Two-dimensional (2-D) gel electrophoresis has been used to map proteins from various cell types in an effort to eventually link such maps to the sequencing of the entire human genome. While this analysis indicates the cellular disposition and expression of proteins, another application of 2-D gels, the analysis of phosphoproteins, can provide much information as to the assembly and "wiring" of the signal transduction circuits within cells which appear to be enervated by phosphate exchange. The preparation and separation of 32P-labeled proteins is described, as well as various analytical methods, including: the variety of gel systems available for specialist types of analyses, comparing 33P- and 32P-labeling of proteins, imaging techniques, phosphoamino analysis, phosphopeptide separation, identifying the amino acid groups that are phosphorylated, and the identification of phosphoproteins on 2-D gels by immunoprecipitation, corunning of purified proteins, comparative mapping and microsequencing, and by Western blotting. Examples (in brackets) are given of applications in which 2-D phosphogels can be applied, which offer advantages over other techniques. These include: (i) identifying in vivo substrates for kinases (protein kinase C activated by phorbol myristate acetate), (ii) investigating cytokine signaling pathways (tumor necrosis factor and interleukin-1), (iii) investigating the effects of drugs on signaling pathways (okadaic acid, menadione and cyclooxygenase inhibitors), (iv) characterization of specific phosphoproteins (heat-shock protein Hsp27 and stathmin), (v) comparing normal and transformed cells (MRC-5 human lung fibroblasts and their SV-40-transformed counterparts, MRC-5 SV1 cells), (vi) purifying phosphoproteins, (vii) investigating the relationship of protein phosphorylation to stages in the cell cycle (stathmin), (viii) investigating protein/protein interactions, (ix) mapping in vitro kinase substrates (protein kinase C, protein kinase A, and mitogen activated protein kinase activated protein kinase 2), and (x) locating and identifying cellular phosphatases (Hsp27 phosphatase). It is possible that the mapping of phosphoproteins can be linked to other 2-D gel databases and that information derived from these can be used in the future to better understand the signaling mechanisms of normal and cancerous cells.
With the completion of the human genome project, analysis of enriched phosphotyrosyl proteins from epidermal growth factor (EGF)-induced phosphotyrosine proteome permits the identification of novel downstream substrates of the EGF receptor (EGFR). Using cICAT-based LC-MS/MS method, we identified and relatively quantified the tyrosine phosphorylation levels of 21 proteins between control and EGF-treated A431 human cervical cancer cells. Of these, Endofin, DCBLD2, and KIAA0582 were validated to be novel tyrosine-phosphorylation targets of EGF signaling and Iressa, a highly selective inhibitor of EGFR. In addition, EGFR activity was shown to be necessary for EGF-induced localization of Endofin, an FYVE domain-containing protein regulated by phosphoinositol lipid and engaged in endosome-mediated receptor modulation. Although several groups have conducted phosphoproteomics of EGF signaling in recent years, our study is the first to identify and validate Endofin, DCBLD2, and KIAA0582 as part of a complex EGF phosphotyrosine signaling network. These novel data will provide new insights into the complex EGF signaling and may have implications on target-directed cancer therapeutics.
Although the activation domains within early growth response gene protein 1 (Egr-1) have been mapped, little is known of the kinases which phosphorylate Egr-1 and how phosphorylation correlates with the transcriptional activity of Egr-1. In this study we report that casein kinase II (CKII) co-immunoprecipitates with Egr-1 from NIH 3T3 cell lysates. The association of Egr-1 and CKII requires the C terminus of Egr-1 and CKII phosphorylates Egr-1 in vitro. The in vitro phosphorylation of Egr-1 by CKII and that induced by serum in vivo was compared by examining the CNBr-digested fragments of the phosphorylated Egr-1. CKII strongly phosphorylates fragments 7 and 10 which cover part of the activation/nuclear localization and DNA binding domains of Egr-1. CKII also phosphorylates, albeit weakly, fragments 5 and 8 which cover part of activation domain and the entire repression domain of Egr-1, respectively. Strong phosphorylation on fragment 10 as well as fragment 5 was also observed in Egr-1 immunoprecipitated from serum-induced, 32 P-labeled cells. CKII phosphorylation of Egr-1 resulted in a decrease of its DNA binding as well as its transcriptional activities. egr-11 (1), also known as krox-24 (2), zif268 (3), Tis8 (4), and NGFI-A (5), is an immediate-early gene. It encodes a transcription factor with three zinc fingers and recognizes a GC-rich sequence, 5Ј-CGCCCCCGC-3Ј (6) which has been identified in the promoter regions of a number of genes. Egr-1 regulates these genes by binding to this consensus sequence. Besides inducing genes involved in cell proliferation, Egr-1 is also implicated in a number of differentiation processes in cardiac (7), neural (1), osteoblast (8), and myeloid cells (9).It is likely that the different effects mediated by Egr-1 in various cells result from its phosphorylation and dephosphorylation (10). Reports that cells treated with kinase and phosphatase inhibitors (11, 12) show differences in their Egr-1 DNA binding ability support this hypothesis. However, little is known about which kinase(s) phosphorylates Egr-1 and if its functional domain(s) is phosphorylated. In addition, the identification of a repressor protein that associates with Egr-1 implies that protein-protein interactions can also regulate its transcriptional activity (13, 14).The protein kinase CKII is an inducible kinase found in both the cytoplasmic and nuclear compartments, although recently CKII was reported to be a predominantly nuclear protein (15). It is a tetrameric Ser/Thr-specific protein kinase complex containing two catalytic (␣, 44 kDa, ␣Ј, 42 kDa) subunits and two regulatory (, 25 kDa) subunits (15). CKII is known to associate with a number of nuclear proteins such as DNA topoisomerase II (16) and FKBP25, the 25-kDa FK506-binding protein (17). CKII also phosphorylates a number of transcription factors and nuclear proteins such as the serum response factor (18), c-Jun (19), and p53 (20). Little is known of the physiological role of CKII or how it regulates the activity of transcription factors in vivo.In this r...
