N‐terminal sequence analysis of the 8 kDa protein in<i>Chlamydomonas reinhardii</i>Localization of the phosphothreonine

Dedner, Norbert; Meyer, Helmut E.; Ashton, Chris; Wildner, Günter F.

doi:10.1016/0014-5793(88)80288-6

Cited by 47 publications

(23 citation statements)

References 26 publications

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“…Despite the generally successful application of vectorial proteomics in this study, we cannot claim that this single approach could identify all protein phosphorylation sites in the algal thylakoid membranes. Particularly we did not detect any phosphorylation of the PSII core subunit PsbH whose phosphorylation was demonstrated in Chlamydomonas cells grown mixotrophically in the presence of high CO 2 (5%) (37). However, it should be emphasized that in all conditions of our study the cells were grown at low CO 2 (0.035%), which may explain this difference.…”

Section: Discussioncontrasting

confidence: 65%

“…To date, protein phosphorylation in photosynthetic membranes of plants and algae has been studied by seven different techniques: (i) radioactive labeling (4,6,8,9,24,25), (ii) detection of the shift in the electrophoretic mobility of individual proteins (24, 26 -28), (iii) immunological analyses with anti-phosphoamino acid antibodies (27)(28)(29)(30), (iv) site-directed mutagenesis of potential protein phosphorylation sites (31)(32)(33), (v) mass spectrometric determination of the masses for intact phosphorylated proteins (34,35), (vi) amino-terminal protein sequencing by Edman degradation (36,37), and (vii) identification and mass spectrometric sequencing of phosphorylated peptides obtained by proteolytic treatment of the membranes (38 -41). None of these techniques is ideal, and the identification of protein phosphorylation events largely depends on the detection method used (28).…”

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confidence: 99%

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Environmentally Modulated Phosphoproteome of Photosynthetic Membranes in the Green Alga Chlamydomonas reinhardtii

Turkina

Kargul

Blanco-Rivero

et al. 2006

Molecular & Cellular Proteomics

108

119

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Mapping of in vivoThe thylakoid membranes in chloroplasts of plants and green algae carry out oxygenic photosynthesis. Two multisubunit pigment-containing protein complexes, photosystem II (PSII) 1 and photosystem I (PSI), located in this membrane system work in series to generate an electrochemical potential gradient of protons across the membrane following vectorial electron flow from PSII to PSI via the cytochrome b 6 f complex. PSII uses light to oxidize water, whereas PSI, via a second photoact, uses reducing equivalents derived from PSII to reduce NADP ϩ to NADPH. The electrochemical potential gradient of protons is used to power conversion of ADP to ATP (1). Several thylakoid membrane proteins that make up the PSII complex and its LHCII (light-harvesting chlorophyll a/b-binding proteins of PSII) antennae undergo light-and redox-dependent phosphorylation (2, 3) as discovered more than 2 decades ago (4 -6). Phosphorylation of LHCII in plants and algae controls photosynthetic state transitions, which optimize efficient use of the absorbed light energy by both photosystems. Thus, in State 1 more energy is transferred to PSII, whereas in State 2 a proportion of the excitation energy is redistributed to PSI (7-9). The essential role of protein phosphorylation in state transitions has recently been proven in the studies using mutants of Arabidopsis thaliana plants and the green alga Chlamydomonas reinhardtii deficient in protein kinases STN7 (9) and Stt7 (8), respectively. However, despite the generally assumed similarity in thylakoid protein phosphorylation between plants and algae and a high homology between the plant STN7 and the algal Stt7 protein kinases (8), the extent of photosynthetic state transitions differs between these species. In plant thylakoids, only 15-20% of LHCII participates in the lateral migration between the photosystems, whereas up to 80% of the excitation energy absorbed by the LHCII antenna can be redistributed from PSII to PSI in green alga (8 -10).

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Section: Discussioncontrasting

confidence: 65%

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confidence: 99%

Environmentally Modulated Phosphoproteome of Photosynthetic Membranes in the Green Alga Chlamydomonas reinhardtii

Turkina

Kargul

Blanco-Rivero

et al. 2006

Molecular & Cellular Proteomics

108

119

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“…Under these conditions, the hydroxyl groups of serine and threonine are much more stable. However, the phosphate groups ofphosphoserine and phosphothreonine are still readily eliminated, leading to the generation of >PhNCS-DTT-dehydroalanine and -dehydroaminobutyric acid, respectively (32). For example, using this approach, the ratio of >PhNCS-DTT-dehydroalanine to serine was found to be 5:1 in the third cycle of sequencing peptide 7, indicating that this site was heavily phosphorylated (Fig.…”

Section: Resultsmentioning

confidence: 98%

“…Symmetrical peaks from both HPLC runs were sequenced in an Applied Biosystems 477A sequencer equipped with a miniaturized reaction cartridge; rapid-cycle chemistry and on-line analysis programs (31) the increase of their dithiothreitol (DTT)/phenylthiohydantoin (>PhNCS) addition products, >PhNCS-DTT-dehydroalanine and >PhNCS-DTT-dehydroaminobutyric acid, respectively (32). In Vitro Phosphorylation of Synthetic Peptides.…”

Section: Methodsmentioning

confidence: 99%

Activation of p70s6k is associated with phosphorylation of four clustered sites displaying Ser/Thr-Pro motifs.

