A “lookup table” schema for synthetic biological patterning

A schema is proposed by which the three-dimensional structure and temporal development of a biological organism might be encoded and implemented via a genetic “lookup table”. In the schema, diffusive morphogen gradients and/or the global concentration of a quickly diffusing signal index sets of kinase genes having promoters with logarithmically diminished affinity for the signal. Specificity of indexing is enhanced via concomitant expression of phosphatases undoing phosphorylation by “neighboring” kinases of greater affinity. Combinations of thus-selected kinases in turn jointly activate, via multiple phosphorylation, a particular enzyme from a virtual, multi-dimensional array thereof, at locations and times specified within the “lookup table”. In principle, such a scheme could be employed to specify arbitrary gross anatomy, surface pigmentation, and/or developmental sequencing, extending the burgeoning toolset of the nascent field of synthetic morphology. A model of two-dimensional surface coloration using this scheme is specified, and LabVIEW software for its exploration is described and made available.     

The human granulocyte-macrophage colony-stimulating factor receptor is capable of initiating signal transduction in NIH3T3 cells.

The ability of the receptor for the hematopoietic cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) to function in non-hematopoietic cells is unknown. NIH3T3 fibroblasts were transfected with cDNAs encoding the alpha and beta subunit of the human GM-CSF receptor and a series of stable transformants were isolated that bound GM-CSF with either low (KD = 860 - > 1000 pM) or high affinity (KD = 20-80 pM). Low affinity receptors were not functional. However, the reconstituted high affinity receptors were found to be capable of activating a number of signal transduction pathways, including tyrosine kinase activity, phosphorylation of Raf-1, and the transient induction of c-fos and c-myc mRNAs. The activation of protein tyrosine phosphorylation by GM-CSF in NIH3T3 cells was rapid (< 1 min) and transient (peaking at 5-20 min) and resulted in the phosphorylation of proteins of estimated molecular weights of 42, 44, 52/53 and 58-60 kDa. Some of these proteins co-migrated with proteins from myeloid cells that were phosphorylated on tyrosine residues in response to GM-CSF. In particular, p42 and p44 were identified as mitogen-activated protein kinases (MAP kinases), and the phosphorylation on tyrosine residues of p42 and p44 MAP kinases occurred at the same time as the phosphorylation of Raf-1. However, despite evidence for activation of many mitogenic signal transduction molecules, GM-CSF did not induce significant proliferation of transfected NIH3T3 cells. These results suggest that murine fibroblasts contain signal transducing molecules that can effectively interact with the human GM-CSF receptor, and that are sufficient to activate at least some of the same signal transduction pathways this receptor activates in myeloid cells, including activation of one or more tyrosine kinase(s). However, the level of activation of signal transduction is either below a threshold of necessary activity or at least one mitogenic signal necessary for proliferation is missing.     

The "Gab" in signal transduction

Tyrosine phosphorylation plays an important role in controlling cellular growth, differentiation and function. Abnormal regulation of tyrosine phosphorylation can result in human diseases such as cancer. A major challenge of signal transduction research is to determine how the initial activation of protein-tyrosine kinases (PTKs) by extracellular stimuli triggers multiple downstream signaling cascades, which ultimately elicit diverse cellular responses. Recent studies reveal that members of the Gab/Dos subfamily of scaffolding adaptor proteins (hereafter, "Gab proteins") play a crucial role in transmitting key signals that control cell growth, differentiation and function from multiple receptors. Here, we review the structure, mechanism of action and function of these interesting molecules in normal biology and disease.     

Paper2sequences: retrieval of sequences listed in a publication

Our web-based tool simplifies the often laborious procedure of retrieving a set of biosequences in a publication or webpage. As a front-end to the Bioperl toolkit, it accepts as an input a list of identifiers. They are specified in an ASCII table (copy-pasted from the publication's PDF or HTML page) and give rise to queries in multiple databases for the protein/nucleic acid data specified. Currently, GenBank, PIR (Protein Information Resource) and Swiss-Prot are supported. For any sequence accession code listed, the database can be specified and, if retrieval fails, automatic lookup for the same code in other databases can be requested. Sequence length information (if specified) and heuristic rules are used to drive the lookup if multiple protein coding sequences (CDS) are part of a single accession. Warnings are issued in cases of ambiguities and inconsistencies. An advanced option enables the user to format the output in whatever format they wish.     

