No description available.

Higher plants cannot move to a more favorable place when the environmental conditions are changing. To adapt to changes in light, temperature and access to water the plants had to evolve special mechanisms at the molecular level. Post-translational modifications of proteins, like phosphorylation, often serve as “on-and-off” switches in regulation of cellular activity and may affect protein-protein interactions. Photosynthesis in higher plants is regulated by reversible protein phosphorylation events, in a unique light- and redox-controlled system. Several biochemical methods are effectively used for characterization of phosphorylated proteins in photosynthetic membranes. Nevertheless, mass spectrometry is the most effective technique when it comes to identification of exact phosphorylation site(s) in the protein sequence, which is the ultimate evidence of protein phosphorylation. The same tandem mass spectrometry analysis identifies other in vivo post-translational modifications as well, such as acetylation of the N-terminus of mature protein. To study membrane proteins is a challenging project. In the present work the “shaving” of surface-exposed part of the membrane proteins, where phosphorylation occur, is used. In combination with mass spectrometry, this technique does not require the use of radioactive labeling or antibodies. The present work in spinach and Arabidopsis thaliana has identified and characterized several known phosphoproteins, new phosphorylation sites in well-known photosynthetic proteins, as well as two phosphoproteins previously unknown to be present in the photosynthetic membrane. Several photosystem II (PSII) core proteins become phosphorylated in their N-termini (D1, D2, CP43, PsbH), process involved in the regulation of the repair cycle of photo-damaged PSII complexes. The protein-protein interactions between PSII and its light harvesting complex (LHCII) seem to be affected by phosphorylation events in the interface area. In higher plants, phosphorylation sites have been identified in LHCII polypeptides, in one of the proteins (CP29) present in the interface area, as well as in the peripheral TSP9 protein. The TSP9 protein is unique among photosynthetic phosphoproteins, since it is a plant-specific soluble protein that becomes triple-phosphorylated in the middle part of the protein. It is also shown that photosystem I (PSI) is subjected to protein phosphorylation. The extrinsic PSI subunit PsaD becomes phosphorylated in its N-terminus. In addition, the latest characterized subunit of PSI, PsaP, is identified as a phosphoprotein. PsaP is an intrinsic protein assembled on the same side of the PSI complex as LHCII attaches. Several kinases are involved in phosphorylation of photosynthetic proteins, some more specific to PSII core proteins whereas others recognize LHCII proteins better. The STN8 kinase does not phosphorylate LHCII proteins, but is involved in the phosphorylation of the PSII core proteins D1, D2, CP43 and PsbH. STN8 is light-activated and is also specific in phosphorylation of threonine-4 (Thr-4) in the PsbH protein, but only after another kinase has phosphorylated Thr-2 first. A common feature of all kinases in plant photosynthetic membranes is the specificity for Thr residues and that the phosphorylation reactions occur in the N-terminal sequence of the proteins, except for the TSP9 protein. Nowadays, research is on the way to solve the complex network of regulation of photosynthetic activity via protein phosphorylation, but far more efforts are needed to get a complete view of the importance of all phosphorylation events and enzymatic specificity.

Diatoms are unicellular photosynthetic algae that display intricately patterned cell walls made of amorphous silicon dioxide (silica). Long-chain polyamines and highly phosphorylated proteins, silaffins and silacidins, are believed to play an important role in biosilica formation. The phosphate moieties on silaffins and silacidins play a significant role in biomineral formation, yet no kinase has been identified that phosphorylates these biomineral forming proteins. This dissertation describes the characterization of a novel kinase from the diatom Thalassiosira pseudonana, tpSTK1, which is upregulated during silica formation. A recombinantly expressed histidine-tagged version of tpSTK1 was capable of phosphorylating recombinant silaffins but not recombinant silacidin in vitro. Through establishing methods for subcellular fraction of T. pseudonana membranes in combination with antibody inhibition assay, it was discovered that native tpSTK1 phosphorylates silaffins but not silacidins in vitro (i.e. it exhibits the same substrate specificity as recombinant tpSTK1). As tpSTK1 is an abundant protein in the ER lumen (~ 0.5 % of total ER protein) it seems highly likely to function as a silaffin kinase in vivo. TpSTK1 lacks clear sequence homologs in non-diatom organisms and is the first molecularly characterized kinase that appears to be involved in biomineralization. The predicted kinase domain (KD) of tpSTK2, the only T. pseudonana homolog of tpSTK1, was recombinantly expressed and tested for phosphorylation activity. Recombinant tpSTK2-KD and native tpSTK2 exhibited detectable activity with myelin basic protein, but did not phosphorylate silaffins or silacidins in vitro. Western blot analysis demonstrated that native tpSTK2 was not present in the ER, but associated with the cytosol and Golgi membrane containing subcellular fractions.

