Quest for the Crypto-phosphoproteome

Protein phosphorylation is one of the most studied post-translational modifications (PTMs). Despite the remarkable advances in phosphoproteomics, a chemically less-stable subset of the phosphosites, which we call the crypto-phosphoproteome, has remained underexplored due to technological challenges. In this Viewpoint, we briefly summarize the current understanding of these elusive protein phosphorylations and identify the missing pieces for future studies.     

Analysis of Site-Specific Phosphorylation of PTEN by Using Enzyme-Catalyzed Expressed Protein Ligation

The activity and localization of PTEN, a tumor suppressor lipid phosphatase that converts the phospholipid PIP3 to PIP2, is governed in part by phosphorylation on a cluster of four Ser and Thr residues near the C terminus. Prior enzymatic characterization of the four monophosphorylated (1p) PTENs by using classical expressed protein ligation (EPL) was complicated by the inclusion of a non-native Cys at the ligation junction (aa379), which may alter the properties of the semisynthetic protein. Here, we apply subtiligase-mediated EPL to create wt 1p-PTENs. These PTENs are more autoinhibited than previously appreciated, consistent with the role of Tyr379 in driving autoinhibition. Alkaline phosphatase sensitivity analysis revealed that these autoinhibited 1p conformations are kinetically labile. In contrast to the Cys mutant 1p-PTENs, which are poorly recognized by an anti-phospho-PTEN antibody, three of the four wt 1p-PTENs are recognized by a commonly used anti-phospho-PTEN antibody.     

Direct Chemical Activation of a Rationally Engineered Signaling Enzyme

Few chemical strategies for activating enzymes have been developed. Here we show that a biarsenical compound (FlAsH) can directly activate a rationally engineered protein tyrosine phosphatase (Shp2 PTP) by disrupting autoinhibitory interactions between Shp2's N‐terminal SH2 domain and its PTP domain. We found that introducing a tricysteine motif at a loop of Shp2's N‐SH2 domain confers affinity for FlAsH; binding of FlAsH to the cysteine‐enriched loop relieves Shp2's inhibitory interdomain interaction and substantially increases the enzyme's PTP activity. Activation of engineered Shp2 is substrate independent and is observed in the contexts of both purified enzyme and complex proteomes. A chemical means for activating Shp2 could be useful for investigating its roles in signaling and oncogenesis, and the loop‐targeting strategy described herein could provide a blueprint for the development of target‐specific activators of other autoinhibited enzymes.     

Assay of ascorbic phosphates and in-vitro availability assay of ascorbic mono-and polyphosphates

A method was devised to assay ascorbic phosphate esters in biological materials by potassium bromoxide hydrolysis followed by determination of the liberated ascorbic acid. For the differential measurement of ascorbic acid and ascorbic phosphate, a spectrophotometric method was employed to screen out the interfering substances based on studies of absorbance curves of 2,4-dinitrophenyl hydrazine derivatives. A variety of vertebrate tissues were examined for phosphatase activity on ascorbic phosphate esters. The results suggest that pigeon kidney, rat liver and several tissues of fishes readily hydrolyse ascorbic monophosphate but not ascorbic polyphosphate. Hydrolysis of ascorbic monophosphate is completed by both phosphatases of intestine, kidney and liver acting at neutral pH and phosphatase of stomach acting at acid pH. Thus, ascorbic monophosphate has the potential to be a source of available vitamin C in vivo, and this explains its antiscorbutic activity in scurvy-prone animals.     

Role of Receptor Protein Tyrosine Phosphatases (RPTPs) in Insulin Signaling and Secretion

Changes in lifestyle in developed countries have triggered the prevalence of obesity and type 2 diabetes mellitus (T2DM) in the latest years. Consequently, these metabolic diseases associated to insulin resistance, and the morbidity associated with them, accounts for enormous costs for the health systems. The best way to face this problem is to identify potential therapeutic targets and/or early biomarkers to help in the treatment and in the early detection. In the insulin receptor signaling cascade, the activities of protein tyrosine kinases and phosphatases are coordinated, thus, protein tyrosine kinases amplify the insulin signaling response, whereas phosphatases are required for the regulation of the rate and duration of that response. The focus of this review is to summarize the impact of transmembrane receptor protein tyrosine phosphatase (RPTPs) in the insulin signaling cascade and secretion, and their implication in metabolic diseases such as obesity and T2DM.     

Cellular inhibition of protein tyrosine phosphatase 1B by uncharged thioxothiazolidinone derivatives

As important regulators of cellular signal transduction, members of the protein tyrosine phosphatase (PTP) family are considered to be promising drug targets. However, to date, the most effective in vitro PTP inhibitors have tended to be highly charged, thus limiting cellular permeability. Here, we have identified an uncharged thioxothiazolidinone derivative (compound 1), as a competitive inhibitor of a subset of PTPs. Compound 1 effectively inhibited protein tyrosine phosphatase 1B (PTP1B) in two cell-based systems: it sensitized wild-type, but not PTP1B-null fibroblasts to insulin stimulation and prevented PTP1B-dependent dephosphorylation of the FLT3-ITD receptor tyrosine kinase. We have also tested a series of derivatives in vitro against PTP1B and proposed a model of the PTP1B-inhibitor interaction. These compounds should be useful in the elucidation of cellular PTP function and could represent a starting point for development of therapeutic PTP inhibitors.