High-throughput screening identified an extract from Streptomyces sp. IM 2096 with inhibitory activity toward several protein tyrosine phosphatases (PTPs). Four 1,2,4-triazine compounds 2096A-D (1-4) were isolated from this extract and their structures elucidated by interpretation of spectroscopic data and confirmed by degradation and synthesis. The novel glycocyamidine derivatives 1 and 2 are diastereomers and may interconvert. Both are inactive in the PTP inhibition assay. Compounds 1 and 2 are unstable and partially decompose to 3 and glycocyamidine (5) at room temperature. Compound 3, known as MSD-92 or 2-methyl-fervenulone, is a broad-specificity PTP inhibitor with comparable potency to vanadate. The imidazo[4, 5-e]-1,2,4-triazine (4), inactive in the PTP-inhibition assay, may be a degradation product of 3.
Folate-mediated one-carbon (1C) metabolism is a major target of many therapies in human diseases. Studies have focused on the metabolism of serine 3-carbon as it serves as a major source for 1C units. The serine 3-carbon enters the mitochondria transferred by folate cofactors and eventually converted to formate and serves as a major building block for cytosolic 1C metabolism. Abnormal glycine metabolism has been reported in many human pathological conditions. The mitochondrial glycine cleavage system (GCS) catalyzes glycine degradation to CO2 and ammonium, while tetrahydrofolate (THF) is converted into 5,10-methylene-THF. GCS accounts for a substantial proportion of whole-body glycine flux in humans, yet the particular metabolic route of glycine 2-carbon recycled from GCS during mitochondria glycine decarboxylation in hepatic or bone marrow 1C metabolism is not fully investigated, due to the limited accessibility of human tissues. Labeled glycine at 2-carbon was given to humans and primary cells in previous studies for investigating its incorporations into purines, its interconversion with serine, or the CO2 production in the mitochondria. Less is known on the metabolic fate of the glycine 2-carbon recycled from the GCS; hence, a model system tracing its metabolic fate would help in this regard. We took the direct approach of isotopic labeling to further explore the in vitro and in vivo metabolic fate of the 2-carbon from [2-13C]glycine and [2-13C]serine. As the 2-carbon of glycine and serine is decarboxylated and catabolized via the GCS, the original 13C-labeled 2-carbon is transferred to THF and yield methyleneTHF in the mitochondria. In human hepatoma cell-lines, 2-carbon from glycine was found to be incorporated into deoxythymidine (dTMP, dT + 1), M + 3 species of purines (deoxyadenine, dA and deoxyguanine, dG), and methionine (Met + 1). In healthy mice, incorporation of GCS-derived formate from glycine 2-carbon was found in serine (Ser + 2 via cytosolic serine hydroxy methyl transferase), methionine, dTMP, and methylcytosine (mC + 1) in bone marrow DNA. In these experiments, labeled glycine 2-carbon directly incorporates into Ser + 1, A + 2, and G + 2 (at C2 and C8 of purine) in the cytosol. It is noteworthy that since the serine 3-carbon is unlabeled in these experiments, the isotopic enrichments in dT + 1, Ser + 2, dA + 3, dG + 3, and Met + 1 solely come from the 2-carbon of glycine/serine recycled from GCS, re-enters the cytosolic 1C metabolism as formate, and then being used for cytosolic syntheses of serine, dTMP, purine (M + 3) and methionine. Taken together, we established model systems and successfully traced the metabolic fate of mitochondrial GCS-derived formate from glycine 2-carbon in vitro and in vivo. Nutritional supply significantly alters formate generation from GCS. More GCS-derived formate was used in hepatic serine and methionine syntheses, whereas more GCS-derived formate was used in dTMP synthesis in the bone marrow, indicating that the utilization and partitioning of GCS-derived 1C unit are tissue-specific. These approaches enable better understanding concerning the utilization of 1C moiety generated from mitochondrial GCS that can help to further elucidate the role of GCS in human disease development and progression in future applications. More studies on GCS using these approaches are underway.
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