Signal transduction pathways implicated in neural recognition molecule L1 triggered neuroprotection and neuritogenesis

The signal transduction pathways involved in adhesion molecule L1-triggered neuritogenesis and neuroprotection were investigated using the extracellular domain of mouse or human L1 in fusion with the Fc portion of human immunoglobulin G or L1 purified from mouse brain by affinity chromatography. Substrate L1-triggered neuritogenesis and neuroprotection depended on distinct but also overlapping signal transduction pathways and on the expression of L1 at the neuronal cell surface. PI3 kinase inhibitors, Src family kinase inhibitors as well as mitogen-activated protein kinase kinase inhibitors reduced both L1-triggered neuritogenesis and neuroprotection. In contrast, fibroblast growth factor receptor inhibitors, a protein kinase A inhibitor, and an inhibitor of cAMP-mediated signal transduction pathways, blocked neuritogenesis, but did not affect L1-triggered neuroprotection. Proteolytic cleavage of L1 or its interaction partners is necessary for both L1-mediated neuritogensis and neuroprotection. Furthermore, L1-triggered neuroprotection was found to be associated with increased phosphorylation of extracellular signal-regulated kinases 1/2, Akt and Bad, and inhibition of caspases. These observations suggest possibilities of differentially targeting signal transduction pathways for L1-dependent neuritogenesis and neuroprotection.     

Rapid increase of the human IFN-gamma receptor phosphorylation in response to human IFN-gamma and phorbol myristate acetate. Involvement of different serine/threonine kinases

Various cell surface receptors are phosphorylated upon binding of their ligand, and this phosphorylation seems to be involved in the signal transduction or in the feedback regulation of this signal. The possibility of a phosphorylation of the human IFN-gamma receptor (hu-IFN-gamma-R) has been investigated with 32P-labeled whole Raji cells and receptor purification either by immunoprecipitation with an anti-hu-IFN-gamma-R polyclonal antiserum or by affinity chromatography. The hu-IFN-gamma-R was found to be phosphorylated at a basal level. Upon incubation of the cells with recombinant hu-IFN-gamma, a dose-dependent two-fold increase of this phosphorylation was observed. Phosphoamino acid analysis by TLC showed that the same amino acids, serine and threonine, are phosphorylated at a basal level and after incubation with hu-IFN-gamma. Protein kinase C and Ca2+/calmodulin-dependent kinase pathways have been reported in some cases to be involved in the signal transduction pathway of hu-IFN-gamma. Both pathways involved the activation of a serine/threonine kinase and therefore we have investigated the possibility of hu-IFN-gamma-R phosphorylation by these kinases. PMA, an activator of protein kinase C, induced a rapid increase of the receptor phosphorylation in Raji cells, whereas the Ca2+ ionophore A23187 did not. PMA-induced hu-IFN-gamma-R phosphorylation was not associated with any effect on expression or inactivation of the receptor. PMA alone did not mimic the hu-IFN-gamma effect in Raji cells as measured by induction of IP-10 gene expression, a high specific marker of hu-IFN-gamma response. But the protein kinase C inhibitors, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7) and staurosporine, reduced this IFN-gamma-induced expression. However, H7 and staurosporine treatment as well as protein kinase C depletion suppressed PMA-induced receptor phosphorylation, whereas constitutive and hu-IFN-gamma-induced phosphorylation remained unchanged. Our results suggest that the serine/threonine kinase involved in the hu-IFN-gamma-R phosphorylation induced by IFN-gamma is different from protein kinase C.     

