Major Phosphoprotein Research Articles

DOK1 and DOK2 regulate CD8 T cell signaling and memory formation without affecting tumor cell killing

Targeting intracellular inhibiting proteins has been revealed to be a promising strategy to improve CD8+ T cell anti-tumor efficacy. Here, we are focusing on intracellular inhibiting proteins specific to TCR signaling: DOK1 and DOK2 expressed in T cells. We hypothesized that depletion of intracellular inhibition checkpoint DOK1 and DOK2 could improve CD8+ T-cell based cancer therapies. To evaluate the role of DOK1 and DOK2 depletion in physiology and effector function of CD8+ T lymphocytes and in cancer progression, we established a transgenic T cell receptor mouse model specific to melanoma antigen hgp100 (pmel-1 TCR Tg) in WT and Dok1/Dok2 DKO (double KO) mice. We showed that both DOK1 and DOK2 depletion in CD8+ T cells after an in vitro pre-stimulation induced a higher percentage of effector memory T cells as well as an up regulation of TCR signaling cascade- induced by CD3 mAbs, including the increased levels of pAKT and pERK, two major phosphoproteins involved in T cell functions. Interestingly, this improved TCR signaling was not observed in naïve CD8+ T cells. Despite this enhanced TCR signaling essentially shown upon stimulation via CD3 mAbs, pre-stimulated Dok1/Dok2 DKO CD8+ T cells did not show any increase in their activation or cytotoxic capacities against melanoma cell line expressing hgp100 in vitro. Altogether we demonstrate here a novel aspect of the negative regulation by DOK1 and DOK2 proteins in CD8+ T cells. Indeed, our results allow us to conclude that DOK1 and DOK2 have an inhibitory role following long term T cell stimulations.

Protein phosphatases in the RNAPII transcription cycle: erasers, sculptors, gatekeepers, and potential drug targets.

The transcription cycle of RNA polymerase II (RNAPII) is governed at multiple points by opposing actions of cyclin-dependent kinases (CDKs) and protein phosphatases, in a process with similarities to the cell division cycle. While important roles of the kinases have been established, phosphatases have emerged more slowly as key players in transcription, and large gaps remain in understanding of their precise functions and targets. Much of the earlier work focused on the roles and regulation of sui generis and often atypical phosphatases-FCP1, Rtr1/RPAP2, and SSU72-with seemingly dedicated functions in RNAPII transcription. Decisive roles in the transcription cycle have now been uncovered for members of the major phosphoprotein phosphatase (PPP) family, including PP1, PP2A, and PP4-abundant enzymes with pleiotropic roles in cellular signaling pathways. These phosphatases appear to act principally at the transitions between transcription cycle phases, ensuring fine control of elongation and termination. Much is still unknown, however, about the division of labor among the PPP family members, and their possible regulation by or of the transcriptional kinases. CDKs active in transcription have recently drawn attention as potential therapeutic targets in cancer and other diseases, raising the prospect that the phosphatases might also present opportunities for new drug development. Here we review the current knowledge and outstanding questions about phosphatases in the context of the RNAPII transcription cycle.

Collapsin Response Mediator Proteins: Their Biological Functions and Pathophysiology in Neuronal Development and Regeneration.

Collapsin response mediator proteins (CRMPs), which consist of five homologous cytosolic proteins, are one of the major phosphoproteins in the developing nervous system. The prominent feature of the CRMP family proteins is a new class of microtubule-associated proteins that play important roles in the whole process of developing the nervous system, such as axon guidance, synapse maturation, cell migration, and even in adult brain function. The CRMP C-terminal region is subjected to posttranslational modifications such as phosphorylation, which, in turn, regulates the interaction between the CRMPs and various kinds of proteins including receptors, ion channels, cytoskeletal proteins, and motor proteins. The gene-knockout of the CRMP family proteins produces different phenotypes, thereby showing distinct roles of all CRMP family proteins. Also, the phenotypic analysis of a non-phosphorylated form of CRMP2-knockin mouse model, and studies of pharmacological responses to CRMP-related drugs suggest that the phosphorylation/dephosphorylation process plays a pivotal role in pathophysiology in neuronal development, regeneration, and neurodegenerative disorders, thus showing CRMPs as promising target molecules for therapeutic intervention.

