Atg38 provides a physical linkage between the Vps15–Vps34 and Atg14–Vps30 subcomplexes to facilitate PI3-kinase complex I formation.

Our ability to model the dynamics of signal transduction networks will depend on accurate methods to quantify levels of protein phosphorylation on a global scale. Here we describe a motif-targeting quantitation method for phosphorylation stoichiometry typing. Proteome-wide phosphorylation stoichiometry can be obtained by a simple phosphoproteomic workflow integrating dephosphorylation and isotope tagging with enzymatic kinase reaction. Proof-of-concept experiments using CK2-, MAPK- and EGFR-targeting assays in lung cancer cells demonstrate the advantage of kinase-targeted complexity reduction, resulting in deeper phosphoproteome quantification. We measure the phosphorylation stoichiometry of >1,000 phosphorylation sites including 366 low-abundance tyrosine phosphorylation sites, with high reproducibility and using small sample sizes. Comparing drug-resistant and sensitive lung cancer cells, we reveal that post-translational phosphorylation changes are significantly more dramatic than those at the protein and messenger RNA levels, and suggest potential drug targets within the kinase–substrate network associated with acquired drug resistance.

No abstract

S-nitrosylation, a post-translational modification of cysteine residues induced by nitric oxide, mediates many physiological functions. Due to the labile nature of S-nitrosylation, detection by mass spectrometry (MS) is challenging. Here, we developed an S-alkylating labeling strategy using the irreversible biotinylation on S-nitrosocysteines for site-specific identification of the S-nitrosoproteome by LC-MS/MS. Using COS-7 cells without endogenous nitric oxide synthase, we demonstrated that the S-alkylating labeling strategy substantially improved the blocking efficiency of free cysteines, minimized the false-positive identification caused by disulfide interchange, and increased the digestion efficiency for improved peptide identification using MS analyses. Using this strategy, we identified total 586 unique S-nitrosylation sites corresponding to 384 proteins in S-nitroso-N-acetylpenicillamine (SNAP)/l-cysteine-treated mouse MS-1 endothelial cells, including 234 previously unreported S-nitrosylated proteins. When the topologies of 84 identified transmembrane proteins were further analyzed, their S-nitrosylation sites were found to mostly face the cytoplasmic side, implying that S-nitrosylation occurs in the cytoplasm. In addition to the previously known acid/basic motifs, the ten deduced consensus motifs suggested that combination of local hydrophobicity and acid/base motifs in the tertiary structure contribute to the specificity of S-nitrosylation. Moreover, the S-nitrosylated cysteines showed preference on beta-strand, having lower relative surface accessibility at the S-nitrosocysteines.

Death-associated protein kinase (DAPK) is a calmodulin-regulated serine/threonine kinase and elicits tumor suppression function through inhibiting cell adhesion/migration and promoting apoptosis. Despite these biological functions, the signaling mechanisms through which DAPK is regulated remain largely elusive. Here, we show that the leukocyte common antigen-related (LAR) tyrosine phosphatase dephosphorylates DAPK at pY491/492 to stimulate the catalytic, proapoptotic, and antiadhesion/antimigration activities of DAPK. Conversely, Src phosphorylates DAPK at Y491/492, which induces DAPK intra-/intermolecular interaction and inactivation. Upon EGF stimulation, a rapid Src activation leads to subsequent LAR downregulation, and these two events act in synergism to inactivate DAPK, thereby facilitating tumor cell migration and invasion toward EGF. Finally, DAPK Y491/492 hyperphosphorylation is found in human cancers in which Src activity is aberrantly elevated. These results identify LAR and Src as a DAPK regulator through their reciprocal modification of DAPK Y491/492 residues and establish a functional link of this DAPK-regulatory circuit to tumor progression.

Background: Bioinformatics tools for automatic processing of biomedical literature are invaluable for both the design and interpretation of large-scale experiments. Many information extraction (IE) systems that incorporate natural language processing (NLP) techniques have thus been developed for use in the biomedical field. A key IE task in this field is the extraction of biomedical relations, such as protein-protein and gene-disease interactions. However, most biomedical relation extraction systems usually ignore adverbial and prepositional phrases and words identifying location, manner, timing, and condition, which are essential for describing biomedical relations. Semantic role labeling (SRL) is a natural language processing technique that identifies the semantic roles of these words or phrases in sentences and expresses them as predicate-argument structures. We construct a biomedical SRL system called BIOSMILE that uses a maximum entropy (ME) machine-learning model to extract biomedical relations. BIOSMILE is trained on BioProp, our semi-automatic, annotated biomedical proposition bank. Currently, we are focusing on 30 biomedical verbs that are frequently used or considered important for describing molecular events.

No abstract

Dinitrosyl-iron complexes (DNICs) are stable carriers for nitric oxide (NO), an important biological signaling molecule and regulator. However, the insolubility of synthetic DNICs, such as Roussin's red ester (RRE), in water has impaired efforts to unravel their biological functions. Here, we report a water-soluble and structurally well-characterized RRE [Fe(mu-SC2H4COOH)(NO)2]2 (DNIC-1) and a {Fe(NO)2}(10) DNIC [(PPh2(Ph-3-SO3Na))2Fe(NO)2] (DNIC-2), their NO-induced protein regulation, and their cellular uptake mechanism using immortalized vascular endothelial cells as a model. Compared with the most common NO donor, S-nitroso-N-acetyl-penicillamine (SNAP), the in vitro NO release assay showed that both DNICs acted as much slower yet higher stoichiometric NO-release agents with low cytotoxicity (IC50 > 1 mM). Furthermore, L-cysteine facilitated NO release from SNAP and DNIC-1, but not DNIC-2, in a dose- and time-dependent manner. EPR spectroscopic analysis showed, for the first time, that intact DNIC-1 can either diffuse or be transported into cells independently and can transform to either paramagnetic protein bound DNIC in the presence of serum or [DNIC-(Cys)2] with excess L-cysteine under serum-free conditions. Both DNICs subsequently induced NO-dependent upregulation of cellular heat shock protein 70 and in vivo protein S-nitrosylation. We conclude that both novel water-soluble DNICs have potential to release physiologically relevant quantities of NO and can be a good model for deciphering how iron-sulfur-nitrosyl compounds permeate into the cell membrane and for elucidating their physiological significance.