Sequence of a rat cDNA encoding the ERK1-MAP kinase

Mitogen-activated protein (MAP) kinases are cytoplasmic and/or nuclear protein kinases which are activated by one or several signal transduction pathways from the cell surface into the nucleus. Their activity is regulated by phosphorylation on Tyr as well as on Ser/Thr residues. A cDNA encoding the rat ERK1 member of the MAP kinase family was isolated and sequenced. The longest cDNA consisted of 1875 nucleotides and coded for a polypeptide of 380 amino acids with a predicted M(r) of 42987.     

Identification of the protein kinases that activate the E3 ubiquitin ligase Pellino 1 in the innate immune system

The E3 ubiquitin ligase Pellino 1 can be interconverted between inactive and active forms by a reversible phosphorylation mechanism. In vitro, phosphorylation and activation can be catalysed by either the IRAKs [IL (interleukin)-1-receptor-associated kinases] IRAK1 and IRAK4, or the IKK {IκB [inhibitor of NF-κB (nuclear factor κB)] kinase}-related kinases [IKK? and TBK1 (TANK {TRAF [TNF (tumour-necrosis-factor)-receptor-associated factor]-associated NF-κB activator}-binding kinase 1)]. In the present study we establish that IRAK1 is the major protein kinase that mediates the IL-1-stimulated activation of Pellino 1 in MEFs (mouse embryonic fibroblasts) or HEK (human embryonic kidney)-293 cells, whereas the IKK-related kinases activate Pellino 1 in TNFα-stimulated MEFs. The IKK-related kinases are also the major protein kinases that activate Pellino 1 in response to TLR (Toll-like receptor) ligands that signal via the adaptors MyD88 (myeloid differentiation primary response gene 88) and/or TRIF [TIR (Toll/IL-1 receptor) domain-containing adaptor protein inducing interferon β]. The present studies demonstrate that, surprisingly, the ligands that signal via MyD88 do not always employ the same protein kinase to activate Pellino 1. Our results also establish that neither the catalytic activity of IRAK1 nor the activation of Pellino 1 is required for the initial transient activation of NF-κB and MAPKs (mitogen-activated protein kinases) that is triggered by IL-1 or TNFα in MEFs, or by TLR ligands in macrophages. The activation of Pellino 1 provides the first direct readout for IRAK1 catalytic activity in cells.     

A tyrosine kinase physically associates with the beta-subunit of the human IL-2 receptor

Cell surface expression of the high affinity IL-2R regulates, in part, the proliferative response occurring in Ag- or mitogen-activated T cells. The functional high affinity IL-2R is composed of at least two distinct ligand-binding components, IL-2R alpha (Tac, p55) and IL-2R beta (p70/75). The IL-2R beta polypeptide appears to be essential for growth signal transduction, whereas the IL-2R alpha protein participates in the regulation of receptor affinity. We have prepared and characterized two mAb, DU-1 and DU-2, that specifically react with IL-2R beta. In vitro kinase assays performed with DU-2 immunoprecipitates, but not anti-IL-2R alpha or control antibody immunoprecipitates, have revealed co-precipitation of a tyrosine kinase enzymatic activity that mediates phosphorylation of IL-2R beta. Because both IL-2R alpha and IL-2R beta lack tyrosine kinase enzymatic domains, these findings strongly suggest that noncovalent association of a tyrosine kinase with the high affinity IL-2R complex. Deletion mutants of the intracellular region of IL-2R beta, lacking either a previously described "critical domain" between amino acids 267 and 322 or the carboxyl-terminal 198 residues (IL-2R beta 88), lacked the ability to co-precipitate this tyrosine kinase activity, as measured by phosphorylation of IL-2R beta in vitro. Both of these mutants also failed to transduce growth-promoting signals in response to IL-2 in vivo. Analysis of the IL-2R beta 88 mutant receptor suggested that a second protein kinase mediating phosphorylation on serine and threonine residues physically interacts with the carboxyl terminus of IL-2R beta. This kinase may be necessary but, alone, appears to be insufficient to support a full IL-2-induced proliferative response. These studies highlight the physical association of protein kinases with the cytoplasmic domain of IL-2R beta and their likely role in IL-2-induced growth signaling mediated through the multimeric high affinity IL-2R complex.     

