Reversible protein phosphorylation plays a pivotal role in the regulation of cellular signaling pathways. Current approaches in phosphoproteomics focus on analysis of the global phosphoproteome in a single cellular state or of receptor stimulation time course experiments, often with a restricted number of time points. Although these studies have provided some insights into newly discovered phosphorylation sites that may be involved in pathways, they alone do not provide enough information to make precise predictions of the placement of individual phosphorylation events within a signaling pathway. Protein disruption and site-directed mutagenesis are essential to clearly define the precise biological roles of the hundreds of newly discovered phosphorylation sites uncovered in modern proteomics experiments. We have combined genetic analysis with quantitative proteomic methods and recently developed visual analysis tools to dissect the tyrosine phosphoproteome of isogenic Zap-70 tyrosine kinase null and reconstituted Jurkat T cells. In our approach, label-free quantitation using normalization to copurified phosphopeptide standards is applied to assemble high density temporal data within a single cell type, either Zap-70 null or reconstituted cells, providing a list of candidate phosphorylation sites that change in abundance after T cell stimulation. Stable isotopic labeling of amino acids in cell culture (SILAC) ratios are then used to compare Zap-70 null and reconstituted cells across a time course of receptor stimulation, providing direct information about the placement of newly observed phosphorylation sites relative to Zap-70. These methods are adaptable to any cell culture signaling system in which isogenic wild type and mutant cells have been or can be derived using any available phosphopeptide enrichment strategy. Molecular & Cellular Proteomics 8:2418 -2431, 2009.The reversible phosphorylation of serine, threonine, and tyrosine residues directly controls many cellular processes, leading to the activation of a coordinated network of additional phosphorylation events across multiple proteins over time. Clearly, there are benefits to individually identifying and characterizing specific components of a particular pathway, such as a phosphorylation site on a given protein, the kinase responsible for the modification, or the proteins interacting subsequently. However, a thorough understanding of these signaling pathways at the molecular level ultimately requires a global, simultaneous evaluation of these phosphorylation events as they occur over time.Currently, the most common method for assessing widescale changes in the proteome is two-dimensional gel electrophoresis (1), but this methodology is relatively low throughput and not optimal for the analysis of low abundance and hydrophobic signaling proteins (2). Recent publications describe alternate approaches for assessing changes in phosphorylation patterns based primarily on LC/MS methodologies (3-8). A variety of promising purification approaches have been dev...

Mast cells play a central role in type I hypersensitivity reactions and allergic disorders such as anaphylaxis and asthma. Activation of mast cells, through a cascade of phosphorylation events, leads to the release of mediators of the early phase allergic response. Understanding the molecular architecture underlying mast cell signaling may provide possibilities for therapeutic intervention in asthma and other allergic diseases. Although many details of mast cell signaling have been described previously, a systematic, quantitative analysis of the global tyrosine phosphorylation events that are triggered by activation of the mast cell receptor is lacking. In many cases, the involvement of particular proteins in mast cell signaling has been established generally, but the precise molecular mechanism of the interaction between known signaling proteins often mediated through phosphorylation is still obscure. Using recently advanced methodologies in mass spectrometry, including automation of phosphopeptide enrichments and detection, we have now substantially characterized, with temporal resolution as short as 10 s, the sites and levels of tyrosine phosphorylation across 10 min of FcRI-induced mast cell activation. These results reveal a far more extensive array of tyrosine phosphorylation events than previously known, including novel phosphorylation sites on canonical mast cell signaling molecules, as well as unexpected pathway components downstream of FcRI activation. M ast cells are regarded as crucial effector cells in allergic reactions and IgE-associated immune responses(1). Activation of mast cells, a critical feature of type I hypersensitive reactions, leads to the release of a wide range of chemical mediators and cytokines that recruit inflammatory cells and regulate inflammatory responses, such as mucus secretion, vasodilation, and bronchoconstriction (2). During activation, the mast cell high-affinity IgE receptor FcRI is cross-linked by allergens through bound IgE, leading to a cascade of signaling events and the release of preformed inflammatory mediators localized in specialized granules, the de novo synthesis and secretion of proinflammatory lipid mediators, and the synthesis and secretion of cytokines and chemokines (3).Some aspects of FcRI-mediated mast cell activation and inhibition pathways have been described previously (4) (Fig. 1). FcRI expressed on mast cells is comprised of three subunits: an IgEbinding ␣ subunit, a signal-amplifying ␤ subunit, and two disulfide-linked signal-initiating ␥ subunits (5). Following FcRI aggregation, the activation of ␤ subunit-bound protein tyrosine kinase Lyn and, subsequently, tyrosine phosphorylation of ITAM regions in the ␤ and ␥ subunits of FcRI, initiates a complex series of intracellular signaling events (6 -10). Phosphorylated ITAMs provide docking sites for Src homology 2 (SH2) 3 domain-containing cytoplasmic tyrosine kinases such as Syk (11), Lyn, and Fyn (12), leading to the activation of Syk (11). The subsequent phosphorylation of linker for activation ...

Lumpy skin disease (LSD) is a re-emerging transboundary viral disease of cattle and buffaloes with severe economic impact and listed as a notifiable disease by the World Organization for Animal Health (OIE, 2017). The severity of clinical signs of LSD varies from subclinical to fatal depending on the virulence of the strains and the cattle breed's susceptibility. Generally, LSD tends to have a mortality of <10% and a morbidity of 0%-90% (OIE, 2017;Sprygin et al., 2018). LSD, first detected in Zambia (1929), was historically restricted and endemic to southern and eastern Africa (Beard, 2016). The first LSD outbreak outside sub-Saharan Africa occurred in Egypt and Israel

Competing positive and negative signaling feedback pathways play a critical role in tuning the sensitivity of T cell receptor activation by creating an ultrasensitive, bistable switch to selectively enhance responses to foreign ligands while suppressing signals from self peptides. In response to T cell receptor agonist engagement, ERK is activated to positively regulate T cell receptor signaling through phosphorylation of Ser59 Lck. To obtain a wide-scale view of the role of ERK in propagating T cell receptor signaling, a quantitative phosphoproteomic analysis of 322 tyrosine phosphorylation sites by mass spectrometry was performed on the human Jurkat T cell line in the presence of U0126, an inhibitor of ERK activation. Relative to controls, U0126-treated cells showed constitutive decreases in phosphorylation through a T cell receptor stimulation time course on tyrosine residues found on upstream signaling proteins (CD3 chains, Lck, ZAP-70), as well as downstream signaling proteins (VAV1, PLCγ1, Itk, NCK1). Additional constitutive decreases in phosphorylation were found on the majority of identified proteins implicated in the regulation of actin cytoskeleton pathway. Although the majority of identified sites on T cell receptor signaling proteins showed decreases in phosphorylation, Tyr598 of ZAP-70 showed elevated phosphorylation in response to U0126 treatment, suggesting differential regulation of this site via ERK feedback. These findings shed new light on ERK’s role in positive feedback in T cell receptor signaling and reveal novel signaling events that are regulated by this kinase, which may fine tune T cell receptor activation.