Anti-elastase and anti-hyaluronidase activity of phosvitin isolated from hen egg yolk

ABSTRACT1. Phosvitin, a major phosphoprotein found in egg yolk, has strong antioxidant activity. Activation of elastase, collagenase, and hyaluronidase by reactive oxygen species are related to the degradation of ECM and skin aging. The objective of this study was to determine the anti-elastase and anti-hyaluronidase activity of phosvitin.2. Elastase from porcine pancreas and hyaluronidase from bovine testes were used to study the inhibitory activity of phosvitin. To elucidate the mechanism of enzyme inhibition, a Lineweaver-Burk plot was constructed.3. Phosvitin inhibited elastase and hyaluronidase activity in a dose-dependent manner. The IC50 value of phosvitin was 31.6 μg/ml and 1,270 μg/ml against elastase and hyaluronidase, respectively. The analysis of elastase and hyaluronidase kinetics indicated that the apparent Michaelis constant (appKm) was increased by phosvitin but the Vmax value was not affected.4. In conclusion, phosvitin exhibited competitive inhibitory activity against elastase and hyaluronidase. Thus, phosvitin could be used as a natural anti-aging agent in the cosmetics industry.

Consequences of Keratin Phosphorylation for Cytoskeletal Organization and Epithelial Functions.

Intermediate filaments are major phosphoproteins. The complex patterns of intermediate filament phosphorylation make up a poorly understood code reflecting cytoskeletal properties and cellular function through an intense crosstalk with multiple signaling pathways. This review focuses on the epithelial keratin intermediate filaments highlighting the tight-knit relationship of keratin phosphorylation and network organization during cell division and apoptosis, and the importance of keratin phosphorylation during epithelial stress responses. The occurrence of keratin phosphorylation in genetic skin diseases and acquired diseases of simple epithelial tissues in liver, pancreas, and colon will be discussed. Finally, we will review the role of keratin phosphorylation in cancer with an emphasis on migration.

Proteomic analysis of quail calcified eggshell matrix: a comparison to chicken and turkey eggshell proteomes.

BackgroundEggshell mineralization in commercially important species such as chicken, turkey or quail is of interest as a general model of calcium carbonate biomineralization. Knowledge of proteins and molecular mechanisms in eggshell assembly may also pave the way to manipulation of thickness of the calcified layer or other features. Comparison of eggshell matrix proteomes of different species may contribute to a better understanding of the mineralization process. The recent publication of the quail genome sequence now enables the proteomic analysis of the quail shell matrix and this comparison with those of chicken and turkey.ResultsThe quail eggshell proteome comprised 622 identified proteins, 311 of which were shared with chicken and turkey eggshell proteomes. Forty-eight major proteins (iBAQ-derived abundance higher than 0.1 % of total identified proteome) together covered 94 % of total proteome mass. Fifteen of these are also among the most abundant proteins in chicken and turkey eggshell matrix. Only three proteins with a percentage higher than 1.0 % of the total had not previously been identified as eggshell matrix proteins. These were an uncharacterized member of the latexin family, an uncharacterized protease inhibitor containing a Kunitz domain, and gastric intrinsic factor. The most abundant proteins were ovocleidin-116, ovalbumin and ovocalyxin-36 representing approximately 31, 13 and 8 % of the total identified proteome, respectively. The major phosphoproteins were ovocleidin-116 and osteopontin. While osteopontin phosphorylation sites were predominantly conserved between chicken and quail sequences, conservation was less in ovocleidin-116.ConclusionsOvocleidin-116 and ovocalyxin-36 are among the most abundant eggshell matrix proteins in all three species of the family Phasianidae analyzed so far, indicating that their presently unknown function is essential for eggshell mineralization. Evidence for other chicken eggshell-specific proteins in quail was inconclusive. Therefore measurement of additional eggshell proteomes, especially from species of different families and preferentially from outside the order Galliformes, will be necessary.Electronic supplementary materialThe online version of this article (doi:10.1186/s12953-015-0078-1) contains supplementary material, which is available to authorized users.