Changes in diacylglycerol labeling, cell shape, and protein phosphorylation distinguish "triggering" from "activation" of human neutrophils

Upon activation neutrophils release reactive oxygen intermediates such as superoxide anion (O2-) which are potent mediators of inflammation. Various agents elicit different responses; N-formylmethionylleucylphenylalanine (fMLP) (0.1 microM) provokes brisk generation of superoxide anion; leukotriene B4 (LTB4, 0.1 microM) is a poor stimulus. In contrast, phorbol myristate acetate (PMA, 1.6 microM) acting directly via protein kinase C is a potent stimulus for O2-. We compared the kinetics of appearance of various "second messengers" with the capacity of these ligands to elicit O2- generation. Kinetic analysis showed a two-phase response to membrane ligands; both an "early" (less than or equal to 15 s) and a "late" (greater than 15 s) increase in [3H]- and [14C]diacylglycerol (DG) was noted in response to fMLP. In contrast, LTB4 elicited only a rapid early increase in DG. The rise in DG evoked by PMA was late. Cytochalasin B increased the late phase of DG labeling elicited by all agonists. Moreover, comparison of increases in [3H]DG versus those of [14C]DG at early and late time points suggested that DG was not formed exclusively from the hydrolysis of polyphosphoinositides. Early increments of DG were also accompanied by addition of plasma membrane (ultrastructural morphometry); the ratio of surface perimeter to area increased rapidly (10 s) and persisted (60 s) in response to fMLP. Increments were more gradual in response to PMA. Kinetic analysis of protein phosphorylation was compared to the early and late increments of DG labeling. A 47,000 Mr protein was phosphorylated with kinetics consistent with the production of O2- and DG in response to fMLP (early and late) and PMA (late). In contrast, LTB4 provoked only early phosphorylation of this protein. The temporal pattern of the formation of diacylglycerol and the phosphorylation of proteins describe a dual signal. The data suggest that neutrophils require not only "triggering" (the rapid generation of a signal) but also "activation" (the maintenance of a signal) to sustain responses.     

