosphorylated proteins and kinases. Interestingly, the placenta contains two types of mitochondria with different sizes. These are 'heavy' mitochondria which sediment at a much lower g force than 'light' mitochondria. Mitochondria were incubated with [gamma32]P-ATP and labelled proteins analysed by electrophoresis and autoradiography. A major protein band of 20 kDa was detected with minor bands at 22, 38 and 85 kDa. The 20 kDa band was attenuated by 83 per cent by the co-incubation of mitochondria with Herbimycin, a tyrosine kinase inhibitor. A 20 kDa protein was also identified using an anti-tyrosine phosphate antibody and detection of this protein was significantly higher in heavy mitochondria as opposed to light mitochondria. Protein kinase A enzyme activity was also detected in mitochondria at a level not significantly different than that found in whole non-fractionated cells. These data indicate that mitochondria from human placenta contains kinase activity and phosphoproteins. These molecules may have functions in signalling systems in this organelle. Phosphoprotein signalling systems may be differentially modulated in heavy mitochondria as compared with light mitochondria.

Adenosine Triphosphate↗
PubMed2004

Open tubular immobilized metal ion affinity chromatography combined with MALDI MS and MS/MS for identification of protein phosphorylation sites.

Protein phosphorylation is one of the most important known posttranslational modifications. Tandem mass spectrometry has become an important tool for mapping out the phosphorylation sites. However, when a peptide generated from the enzymatic or chemical digestion of a phosphoprotein is highly phosphorylated or contains many potential phosphorylation residues, phosphorylation site assignment becomes difficult. Separation and enrichment of phosphopeptides from a digest mixture is desirable and often a critical step for MS/MS-based site determination. In this work, we present a novel open tubular immobilized metal ion affinity chromatography (OT-IMAC) method, which is found to be more effective and reproducible for phosphopeptide enrichment, compared to a commonly used commercial product, Ziptip from Millipore. A strategy based on a combination of OT-IMAC, sequential dual-enzyme digestion, and matrix-assisted laser desorption/ionization (MALDI) quadrupole time-of-flight tandem mass spectrometry for phosphoprotein characterization is presented. It is shown that MALDI MS/MS with collision-induced dissociation can be very effective in generating fragment ion spectra containing rich structural information, which enables the identification of phosphorylation sites even from highly phosphorylated peptides. The applicability of this method for real world applications is demonstrated in the characterization and identification of phosphorylation sites of a Na(+)/H(+) exchanger fusion protein, His182, which was phosphorylated in vitro using the kinase Erk2.

Journal Article↗
PubMed1989

Intracellular protein phosphorylation in oat (Avena sativa L.) protoplasts by phytochrome action. 1. Measurement of action spectra for the protein phosphorylation.

The effects of red light and wavelength dependency of the protein phosphorylation in oat protoplasts were investigated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. Red light (660 nm) irradiation of the protoplasts increased the phosphorylation of 15 different proteins, and the phosphorylation of 2 proteins (27 KDa, 32 KDa) out of 15 were observed to be dependent on the wavelength of the irradiating light. The phosphorylation densities of these two proteins increased up to two or three hundred percent during a three-minute period of irradiation. The phosphorylation of these two proteins revealed a red/far-red photoreversibility of phytochrome. When a calcium ion chelator (2 mM EGTA) was added into the cell suspension, the phosphorylations of all the proteins were reduced about 200%. These findings suggest that phytochrome action and Ca2+ influx are certainly involved in the in vivo phosphorylation of proteins in oat protoplasts.

Autoradiography↗
PubMed2000

Inhibitors of protein phosphorylation including the retinoblastoma protein induce germination of Candida albicans.

It has been previously shown that the induction of germination in Candida albicans occurs following its cessation of growth as a yeast. Similarly, mammalian cells undergo a differentiation process that is preceded by a growth cessation associated with a hypophosphorylation of proteins of the retinoblastoma gene family. It is postulated that a similar type of mechanism may be operative in C. albicans and protein phosphorylation inhibitors: forskolin (stimulates cyclic adenosine monophosphate production), okadaic acid (phosphatase inhibitor) and D-erythro-sphingosine (retinoblastoma protein phosphorylation inhibitor) have been used to further strengthen this hypothesis. Okadaic acid (1-1000 nM) and D-erythro-sphingosine (100 microM) significantly inhibited the growth of yeast cells of C. albicans. D-Erythro-sphingosine at 1000 microM was candidicidal. Forskolin did not significantly affect growth. Exponentially grown C. albicans pretreated with forskolin (10 microM), okadaic acid (1000 nM) or D-erythro-sphingosine (100 microM) readily germinated. In comparison, when these inhibitors were incorporated in the same medium, germination of exponentially grown cells did not occur. These results suggest that protein dephosphorylation may be necessary at an early stage of the yeast-hyphae transition in C. albicans.

Candida albicans↗
PubMed1982

Calmodulin and acidic compounds alter basic protein phosphorylation by the protein kinase from human platelets.