DSPP Contains an IRES Element Responsible for the Translation of Dentin Phosphophoryn

The major phosphoprotein in dentin is the aspartic acid and serine-rich protein called dentin phosphophoryn (DPP). DPP appears to be synthesized as a part of a larger compound protein, dentin sialophosphoprotein (DSPP). DSPP has never been isolated or detected in dentin extracts. It is now evident that DSPP is a chimeric protein composed of 3 parts: dentin sialoprotein (DSP), DPP, and dentin glycoprotein (DGP). Previous reports have suggested that the BMP1 protease is responsible for processing DSPP. However, unequal amounts of these products are present in the dentin matrix. Here, we provide evidence for an internal ribosome entry site in the DSPP gene that directs the synthesis of DPP. This mechanism would account for unequal amounts of intracellular DSP and DPP. The internal ribosomal entry site (IRES) activity varied in different cell types, suggesting the presence of additional regulatory elements during the translational regulation of DPP. Further, we provide evidence that DPP is transported to the extracellular matrix (ECM) through exosomes. Using tissue recombination and lentivirus-mediated gain-of-function approaches, we also demonstrate that DPP is essential for the formation of well-defined tooth structures with mineralized dentin matrix.

Interaction between titanium and phosphoproteins revealed by chromatography column packed with titanium beads

The biochemical mechanism behind the strong binding between titanium and living bone has not been fully elucidated, in spite of worldwide clinical application of this phenomenon. We hypothesized that one of the core mechanisms may reside in the interaction between certain proteins in the host tissues and the implanted titanium. To verify the interaction between titanium and proteins, we chose the technique of chromatography in that titanium spherical beads (45 μm) were packed into a column to obtain a bed volume of 16×50 mm, which was eluted with phosphate buffered saline (PBS) and a straight gradient system made by using PBS and 25 mM NaOH. Fetal calf serum, albumin, lysozyme, casein, phosvitin and dentin phosphoprotein (phosphophoryn) were applied to the column. Most part of albumin and lysozyme eluted with the breakthrough peak, indicating practically no affinity to titanium. Fetal bovine serum also eluted mostly as the breakthrough peak, but distinct retained peak was observed. On the other hand, α-casein, phosvitin and phosphophoryn exhibited a distinct retained peak separated from the breakthrough peak. We proposed that phosphate groups (phosphoserines) in the major phosphoproteins, α-casein, phosvitin and phosphophoryn may be involved in the binding of these proteins with titanium.

14-3-3 phosphoprotein interaction networks - does isoform diversity present functional interaction specification?

The 14-3-3 proteins have emerged as major phosphoprotein interaction proteins and thereby constitute a key node in the Arabidopsis Interactome Map, a node through which a large number of important signals pass. Throughout their history of discovery and description, the 14-3-3s have been described as protein families and there has been some evidence that the different 14-3-3 family members within any organism might carry isoform-specific functions. However, there has also been evidence for redundancy of 14-3-3 function, suggesting that the perceived 14-3-3 diversity may be the accumulation of neutral mutations over evolutionary time and as some 14-3-3 genes develop tissue or organ-specific expression. This situation has led to a currently unresolved question – does 14-3-3 isoform sequence diversity indicate functional diversity at the biochemical or cellular level? We discuss here some of the key observations on both sides of the resulting debate, and present a set of contrastable observations to address the theory functional diversity does exist among 14-3-3 isoforms. The resulting model suggests strongly that there are indeed functional specificities in the 14-3-3s of Arabidopsis. The model further suggests that 14-3-3 diversity and specificity should enter into the discussion of 14-3-3 roles in signal transduction and be directly approached in 14-3-3 experimentation. It is hoped that future studies involving 14-3-3s will continue to address specificity in experimental design and analysis.

Development of an off-line capillary column IMAC phosphopeptide enrichment method for label-free phosphorylation relative quantification