Plants, green algae and cyanobacteria perform photosynthetic conversion of sunlight into chemical energy in the permanently changing natural environment. For successful survival and growth photosynthetic organisms have developed complex sensing and signaling acclimation mechanisms. The environmentally dependent protein phosphorylation in photosynthetic membranes is implied in the adaptive responses; however, the molecular mechanisms of this regulation are still largely unknown. We used a mass spectrometry-based approach to achieve a comprehensive mapping of the in vivo protein phosphorylation sites within photosynthetic membranes from the green alga Chlamydomonas reinhardtii subjected to distinct environmental conditions known to affect the photosynthetic machinery. The state transitions process regulating the energy distribution between two photosystems, involves the temporal functional coupling of phosphorylated light-harvesting complexes II (LHCII) to photosystem I (PSI). During state transitions several of the thylakoid proteins undergo redox-controlled phosphorylation-dephosphorylation cycles. This work provided evidences suggesting that redox-dependent phosphorylation-induced structural changes of the minor LHCII antenna protein CP29 determine the affinity of LHCII for either of the two photosystems. In state 1 the doubly phosphorylated CP29 acts as a linker between the photosystem II (PSII) core and the trimeric LHCII whereas in state 2 this quadruply phosphorylated CP29 would migrate to PSI on the PsaH side and provide the docking of LHCII trimers to the PSI complex. Moreover, this study revealed that exposure of Chlamydomonas cells to high light stress caused hyperphosphorylation of CP29 at seven distinct residues and suggested that high light-induced hyperphosphorylation of CP29 may uncouple this protein together with LHCII from both photosystems to minimize the damaging effects of excess light. Reversible phosphorylation of the PSII reaction center proteins was shown to be essential for the maintenance of active PSII under high light stress. Particularly dephosphorylation of the light-damaged D1 protein, a central functional subunit of the PSII reaction center, is required for its degradation and replacement. We found in the alga the reversible D1 protein phosphorylation, which until our work, has been considered as plant-specific. We also discovered specific induction of thylakoid protein phosphorylation during adaptation of alga to limiting environmental CO2. One of the phosphorylated proteins has five phosphorylation sites at both serine and treonine residues. The discovered specific low-CO2- and redox-dependent protein phosphorylation may be an early adaptive and signalling response of the green alga to limitation in inorganic carbon. This work provides the first comprehensive insight into the network of environmentally regulated protein phosphorylation in algal photosynthetic membranes.

The 2-micron circle of Saccharomyces cerevisiae is one of a small number of similar DNA plasmids found only in budding yeast. To understand how this cryptic parasite persists, despite conferring no advantage to the host, I investigated the plasmid-encoded Rep1 and Rep2 proteins. Interaction of Rep1 and Rep2 with each other and with the plasmid STB locus is required for equal partitioning of plasmid copies at mitosis. The Rep proteins also repress expression of Flp, the recombinase that mediates plasmid copy-number amplification. In this study, absence of Rep1 and Rep2, or over-expression of the plasmid-encoded Raf antirepressor, increased expression of a longer, novel FLP transcript. Translation of this mRNA may explain elevated Flp activity at low plasmid copy number. Raf competed for Rep2 selfassociation and interaction with Rep1, suggesting the mechanism of Raf anti-repression. Deletion analysis identified a target site for Rep protein repression of FLP that is also repeated in the STB locus, suggesting this as the sequence required for Rep protein association with both regions of the plasmid. Distinct roles for Rep1 and Rep2 were identified; Rep1 was found to depend on Rep2 for post-translational stability, with Rep2 dependent on Rep1 for stable association with STB. Lysine-to-arginine substitutions in Rep1 and Rep2 impaired their association with the host covalent-modifier protein SUMO, suggesting these were sites of sumoylation. The substitutions did not affect interaction of the Rep proteins with each other or their stability but did perturb plasmid inheritance, suggesting that Rep protein sumoylation contributes to their plasmid partitioning function. When Rep1 was mutant, both Rep proteins lost their normal localization to the nuclear foci where 2-micron plasmids cluster, and were impaired for association with STB, supporting this as the cause of defective plasmid inheritance. The potential sumoylation-dependent association of the Rep proteins with the 2-micron plasmid partitioning locus suggests the plasmid has acquired a strategy common to eukaryotic viral and host genomes that depend on sumoylation of their segregation proteins for faithful inheritance. Collectively, my results shed light on how the 2-micron plasmid maintains the delicate balance of persisting without harming its host.