A cyclic nucleotide-independent protein kinase of human platelets, which phosphorylated histones, myelin basic protein and protamine and did not catalyze the phosphorylation of acidic proteins such as casein, phosvitin and myosin light chain, has been purified approx. 1,500-fold from the crude extract by steps of DEAE-cellulose, Sephadex G-200, hydroxylapatite and phosphoryl cellulose column chromatography. The substrate phosphorylation by this kinase was markedly enhanced by calmodulin even in the absence of Ca2+, when mixed histone was used as a substrate. The interaction of the kinase with mixed histone resulted in an irreversible inactivation of the enzyme. Calmodulin prevented this inactivation, and this compound produced an apparent increase in histone phosphorylation by the kinase. It should be noted that acidic polypeptides such as troponin-C, phospholipids and nucleic acids have a similar ability. The addition of Ca2+ reduced the effect of calmodulin more than the effects of other acidic compounds.

Blood Platelets↗
PubMed2002

The determination of protein phosphorylation on electrophoresis gel blots by laser ablation inductively coupled plasma-mass spectrometry.

Laser ablation interfaced with inductively coupled plasma-mass spectrometry is described as a new method to determine the presence of phosphorylated proteins on electrophoresis gel blots. The method was applied to the phosphoprotein beta-casein with good detection levels being observed at 16 pmole. Attempts at using the technique to detect beta-casein on electrophoresis gels are also described.

Caseins↗
PubMed2001

Use of antibodies for detection of phosphorylated proteins separated by two-dimensional gel electrophoresis.

Protein phosphorylation and dephosphorylation are key regulatory mechanisms in prokaryotic and eukaryotic cells. Considering the role of phosphorylation in many human diseases, it appears a major challenge to refine on the methods to analyze the phospho-proteome. Here we review the use of monoclonal antibodies directed against specific phosphorylated amino acid residues to visualize phosphoproteins separated by two-dimensional gel electrophoresis. Strategies are described how this method can successfully be applied to create phospho-proteome maps of mammalian cells.

Animals↗
PubMed2006

Total tau protein, phosphorylated tau (181p) protein, beta-amyloid(1-42), and beta-amyloid(1-40) in cerebrospinal fluid of patients with dementia with Lewy bodies.

The intra vitam diagnosis of different dementias is still based on clinical grounds. So far, no technical investigations have been available to support these diagnoses. For tau protein and beta-amyloid(1-42) in cerebrospinal fluid (CSF), promising results for the diagnosis of Alzheimer's disease (AD) have been reported; however, their differential diagnostic spectrum is limited, as was recently shown for dementia with Lewy bodies (DLB) and for AD. Therefore, further marker proteins have to be established to ameliorate, support, and differentiate these clinical diagnoses. We evaluated beta-amyloid(1-40) and phosphorylated tau protein (181p), in addition to total tau protein and beta-amyloid(1-42), in 20 patients with DLB, 34 AD patients, and 20 non-demented neurological controls (NDCs). All markers could differentiate between the dementia groups (AD, DLB) and the controls. AD and DLB could be differentiated only by levels of total tau protein and by the ratio total tau protein/phosphorylated tau protein. However, values still overlapped markedly. In some cases, tau protein levels in CSF may contribute to the clinical distinction between DLB and AD, but the value of the markers is still limited, especially because of mixed pathology. We conclude that more specific markers have to be established to differentiate between these diseases.

Aged↗
PubMed2002

Auxin stimulates S6 ribosomal protein phosphorylation in maize thereby affecting protein synthesis regulation.

Auxin is known to stimulate protein synthesis in many plant tissues, but the mechanisms involved in this process are unknown. The present research inquires whether auxin might regulate selective translation of mRNAs by inducing S6 ribosomal protein phosphorylation on the 40S ribosomal subunit in maize (Zea mays L.). Maize embryonic axes auxin-stimulated by natural (IAA) or synthetic (Dicamba or 1-NAA) auxins, selectively increased ribosomal protein synthesis. This effect was not reproduced by auxin inactive analogue 2-NAA. Enhanced S6 ribosomal protein phosphorylation on the 40S ribosomal subunit was also observed after auxin stimulation, as measured by [32P] incorporation into this protein. This increment did not occur when stimulation was performed with the inactive auxin analogue. Further, increased recruitment into polysomes of two 5'TOP-like mRNAs, encoding for the initiation translation factor eIF-iso4E and the S6 ribosomal protein, was also found after auxin stimulation of maize axes. A positive correlation was established between the levels of S6 ribosomal protein phosphorylation and the S6 ribosomal protein transcript recruitment into polysomes by means of okadaic acid or heat shock application to maize axes. These data indicate that auxin stimulates S6 ribosomal protein phosphorylation on maize ribosomes, concomitant to the recruitment of specific mRNAs (5'TOP-like mRNAs) into polysomes for translation. It is proposed that by this mechanism auxin regulate the synthesis of specific proteins in maize tissues.