Immobilized metal affinity chromatography (IMAC) and metal oxide type affinity chromatography (MOAC) techniques have been widely used for mass spectrometry-based phosphorylation analysis. Unlike MOAC techniques, IMAC requires rather complete removals of buffering reagents, salts and high concentrations of denaturant prior to sample loading in order for the successful enrichment of phosphopeptides. In this study, a simple off-line capillary column-based IMAC phosphopeptide enrichment method can shorten sample preparation time by eliminating the speed-vac step from the desalting process. Tryptic digest peptide samples containing 2 M urea can be directly processed and the entire IMAC procedure can be completed within 6 h. When tryptic digest peptide samples prepared from mouse whole brain tissues were analyzed using our method, an average of 249 phosphoproteins and 463 unique phosphopeptides were identified from single 2-h RPLC–MS/MS analysis (∼88% specificity). An additional advantage of this method is the significantly improved reproducibility of the phosphopeptide enrichment results. When four independent phosphopeptide enrichment experiments were carried out, the peak areas of phosphopeptides identified among four enrichment experiments were relatively similar (less than 16.2% relative standard dev.). Because of this increased reproducibility, relative phosphorylation quantification analysis of major phosphoproteins appears to be feasible without the need for stable isotope labeling techniques.

Abstract 2967: Nucleolin phosphorylation by CK2 modulates its role in cell cycle checkpoint activation

Abstract Nucleolin is a multifunctional abundant nucleolar protein and its gene expression is transactivated by the proto-oncogene c-myc during exponential cellular growth. This non-ribosomal protein is a major nucleolar phosphoprotein which is involved in ribosomal RNA processing, RNA polymerase I-catalyzed transcription, and the regulation of apoptosis, demonstrating both positive and negative effects on cell proliferation. During an unperturbed cell-cycle, interphase (CK2) and mitotic (Cdc2) kinases regulate nucleolin phosphorylation, modulating its function and nucleolar localization. Following cellular stress, nucleolin has been shown to inhibit chromosomal replication and to relay nucleolar stress signals mediated by its re-localization within the nucleus. Nucleolin also interacts with prominent components of the ARF (Alternate Reading Frame)/p53 checkpoint pathway that controls growth upon oncogenic stress signals. We have previously demonstrated that nucleolin binds to the p53-antagonist Hdm2, inhibits Hdm2-mediated degradation of p53 and causes p53-mediated apoptosis in an ARF-independent manner. Here we further dissect role/s of nucleolin phosphorylation in regulating the ARF/p53 checkpoint. We have generated a novel system by engineering human osteocarcinoma (NARF6) cells to induce expression of nucleolin-wt (wild-type) or -S6A (a phospho-mutant with six consensus site serines mutated to alanine, impairing phosphorylation by CK2). This novel system can control expression of both nucleolin and ARF by different promoters, allowing us to dissect the downstream events that are solely nucleolin- or ARF- dependent, or regulated by both. We found that increased levels of nucleolin-S6A significantly decreased ARF protein levels as compared to nucleolin-wt. Nucleolin-S6A was significantly more nucleoplasmic and, interestingly, cells that express S6A lacked nucleolar ARF expression. Although a dramatic loss in both ARF and p21 protein levels is evident with nucleolin-S6A expression, there was a net increase in p53 levels. Significantly, nucleolin-S6A expression caused an increase in the fraction of G1 cells in an ARF-independent manner. Thus, reduction of nucleolin phosphorylation by CK2 acts as a molecular switch causing the sub-nuclear re-localization of both nucleolin and ARF. These events lead to p53 activation, and the control of growth in both normal and stressed cells. These results also provide validation of our unique cellular system that allows us to manipulate nucleolin wt and mutant levels, facilitating the study of its physiological functions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2967. doi:10.1158/1538-7445.AM2011-2967

Quantitative genetic study of the circulating osteopontin in community-selected families

The study assessed contribution of genetic factors to variability of osteopontin (OPN) levels. Evidence of association of OPN levels with polymorphisms in its structural gene and integrin-binding sialoprotein gene loci was obtained. The results motivate research of OPN-related proteins and genes with respect to biomineralization and other biological processes. OPN is a major phosphoprotein in bone, which plays key role in regulation of bone mineralization process. It is considered as a promising biomarker for osteoarthritis and osteoporosis, and various other pathological conditions. However, the contribution of genetics and other confounding factors to OPN circulating levels variation in general population has never been specifically determined. The main aims of the present study included (1) evaluation of the putative genetic and familial factors' effect on OPN variability and (2) testing the hypothesis that OPN plasma levels are associated with the genetic polymorphisms in its structural gene locus (SPP1) and in integrin-binding sialoprotein gene locus (IBSP). To address these questions, we used a family-based sample of 925 apparently healthy Caucasian individuals. Association of OPN levels with three SNPs in each of the two selected gene loci was explored using pedigree disequilibrium tests. Some 58% and 13% of the OPN levels variability were attributable to genetic factors and common spouse environment, respectively. Three SNPs showed nominally significant association with OPN (p < 0.05). Of these, rs2616262 linked to IBSP promoter region remained significant after correction for multiple testing (p = 0.003). Significant association of this SNP and rs10516799 (distal segment of SPP1) with OPN was confirmed in several statistical tests. Using a special modification of variance component analysis, we examined gene-gene and gene-sex interaction effects, but found non-significant confirmation for these hypotheses. Further studies are required to confirm the observed results and to explore the underlying molecular and physiological mechanisms.