Large-scale Proteomics Analysis of the Human Kinome

Members of the human protein kinase superfamily are the major regulatory enzymes involved in the activity control of eukaryotic signal transduction pathways. As protein kinases reside at the nodes of phosphorylation-based signal transmission, comprehensive analysis of their cellular expression and site-specific phosphorylation can provide important insights into the architecture and functionality of signaling networks. However, in global proteome studies, low cellular abundance of protein kinases often results in rather minor peptide species that are occluded by a vast excess of peptides from other cellular proteins. These analytical limitations create a rationale for kinome-wide enrichment of protein kinases prior to mass spectrometry analysis. Here, we employed stable isotope labeling by amino acids in cell culture (SILAC) to compare the binding characteristics of three kinase-selective affinity resins by quantitative mass spectrometry. The evaluated pre-fractionation tools possessed pyrido[2,3-d]pyrimidine-based kinase inhibitors as immobilized capture ligands and retained considerable subsets of the human kinome. Based on these results, an affinity resin displaying the broadly selective kinase ligand VI16832 was employed to quantify the relative expression of more than 170 protein kinases across three different, SILAC-encoded cancer cell lines. These experiments demonstrated the feasibility of comparative kinome profiling in a compact experimental format. Interestingly, we found high levels of cytoplasmic and low levels of receptor tyrosine kinases in MV4–11 leukemia cells compared with the adherent cancer lines HCT116 and MDA-MB-435S. The VI16832 resin was further exploited to pre-fractionate kinases for targeted phosphoproteomics analysis, which revealed about 1200 distinct phosphorylation sites on more than 200 protein kinases. This hitherto largest survey of site-specific phosphorylation across the kinome significantly expands the basis for functional follow-up studies on protein kinase regulation. In conclusion, the straightforward experimental procedures described here enable different implementations of kinase-selective proteomics with considerable potential for future signal transduction and kinase drug target analysis.Reversible protein phosphorylation represents the most common type of post-translational modification (PTM)¹ in eukaryotic organisms. A plethora of studies on a large variety of proteins have established that site-specific phosphorylation events fulfill key functions in the activity control of signaling cascades and networks (1). Cellular protein phosphorylation is controlled by more than 500 members of the protein kinase superfamily, which comprises one of the largest enzyme families encoded by the human genome (2). Protein kinases represent the key elements in phosphorylation-based signal transmission. Aberrant protein kinase expression and/or activity, often because of gene amplification or mutational changes, is involved in pathological processes leading to malignant transformation and tumor development (3). Therefore, protein kinases have emerged as a major class of drug targets for therapeutic intervention (4–6). Given the diversity of molecular mechanisms related to de-regulated kinase function in human cancers, proteomic approaches could significantly enhance our understanding of disease-relevant kinase function and also help to optimize and adjust therapeutic strategies. In addition to assessing protein expression, the analysis of site-specific phosphorylations on protein kinases is of particular relevance, as these PTMs can be indicative of their cellular catalytic activities (7, 8). Protein kinases can not only modulate each other''s functions and activities through site-specific phosphorylation events, but often also undergo site-specific autophosphorylation once they get activated (9). Thus, the comprehensive assessment of kinase-derived phosphopeptides can provide important insights into the regulation of these key players in phosphorylation-controlled signaling.Regulatory enzymes such as protein kinases are often expressed at low cellular levels. This can impede their detection by LC-MS in highly complex peptide mixtures derived from total cell or tissue extracts. These analytical challenges are further aggravated in phosphoproteomic experiments due to the fact that many phosphopeptide species result from sub-stoichiometric phosphorylation events (10). Consequently, phosphopeptide isolation methods have proven to be essential. Among others, techniques such as immobilized metal affinity chromatography or enrichment by means of titanium dioxide (TiO₂)-coated beads have found widespread use in MS-based phosphoproteomics (11–13). In addition, to reduce initial sample complexity, either protein fractionation by gel electrophoresis or peptide separation by strong cation exchange chromatography is typically included in contemporary phosphoproteomics workflows (14–16). These separation techniques in combination with LC-MS on state-of-the-art mass spectrometers enabled the identification of thousands of phosphorylation sites from total cellular extracts (15, 17, 18). Despite these impressive advances, such large-scale efforts require considerable instrument time, and the current methodology is still not comprehensive across the full dynamic range of the entire phosphoproteome. This creates the rationale for sub-proteome analyses to achieve high coverage and analytical sensitivity, which is particularly relevant for members of the protein kinase enzyme family.To date, the only pre-fractionation techniques permitting the enrichment of more than a few protein kinases are affinity capture methods relying on immobilized and kinase-selective small molecule inhibitors (19–21). We and others have demonstrated that combinations of such kinase inhibitor resins efficiently pre-fractionate kinases for subsequent phosphorylation analysis (7, 22, 23). Ideally, capture molecules for kinase proteomics have two properties. First, they should exhibit high non-selectivity within the kinase superfamily. Second, they should efficiently discriminate between protein kinases and other classes of cellular proteins under the biochemical conditions of the pre-fractionation procedure.In our efforts to characterize affinity reagents fulfilling these criteria, we quantitatively compared a selection of immobilized pyrido[2,3-d]pyrimidine-based inhibitors with respect to their proteome-wide kinase binding properties. Based on this assessment, an affinity matrix displaying the small molecule VI16832 was used as an enrichment tool for the comparative expression analysis of protein kinases in different cancer cell lines. The highly efficient VI16832 affinity resin further enabled a large-scale phosphoproteomics survey resulting in the identification and confident assignment of about 1200 phosphorylation sites on more than 200 distinct protein kinases.     