Journal Article↗
PubMed1986

Studies on the methods for the determination of phosphorylation sites in highly phosphorylated peptides or proteins: phosphorylation sites of hen egg white riboflavin binding protein.

To determine the phosphate binding sites in hen egg white riboflavin binding protein (RBP), a highly phosphorylated peptide, which consisted of 23 amino acid residues including eight phosphoserines, was isolated from the tryptic digest of reduced and carboxymethylated RBP. The conditions of the beta-elimination-addition reaction to convert phosphoserine residues in the peptide to cysteic acids, S-methylcysteines, alanines, and beta-methylaminoalanines (DL-alpha-amino-beta-methylamino propionic acid) were examined. These converted peptides were purified by HPLC and subjected to Edman degradation. The results of Edman degradation indicated that the S-methylcysteine derivative of the peptide gave the most satisfactory result for determining the phosphate binding sites in the peptide. The phosphorylation sites of the peptide determined by the method mentioned above are as follows: His182-Leu-Leu-Ser185-Glu-Ser(P)-Ser(P)-Glu-Glu190-Ser (P)-Ser(P)-Ser(P)-Met-Ser195(P)-Ser(P)-Ser(P)-Glu-Glu-. These studies indicated that the conversion of phosphoserines in phosphoproteins to S-methylcysteines followed by Edman analysis was a useful method for the elucidation of the phosphorylation sites in phosphopeptides.

Amino Acid Sequence↗
PubMed1989

Protein phosphorylation in astrocytes mediated by protein kinase C: comparison with phosphorylation by cyclic AMP-dependent protein kinase.

The protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), has been found recently to transform cultured astrocytes from flat, polygonal cells into stellate-shaped, process-bearing cells. Studies were conducted to determine the effect of PMA on protein phosphorylation in astrocytes and to compare this pattern of phosphorylation with that elicited by dibutyryl cyclic AMP (dbcAMP), an activator of the cyclic AMP-dependent protein kinase which also affects astrocyte morphology. Exposure to PMA increased the amount of 32P incorporation into several phosphoproteins, including two cytosolic proteins with molecular weights of 30,000 (pI 5.5 and 5.7), an acidic 80,000 molecular weight protein (pI 4.5) present in both the cytosolic and membrane fractions, and two cytoskeletal proteins with molecular weights of 60,000 (pI 5.3) and 55,000 (pI 5.6), identified as vimentin and glial fibrillary acidic protein, respectively. Effects of PMA on protein phosphorylation were not observed in cells depleted of protein kinase C. In contrast to the effect observed with PMA, treatment with dbcAMP decreased the amount of 32P incorporation into the 80,000 protein. Like PMA, treatment with dbcAMP increased the 32P incorporation into the proteins with molecular weights of 60,000, 55,000 and 30,000, although the magnitude of this effect was different. The effect of dbcAMP on protein phosphorylation was still observed in cells depleted of protein kinase C. The results suggest that PMA, via the activation of protein kinase C, can alter the phosphorylation of a number of proteins in astrocytes, and some of these same phosphoproteins are also phosphorylated by the cyclic AMP-dependent mechanisms.

Animals↗
PubMed1993

Erythropoietin induces the association of phosphatidylinositol 3'-kinase with a tyrosine-phosphorylated protein complex containing the erythropoietin receptor.

Stimulation of sensitive cells with erythropoietin results in rapid induction of protein tyrosine phosphorylation. Other than tyrosine phosphorylation of one chain of the erythropoietin receptor, the identities of the remaining tyrosine-phosphorylated proteins are undefined. In this report, we demonstrate that the stimulation of the erythropoietin-sensitive human UT7 cells by erythropoietin rapidly resulted in the appearance of phosphatidylinositol 3-kinase activity in anti-phosphotyrosine immunoprecipitates. Erythropoietin action was rapid, detectable after as early as 1 min stimulation, transient, returning to control level after 30 min stimulation and was observed using the erythropoietin concentrations able to stimulate the cell proliferation. Anti-(phosphatidylinositol 3-kinase) antibodies specifically immunoprecipitated 125I-erythropoietin bound to its receptor, strongly suggesting that phosphatidylinositol 3-kinase associated with a protein complex containing the activated erythropoietin receptor. To confirm this result, phosphatidylinositol 3-kinase was immunoprecipitated from erythropoietin-stimulated cells using mild conditions followed by Western analysis using anti-phosphotyrosine antibodies. Five tyrosine phosphorylated proteins were revealed: the cloned chain of the erythropoietin receptor, the regulatory subunit of phosphatidylinositol 3-kinase and three unidentified proteins of 111, 97 and 64 kDa. None of these tyrosine phosphorylated proteins was detected in anti-(phosphatidylinositol 3-kinase) immunoprecipitates from unstimulated cells. Thus, our results show that phosphatidylinositol 3-kinase associates with a tyrosine-phosphorylated protein complex containing the activated erythropoietin receptor.

Cell Line↗