Stabilization of amorphous calcium carbonate by phosphate rich organic matrix proteins and by single phosphoamino acids

Stable amorphous calcium carbonate (ACC) is a unique material produced naturally exclusively as a biomineral. It was demonstrated that proteins extracted from biogenic stable ACC induce and stabilize synthetic ACC in vitro. Polyphosphate molecules were similarly shown to induce amorphous calcium carbonate formation in vitro. Accordingly, we tested the hypothesis that biogenic ACC induction and stabilization is mediated by the phosphorylated residues of phosphoproteins. We show that extracellular organic matrix extracted from gastroliths of the red claw crayfish Cherax quadricarinatus induce stable ACC formation in vitro. The proteinaceous fraction of this organic matrix is highly phosphorylated and is incorporated into the ACC mineral phase during precipitation. We have identified the major phosphoproteins of the organic matrix and showed that they have high calcium binding capacity. Based on the above, in vitro precipitation experiments with single phosphoamino acids were performed, indicating that phosphoserine or phosphothreonine alone can induce the formation of highly stable ACC. The results indicate that phosphoproteins may play a major role in the control of ACC formation and stabilization and that their phosphoamino acid moieties are key components in this process.

Frequency of P/S(XX)P Duplication and FRFE, Absence of LYP in P6&lt;i&gt;Gag&lt;/i&gt; of Indian Human Immunodeficiency Virus-1 Subtype C Isolates

Problem statement: The presence of a p6 domain at the C-terminus of the Gag polyprotein is a characteristic feature of HIV-1 and other primate lentiviruses. The p6Gag protein is considered as a major phosphoprotein in mature HIV-1 virions and is involved in the viral assembly and budding process. Sequence variation in the p6gag region in HIV-1 has been associated with changes in viral replication capacity and antiretroviral drug susceptibility. Approach: We examined sequence variation in the HIV-1 p6gag region using 38 isolates or infected Peripheral Blood Mononuclear Cells of HIV-1 subtype C and 20 additional Indian subtype C gag sequences from the Los Alamos database. Different patterns of insertions and deletions were observed in different motifs present in the p6 region. PTAP duplication was found in three strains. Results: We found PAP/TAP/LEPTAP/LPTVPTAP/ PAVPAAP like insertions at PTAP duplication site. KQE motif (84%) was observed as one of the most conserved motif in p6Gag while LYP motif was absolutely absent in Indian clade C sequences. Insertion of T/H/TT /PYRE/PYKE/EPKDRE was found instead of LYP motif. Conclusion: Further studies are needed to determine whether p6gag polymorphisms found in different motifs influence viral replication capacity, antiretroviral drug susceptibility, or other phenotypic properties of these strains.

Tear lipocalin is the predominant phosphoprotein in human tear fluid

Proteins are very important components in tears. Their phosphorylation is an important posttranslational modification affecting biological activity. Using proteomic techniques, this study was designed to analyze phosphoproteins found in open eye basal tears from normal human subjects. Proteins in tear samples were separated in 1-dimensional (1D) and 2-dimensional (2D) gels and phosphoproteins were selectively stained with Pro-Q diamond dye before visualization of all proteins using Sypro Ruby. Potential phosphoproteins in 2D gels were identified by liquid chromatography-mass spectrometry (LC-MS/MS) after trypsin digestion and phosphopeptide enrichment using titanium dioxide (TiO 2) columns. The tryptic digests of the tear samples were also analyzed to identify phosphoproteins directly by LC-MS/MS after phosphopeptide enrichment. The major phosphoprotein stained by Pro-Q diamond in the gels and identified by LC-MS/MS from the spots was tear lipocalin. Tear lipocalin was separated into 3 different isoforms and one phosphorylation site (serine at position 24) was identified in one of the isoforms. Prolactin-induced protein, nucleobindin-2 and lipophilin C were also stained with Pro-Q diamond although no phosphorylated peptides from these proteins could be found using LC-MS/MS. Direct analysis of the tear tryptic digests by LC-MS/MS identified a further 12 potential phosphoproteins with tear lipocalin predominant. Four phosphorylation sites (position 24 (serine), 32 (serine), 34 (threonine) and 36 (tyrosine)) were identified for tear lipocalin using this method. These results indicate that tear lipocalin is the predominant phosphoprotein in normal human basal tears. Nucleobindin-2, prolactin-induced protein and lipophilin C also appear to be phosphorylated in basal tear samples.