Ferrari

Bannwarth

Morley

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

158

128

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Partial amino acid sequences were obtained from 22 internal tryptic peptides of rat liver p70' (Mr 70000 ribosomal protein S6 kinase), 3 of which were found to contain phosphorylated residues. To determine whether these sites were asoated with p70" activation, the kinase was labeled to high specific activity with 32P1 in Swiss mouse 3T3 cells. By sequential cleavage with CNBr and endoproteinase Lys-C followed by two-dimensonal tryptic peptide analysis, it could be shown that all of the sites were located in a small endoproteinase Lys-C peptide ofMr 2400. Analysis of the p70"" protein sequence revealed a single candidate that could represent this peptide. Three tryptic peptides derived from the endoproteinase Lys-C fragment were chosen by a newly described computer program as the most likely candidates to contain the in vvo sites of phosphorylation. Synthetic peptides based on these sequences were phosphorylated either chemically or enzymatically and found to comigrate by two-dimensional thin-layer electrophoresis/chromatography with the four major in vivo labeled tryptic phosphopeptides. Three of the phosphorylation sites in these peptides were equivalent to those sequenced in the rat liver p7O"". In addition, all four sites display the motif Ser/Thr-Pro, typical of cell cycle-regulated sites, and are clustered in a putative autoinhibitory domain of the enzyme.Growth factors induce quiescent cells in culture to reenter the cell cycle, replicate their DNA, and divide through a complex array ofbiochemical responses (1). In most cases this process is initiated through specific ligand-activated receptor tyrosine kinases at the cell surface but is orchestrated intracellularly by a network of activated serine/threonine kinases (2). The mechanisms by which tyrosine kinases activate serine/ threonine kinases are still unclear, with the possible exception of protein kinase C (2, 3). One of the many early obligatory responses elicited by growth factors is the activation and maintenance of high rates of protein synthesis, which is required for reentry and transit through the G1 phase of the cell cycle (4, 5). This event is associated with increased phosphorylation of serine/threonine residues in a number of specific translational components, including 40S ribosomal protein S6 (6, 7). In vitro and in vivo studies have indicated that phosphorylation at five serine residues at the carboxyl terminus of S6 is required for the activation of protein synthesis (8-11). The kinase mediating this event was first detected in extracts of quiescent cells stimulated to proliferate by serum or epidermal growth factor (12, 13). Purification of this S6 kinase activity eventually revealed an enzyme of Mr 70,000, referred to as p70s6k (14,15), which is highly specific for ribosomal protein S6 (14), biphasically activated (16, 17), and selectively inactivated by type 2A phosphatase (18). The p70s"s phosphorylates four and possibly all five ofthe S6 sites observed in vivo (19), with recognition of the substrate absolutely dependent o...

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“…Radioactive labeling was done with [y. '~P]ATP (NEN Du Pont) at 10 mCi/ml, radioactive t~ptic peptides were generated, isolated by HPLC, and sequenced as described [3,30], For details on sequencing methods see [8,29].…”

Section: Methodsmentioning

confidence: 99%

The Alzheimer‐like phosphorylation of tau protein reduces microtubule binding and involves Ser‐Pro and Thr‐Pro motifs

et al. 1992

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Tau protein can be transformed into an Alzheimer-like state by pho~phorylation with a kinase activity from brain EMBO J, 11, 1593EMBO J, 11, -1597], Here we show that the phosphorylation at Set-Pro motifs strongly decreases tau's affinity for mierotubul~s. The major reduction occurs during the first of the three main stages of phosphorylation, The data explain the lower stability of microtubules resulting from the pathological tau phosphorylation,

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N‐terminal sequence analysis of the 8 kDa protein inChlamydomonas reinhardiiLocalization of the phosphothreonine

Cited by 47 publications

References 26 publications

Environmentally Modulated Phosphoproteome of Photosynthetic Membranes in the Green Alga Chlamydomonas reinhardtii

Environmentally Modulated Phosphoproteome of Photosynthetic Membranes in the Green Alga Chlamydomonas reinhardtii

Activation of p70s6k is associated with phosphorylation of four clustered sites displaying Ser/Thr-Pro motifs.

The Alzheimer‐like phosphorylation of tau protein reduces microtubule binding and involves Ser‐Pro and Thr‐Pro motifs

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