Activation of insulin-epidermal growth factor (EGF) receptor chimerae regulates EGF receptor binding affinity

Cell surface tyrosine kinase receptors are subject to a rapid activation by their ligand, which is followed by secondary regulatory processes. The IHE2 cell line is a unique model system to study the regulation of EGF binding to EGF receptors after activation of the EGF receptor kinase. IHE2 cells express both a chimeric insulin-EGF receptor kinase (IER) and a kinase-deficient EGF receptor (HER K721A). We have previously reported that IER is an insulin-responsive EGF receptor tyrosine kinase that activates one or several serine/threonine kinases, which in turn phosphorylate(s) the unoccupied HER K721A. In this article we show that insulin through IER activation induces a decrease in 125I-EGF binding to IHE2 cells. Scatchard analysis indicates that, as for TPA, the effect of insulin can be accounted for by a loss of the high affinity binding of EGF to HER K721A. Since this receptor transmodulation persists in protein kinase C downregulated IHE2 cells, it is likely to be due to a mechanism independent of protein kinase C activation. Using an in vitro system of 125I-EGF binding to transmodulated IHE2 membranes, we illustrate that the inhibition of EGF binding induced by IER activation is related to the phosphorylation state of HER K721A. Further, studies with phosphatase 2A, or at a temperature (4 degrees C) where only IER is functional, strongly suggest that the loss of high affinity EGF binding is related to the serine/threonine phosphorylation of HER K721A after IER activation. Our results provide evidence for a "homologous desensitization" of EGF receptor binding after activation of the EGF receptor kinase of the IER receptor.     

Conserved spatial patterns across the protein kinase family

Protein kinases are a large family of enzymes heavily involved in signal transduction, regulation of metabolism, and control of cell growth and differentiation. These functions require precise recognition of widely diverse signals and substrates, and very detailed control of protein kinase activity. Large molecules interact primarily through recognition of surface features. Comparison of surfaces is complicated by both sequence diversity and conformational variability, including multiple possible rotameric states of side chains. We used a recently developed method of protein surface comparison to compare different serine/threonine and tyrosine kinases. As we have shown, two hydrophobic cores inside a protein kinase molecule are connected by a unique formation, called the "spine". It exists only in the active conformation of protein kinases and is dynamically disassembled during the inactivation process. Detection of such structures by any other method was not possible as the residues which comprise the spine do not form any sequence or 3D motifs in a traditional sense.     

G protein-coupled receptor kinase 6A phosphorylates the Na(+)/H(+) exchanger regulatory factor via a PDZ domain-mediated interaction.

The Na(+)/H(+) exchanger regulatory factor (NHERF) is constitutively phosphorylated in cells, but the site(s) of this phosphorylation and the kinase(s) responsible for it have not been identified. We show here that the primary site of constitutive NHERF phosphorylation in human embryonic kidney 293 (HEK-293) cells is Ser(289), and that the stoichiometry of phosphorylation is near 1 mol/mol. NHERF contains two PDZ domains that recognize the sequence S/T-X-L at the carboxyl terminus of target proteins, and thus we examined the possibility that kinases containing this motif might associate with and phosphorylate NHERF. Overlay experiments and co-immunoprecipitation studies revealed that NHERF binds with high affinity to a splice variant of the G protein-coupled receptor kinase 6, GRK6A, which terminates in the motif T-R-L. NHERF does not associate with GRK6B or GRK6C, alternatively spliced variants that differ from GRK6A at their extreme carboxyl termini. GRK6A phosphorylates NHERF efficiently on Ser(289) in vitro, whereas GRK6B, GRK6C, and GRK2 do not. Furthermore, the endogenous "NHERF kinase" activity in HEK-293 cell lysates is sensitive to treatments that alter the activity of GRK6A. These data suggest that GRK6A phosphorylates NHERF via a PDZ domain-mediated interaction and that GRK6A is the kinase in HEK-293 cells responsible for the constitutive phosphorylation of NHERF.     