Phosphorylation of tropomyosin extends cooperative binding of myosin beyond a single regulatory unit

Tropomyosin (Tm) is one of the major phosphoproteins comprising the thin filament of muscle. However, the specific role of Tm phosphorylation in modulating the mechanics of actomyosin interaction has not been determined. Here we show that Tm phosphorylation is necessary for long-range cooperative activation of myosin binding. We used a novel optical trapping assay to measure the isometric stall force of an ensemble of myosin molecules moving actin filaments reconstituted with either natively phosphorylated or dephosphorylated Tm. The data show that the thin filament is cooperatively activated by myosin across regulatory units when Tm is phosphorylated. When Tm is dephosphorylated, this "long-range" cooperative activation is lost and the filament behaves identically to bare actin filaments. However, these effects are not due to dissociation of dephosphorylated Tm from the reconstituted thin filament. The data suggest that end-to-end interactions of adjacent Tm molecules are strengthened when Tm is phosphorylated, and that phosphorylation is thus essential for long range cooperative activation along the thin filament.

Role of the major ecto‐phosphoprotein in sperm flagellar motility using a cell electroporation method

Previous studies from our laboratory have identified MPS, a 100-kDa protein, as the major phosphoprotein substrate of caprine sperm ecto-cyclic AMP independent protein kinase. In this study the isolated (32)P-labelled MPS has been incorporated into mature caprine (Capra indicus) cauda-epididymal spermatozoa with the help of cell electroporation technique to investigate the effect of MPS on sperm flagellar motility. The optimum conditions for electroporation of sperm cells consisted of exposure of 0.2 ml of sperm cells (2 x 10(8)/ml) to external electric field of intensity 1.5 kV/cm and capacitation of 25 microF at 4 degrees C and post-pulse incubation at 37 degrees C for 1 hr. when nearly 50% of the cells lost motility. Scanning electron micrographs (SEM) demonstrate the formation of micro-pores and local osmotic swelling in the electroporated spermatozoa. MPS incorporation was maximal when its concentration was 30 microg/ml (300 pmol) in the medium and when the post-pulse incubation time was 60 min. At maximum (75%) MPS incorporation, total and forward motility increments were also maximum: 34% (P < 0.01) and 32% (P < 0.01), respectively. The subcellular fractionation data show that major portion of the introduced MPS was bound to the plasma-membrane of spermatozoa. The 32P-labelled electrophoresed intact spermatozoa lost radioactivity due to the action of the endogenous ecto-phosphoprotein phosphatase. Therefore MPS is primarily localised on the sperm external surface leaving its phosphate group(s) oriented in the extracellular medium. The data provided further evidence to strengthen the view that MPS is an ecto-phosphoprotein and that it plays an important role in the regulation of sperm flagellar motility.

The Phosphoproteome of aChlamydomonas reinhardtiiEyespot Fraction Includes Key Proteins of the Light Signaling Pathway