京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第23551号 / 生博第462号 / 新制||生||62(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 中野 雄司, 教授 見学 美根子, 教授 千坂 修 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Small heat shock proteins (hsp) have been implicated in mediation of classic preconditioning in the rabbit. Hsp27 is a terminal substrate of the p38 MAPK cascade. One and 2D gel electrophoresis and immunoblotting of cell fractions was used to determine p38 MAPK and hsp27 phosphorylation levels, respectively, during in vitro ischemia in control, calyculin A (Cal A)-treated (protein phosphatase inhibitor), SB203580-treated (p38MAPK inhibitor) and preconditioned (IPC) isolated adult rabbit cardiomyocytes. The dual phosphorylation of p38 MAPK was increased by early ischemia (30-60 min), after which there was a loss of total cytosolic p38 MAPK. The ischemic increase of p38 MAPK dual phosphorylation was enhanced by IPC. Cal A strongly activated dual phosphorylation of p38 MAPK in oxygenated cells and this was maintained into early ischemia. SB203580 inhibited the dual phosphorylation of p38 MAPK and attenuated the loss of total cytosolic p38 MAPK. In each protocol, ischemia translocated hsp27 from the cytosolic fraction to the cytoskeletal fraction at similar rates and extents. Hsp27 phosphorylation was quantitated as the fraction of diphosphorylated hsp27, based on IEF mobility shifts of hsp27 phosphorylation isoforms. In oxygenated control cells, cytosolic and cytoskeletal hsp27 was highly phosphorylated. After 90 min ischemia, cytoskeletal hsp27 was markedly dephosphorylaled. Cal A slightly increased control cytoskeletal hsp27 phosphorylation. During ischemic incubation, Cal A blocked ischemic dephosphorylation. SB203580 accelerated ischemic hsp27 dephosphorylation and injury. IPC insignificantly decreased the initial rate of ischemic dephosphorylation of hsp27, but not the extent of dephosphorylation in later ischemia. Phosphorylation is regulated by both kinase and phosphatase activities. IPC protection was not correlated with a significant increase in cytosolic or cytoskeletal hsp27 phosphorylation levels during prolonged (> 60-90 min) ischemia.

No description available.