Protein phosphatase-1 and insulin action

Protein Phosphatase-1 (PP-1) appears to be the key component of the insulin signalling pathway which is responsible for bridging the initial insulin-simulated phosphorylation cascade with the ultimate dephosphorylation of insulin sensitive substrates. Dephosphorylations catalyzed by PP-1 activate glycogen synthase (GS) and simultaneously inactivate phosphorylase a and phosphorylase kinase promoting glycogen synthesis. Our in vivo studies using L6 rat skeletal muscle cells and freshly isolated adipocytes indicate that insulin stimulates PP-1 by increasing the phosphorylation status of its regulatory subunit (PP-1_G). PP-1 activation is accompanied by an inactivation of Protein Phosphatase-2A (PP-2A) activity. To gain insight into the upstream kinases that mediate insulin-stimulated PP-1_G phosphorylation, we employed inhibitors of the ras/MAPK, PI3-kinase, and PKC signalling pathways. These inhibitor studies suggest that PP-1_G phosphorylation is mediated via a complex, cell type specific mechanism involving PI3-kinase/PKC/PKB and/or the ras/MAP kinase/Rsk kinase cascade. cAMP agonists such as SpcAMP (via PKA) and TNF- (recently identified as endogenous inhibitor of insulin action via ceramide) block insulin-stimulated PP-1_G phosphorylation with a parallel decrease of PP-1 activity, presumably due to the dissociation of the PP-1 catalytic subunit from the regulatory G-subunit. It appears that any agent or condition which interferes with the insulin-induced phosphorylation and activation of PP-1, will decrease the magnitude of insulin's effect on downstream metabolic processes. Therefore, regulation of the PP-1_G subunit by site-specific phosphorylation plays an important role in insulin signal transduction in target cells. Mechanistic and functional studies with cell lines expressing PP-1_G subunit site-specific mutations will help clarify the exact role and regulation of PP-1_G site-specific phosphorylations on PP-1 catalytic function.     

Stimulation of A(2A) adenosine receptor phosphorylation by protein kinase C activation: evidence for regulation by multiple protein kinase C isoforms.

Activation of the A(2A) adenosine receptor (A(2A)AR) contributes to the neuromodulatory and neuroprotective effects of adenosine in the central nervous system. Here we demonstrate that, in rat C6 glioma cells stably expressing an epitope-tagged canine A(2A)AR, receptor phosphorylation on serine and threonine residues can be increased by pretreatment with either the synthetic protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) or endothelin 1, which increases PKC activity via binding to endogenous endothelin(A) receptors. Under conditions in which PMA was maximally effective, activation of other second messenger-regulated kinases was without effect. While basal and PMA-stimulated phosphorylation were unaffected by the A(2A)AR-selective antagonist ZM241385, they were both blocked by GF109203X (a selective inhibitor of conventional and novel PKC isoforms) and rottlerin (a PKCdelta-selective inhibitor) but not Go6976 (selective for conventional PKC isoforms). However, coexpression of the A(2A)AR with each of the alpha, betaI, and betaII isoforms of PKC increased basal and PMA-stimulated phosphorylation. Mutation of the three consensus PKC phosphorylation sites within the receptor (Thr298, Ser320, and Ser335) to Ala failed to inhibit either basal or PMA-stimulated phosphorylation. In addition, phosphorylation of the receptor was not associated with detectable changes in either its signaling capacity or cell surface expression. These observations suggest that multiple PKC isoforms can stimulate A(2A)AR phosphorylation via activation of one or more downstream kinases which then phosphorylate the receptor directly. In addition, it is likely that phosphorylation controls interactions with regulatory proteins distinct from those involved in the classical cAMP signaling pathway utilized by this receptor.     

Large-scale phosphotyrosine proteomic profiling of rat renal collecting duct epithelium reveals predominance of proteins involved in cell polarity determination