Flagellate green algae have developed a visual system, the eyespot apparatus, which allows the cell to phototax. In a recent proteomic approach, we identified 202 proteins from a fraction enriched in eyespot apparatuses of Chlamydomonas reinhardtii. Among these proteins, five protein kinases and two protein phosphatases were present, indicating that reversible protein phosphorylation occurs in the eyespot. About 20 major phosphoprotein bands were detected in immunoblots of eyespot proteins with an anti-phosphothreonine antibody. Toward the profiling of the targets of protein kinases in the eyespot fraction, we analyzed its phosphoproteome. The solubilized proteins of the eyespot fraction were treated with the endopeptidases LysC and trypsin prior to enrichment of phosphopeptides with immobilized metal-ion affinity chromatography. Phosphopeptides were analyzed by nano-liquid chromatography-electrospray ionization-mass spectrometry (MS) with MS/MS as well as neutral-loss-triggered MS/MS/MS spectra. We were able to identify 68 different phosphopeptides along with 52 precise in vivo phosphorylation sites corresponding to 32 known proteins of the eyespot fraction. Among the identified phosphoproteins are enzymes of carotenoid and fatty acid metabolism, putative signaling components, such as a SOUL heme-binding protein, a Ca(2+)-binding protein, and an unusual protein kinase, but also several proteins with unknown function. Notably, two unique photoreceptors, channelrhodopsin-1 and channelrhodopsin-2, contain three and one phosphorylation sites, respectively. Phosphorylation of both photoreceptors occurs in the cytoplasmatic loop next to their seven transmembrane regions in a similar distance to that observed in vertebrate rhodopsins, implying functional importance for regulation of these directly light-gated ion channels relevant for the photoresponses of C. reinhardtii.

The cytoplasmic phosphoproteome of the Gram‐negative bacterium Campylobacter jejuni: Evidence for modification by unidentified protein kinases

We have undertaken a comprehensive analysis of cytoplasmic protein phosphorylation in Campylobacter jejuni by mass spectrometric identification of phosphoproteins and localization of the sites of modification by phosphopeptide analyses. Cell extracts, enriched for phosphoproteins using Fe(III) IMAC or commercial phosphoprotein purification kits, were analyzed by 1-D and 2-D SDS-PAGE and subjected to mass fingerprinting by in-gel tryptic digestion and MALDI-TOF MS. Fifty-eight phosphopeptides were identified from 1-D gel bands by nano-LC-MS/MS and automated searching in a C. jejuni ORF database resulting in the unequivocal identification of 36 phosphoproteins of diverse function. In addition to elongation factors and chaperonins, which have been reported to be phosphorylated in other bacteria, the major phosphoproteins included bacterioferritin and superoxide dismutase. The sequences around the phosphorylated Ser and Thr residues are indicative of specific kinases being responsible for some of the modifications. However, many of the other identified proteins are enzymes that have phosphorylated substrates, including ATP, hence other modifications may arise from autophosphorylation. Comparative analyses of IMAC extracts from the Escherichia coli strain AD202 and Helicobacter pylori resulted in the identification of homologs of six of the C. jejuni phosphoproteins, though their overall phosphoproteome maps were distinctly different.

Direct Protein Kinase C-dependent Phosphorylation Regulates the Cell Surface Stability and Activity of the Potassium Chloride Cotransporter KCC2

The potassium chloride cotransporter KCC2 plays a major role in the maintenance of transmembrane chloride potential in mature neurons; thus KCC2 activity is critical for hyperpolarizing membrane currents generated upon the activation of gamma-aminobutyric acid type A and glycine (Gly) receptors that underlie fast synaptic inhibition in the adult central nervous system. However, to date an understanding of the cellular mechanism that neurons use to modulate the functional expression of KCC2 remains rudimentary. Using Escherichia coli expression coupled with in vitro kinase assays, we first established that protein kinase C (PKC) can directly phosphorylate serine 940 (Ser(940)) within the C-terminal cytoplasmic domain of KCC2. We further demonstrated that Ser(940) is the major site for PKC-dependent phosphorylation for full-length KCC2 molecules when expressed in HEK-293 cells. Phosphorylation of Ser(940) increased the cell surface stability of KCC2 in this system by decreasing its rate of internalization from the plasma membrane. Coincident phosphorylation of Ser(940) increased the rate of ion transport by KCC2. It was further evident that phosphorylation of endogenous KCC2 in cultured hippocampal neurons is regulated by PKC-dependent activity. Moreover, in keeping with our recombinant studies, enhancing PKC-dependent phosphorylation increased the targeting of KCC2 to the neuronal cell surface. Our studies thus suggest that PKC-dependent phosphorylation of KCC2 may play a central role in modulating both the functional expression of this critical transporter in the brain and the strength of synaptic inhibition.