Three distinct families of PTPs, the conventional (cPTPs), low molecular weight (LMW PTPs), and Cdc25 PTPs, have converged upon a common catalytic mechanism and active site sequence, mainly, the phosphate-binding loop encompassing the PTP signature motif (H/V)<b>C</b>(X)₅<b>R</b>(S/T) and an essential Asp residue on a surface loop. There is little sequence similarity among the three families of phosphatases. All known LMW PTP remove phosphoryl groups esterified to the hydroxyl amino acid: tyrosine, whereas all members of the Cdc25 family are dual-specificity protein phosphatases that dephosphorylate all the hydroxyl amino acids: tyrosine, serine and threonine. The cPTP family primarily functions as tyrosine phosphatases, but it also includes dual-specific members. ORFs encoding potential cPTPs have been identified in five archaeal species: <i>Methanobacterium thermoautotrophicum</i>, <i>Methanococcus jannaschii</i>, <i>Thermococcus kodakaraensis</i>, <i>Pyrococcus horikoshii</i>, and <i>S. solfataricus</i>. Only one has been partially characterized, <i>Tk</i>-PTP from <i>T. kodakaraensis</i>. Hence, our current body of knowledge concerning the functional properties and physiological roles of these enzymes remains fragmented. The genome of <i>S. solfataricus</i> encodes a single conventional protein tyrosine phosphatase, SsoPTP. SsoPTP is the smallest known archaeal PTP (18.3 kDa) with a primary amino acid sequence that conforms to the cPTP protein tyrosine phosphatase paradigm, HCX₅R(S/T). Relatively little is known about its mode of action " whether it follows the conventional PTP mechanism or employs a different route for catalysis " or its physiological role. ORF <i>sso2453</i> from the genome of <i>Sulfolobus solfataricus</i>, encoding a protein tyrosine phosphatase, was cloned and its recombinant protein product, SsoPTP, was expressed in <i>E. coli</i> and purified by immobilized metal affinity chromatography. SsoPTP displayed the ability to dephosphorylate protein-bound phosphotyrosine as well as protein-bound phosphoserine/phosphothreonine. SsoPTP hydrolyzed both isomers of naphthyl phosphate, an indication of dual specificity. The four conserved residues within the presumed active site sequence: Asp⁶⁹, His⁹⁵, and Arg¹⁰², and the invariant Gln¹³⁹ residue were essential for catalysis, as it was predicted for the established members of the PTP family in both bacteria and eukaryotes. A substrate trapping protein variant, SsoPTP-C96S/D69A, was constructed to isolate possible SsoPTP substrates present in <i>S. solfataricus</i> cell lysates. Several potential substrates were isolated and identified by mass spectroscopy. / Ph. D.

Protein phosphorylation plays critical roles in diverse cellular functions, including cell cycle, growth, differentiation, and apoptosis. Deregulated phospho-signaling is often associated with many human diseases and cancers. Despite tremendous efforts to investigate the molecular mechanisms that control the functionality of phospho-signaling pathways, only limited progress has been made on describing the temporal and spatial profiles of cellular protein phosphorylation. The main challenges associated with the study of phospho-signaling processes in cells are related to the short time-scale of certain phosphorylation and dephosphorylation events, the low abundance of the phosphorylated protein forms as compared to their non-phosphorylated counterparts, the complicated and time-consuming sample preparation methods that are accompanying such type of work, and, last, the performance of the detection methods that are suitable for assessing protein phosphorylation. To tackle the challenges associated with the investigation of protein phosphorylation in cells, our objective was to develop a combined mass spectrometry (MS) and microfluidics strategy that enables fast sampling and sensitive detection of key signaling phosphoproteins in complex biological samples. MS is the most widely used analytical tool in the field of proteomics due to its high sensitivity, specificity, and throughput. Microfluidics has been proven as a suitable platform for handling small volumes of scarce samples, being also amenable to automation, integration, and multiplexing. To achieve our objective, this study was conducted in multiple steps: (1) We performed a comprehensive analysis of the factors that affect the performance of mass spectrometry detection (i.e., sensitivity, reproducibility, ability to accurately identify a large number of proteins from complex samples), when used in conjunction with technologies that are conducted in a non-standard fashion, on short time-scales; (2) We developed and evaluated a miniaturized strategy for rapid proteolytic digestion and phosphopeptide enrichment; (3) We demonstrated sensitive detection and quantification of phosphopeptides from complex biological samples using multiple reaction monitoring mass spectrometry (MRM-MS) and microfluidic sample processing; and (4) We developed a microfluidic platform for handling and processing cells that enables the investigation of biological processes that occur on short time-scales, and that can be integrated with the devices developed for the analysis of phospho-proteins. SKBR3 cells were used as a model system for developing and demonstrating the microfluidic chips. The detection and quantification of phospho-proteins involved in MAPK (mitogen activated protein kinase) signaling pathways was achieved at the low nM level. Overall, this study demonstrates proof-of-concept applicability of a microfluidics-MS strategy for monitoring phosphorylation processes in signaling networks. / PHD