Although extensive phosphoproteomic information is available for renal epithelial cells, previous emphasis has been on phosphorylation of serines and threonines with little focus on tyrosine phosphorylation. Here we have carried out large-scale identification of phosphotyrosine sites in pervanadate-treated native inner medullary collecting ducts of rat, with a view towards identification of physiological processes in epithelial cells that are potentially regulated by tyrosine phosphorylation. The method combined antibody-based affinity purification of tyrosine phosphorylated peptides coupled with immobilized metal ion chromatography to enrich tyrosine phosphopeptides, which were identified by LC-MS/MS. A total of 418 unique tyrosine phosphorylation sites in 273 proteins were identified. A large fraction of these sites have not been previously reported on standard phosphoproteomic databases. All results are accessible via an online database: http://helixweb.nih.gov/ESBL/Database/iPY/. Analysis of surrounding sequences revealed four overrepresented motifs: [D/E]xxY*, Y*xxP, DY*, and Y*E, where the asterisk symbol indicates the site of phosphorylation. These motifs plus contextual information, integrated using the NetworKIN tool, suggest that the protein tyrosine kinases involved include members of the insulin- and ephrin-receptor kinase families. Analysis of the gene ontology (GO) terms and KEGG pathways whose protein elements are overrepresented in our data set point to structures involved in epithelial cell-cell and cell-matrix interactions ("adherens junction," "tight junction," and "focal adhesion") and to components of the actin cytoskeleton as major sites of tyrosine phosphorylation in these cells. In general, these findings mesh well with evidence that tyrosine phosphorylation plays a key role in epithelial polarity determination.     

Ethanol inhibits L1 cell adhesion molecule tyrosine phosphorylation and dephosphorylation and activation of pp60src

Fetal alcohol syndrome is a leading cause of mental retardation. The neuropathology found in patients with fetal alcohol syndrome overlaps with those with mutations in the gene for cell adhesion molecule (L1). We have previously shown that L1-mediated neurite outgrowth and L1 activation of extracellular receptor kinases 1/2 are inhibited at low concentrations of ethanol. One possible mechanism for this effect is through disruption of a tyrosine-based sorting signal, Y(1176)RSLE, on the cytoplasmic domain of L1. Our goal was to determine if ethanol inhibited the sorting signal or its phosphorylation state. Using cerebellar granule neurons and dorsal root ganglion neurons, we found that ethanol had no effect on L1 distribution to the growth cone or its ability to be expressed on the cell surface as determined by confocal microscopy. In cerebellar granule neurons, clustering of L1 resulted in increased dephosphorylation of Y(1176), increased L1 tyrosine phosphorylation, and an increase in the activation of pp60^src as measured by immunoblot. All changes were inhibited by 25 mM ethanol. Using PP2 to inhibit pp60^src activation resulted in inhibition of increases in L1 tyrosine and extracellular receptor kinases 1/2 phosphorylation, and Y(1176) dephosphorylation. We conclude that ethanol disrupts L1 trafficking/signaling following its expression on the surface of the growth cone, and prior to its activation of pp60^src.     

Temporal analysis of sucrose-induced phosphorylation changes in plasma membrane proteins of Arabidopsis

Sucrose is the main product of photosynthesis and the most common transport form of carbon in plants. In addition, sucrose is a compound that serves as a signal affecting metabolic flux and development. Here we provide first results of externally induced phosphorylation changes of plasma membrane proteins in Arabidopsis. In an unbiased approach, seedlings were grown in liquid medium with sucrose and then depleted of carbon before sucrose was resupplied. Plasma membranes were purified, and phosphopeptides were enriched and subsequently analyzed quantitatively by mass spectrometry. In total, 67 phosphopeptides were identified, most of which were quantified over five time points of sucrose resupply. Among the identified phosphorylation sites, the well described phosphorylation site at the C terminus of plasma membrane H(+)-ATPases showed a relative increase in phosphorylation level in response to sucrose. This corresponded to a significant increase of proton pumping activity of plasma membrane vesicles from sucrose-supplied seedlings. A new phosphorylation site was identified in the plasma membrane H(+)-ATPase AHA1 and/or AHA2. This phosphorylation site was shown to be crucial for ATPase activity and overrode regulation via the well known C-terminal phosphorylation site. Novel phosphorylation sites were identified for both receptor kinases and cytosolic kinases that showed rapid increases in relative intensities after short times of sucrose treatment. Seven response classes were identified including non-responsive, rapid increase (within 3 min), slow increase, and rapid decrease. Relative quantification of phosphorylation changes by phosphoproteomics provides a means for identification of fast responses to external stimuli in plants as a basis for further functional characterization.