Molecular Basis of Gain-of-Function LEOPARD Syndrome-Associated SHP2 Mutations

The Src homology 2 (SH2) domain-containing protein tyrosine phosphatase 2 (SHP2) is a critical signal transducer downstream of growth factors that promotes the activation of the RAS-ERK1/2 cascade. In its basal state, SHP2 exists in an autoinhibited closed conformation because of an intramolecular interaction between its N-SH2 and protein tyrosine phosphatase (PTP) domains. Binding to pTyr ligands present on growth factor receptors and adaptor proteins with its N-SH2 domain localizes SHP2 to its substrates and frees the active site from allosteric inhibition. Germline mutations in SHP2 are known to cause both Noonan syndrome (NS) and LEOPARD syndrome (LS), two clinically similar autosomal dominant developmental disorders. NS-associated SHP2 mutants display elevated phosphatase activity, while LS-associated SHP2 mutants exhibit reduced catalytic activity. A conundrum in how clinically similar diseases result from mutations to SHP2 that have opposite effects on this enzyme’s catalytic functionality exists. Here we report a comprehensive investigation of the kinetic, structural, dynamic, and biochemical signaling properties of the wild type as well as all reported LS-associated SHP2 mutants. The results reveal that LS-causing mutations not only affect SHP2 phosphatase activity but also induce a weakening of the intramolecular interaction between the N-SH2 and PTP domains, leading to mutants that are more readily activated by competing pTyr ligands. Our data also indicate that the residual phosphatase activity associated with the LS SHP2 mutant is required for enhanced ERK1/2 activation. Consequently, catalytically impaired SHP2 mutants could display gain-of-function properties because of their ability to localize to the vicinity of substrates for longer periods of time, thereby affording the opportunity for prolonged substrate turnover and sustained RAS-ERK1/2 activation

Cardiovascular Safety During Treatment With Baricitinib in Rheumatoid Arthritis

OBJECTIVE: To assess the frequency of cardiovascular and venous thromboembolic events in clinical studies of baricitinib, an oral, selective JAK1 and JAK2 inhibitor approved in more than 50 countries for the treatment of moderately-to-severely active rheumatoid arthritis (RA). METHODS: Data were pooled from 9 RA studies. Placebo comparison up to 24 weeks included data from 6 studies. Randomized dose comparison between baricitinib doses of 2 mg and 4 mg used data from 4 studies and from the associated long-term extension study. The data analysis set designated "All-bari-RA" included all baricitinib exposures at any dose. RESULTS: Overall, 3,492 RA patients received baricitinib (7,860 patient-years of exposure). No imbalance compared to the placebo group was seen in the incidence of major adverse cardiovascular events (MACE) (incidence rates [IRs] of 0.5 per 100 patient-years for placebo and 0.8 per 100 patient-years for 4 mg baricitinib), arterial thrombotic events (ATE) (IRs of 0.5 per 100 patient-years for placebo and 0.5 per 100 patient-years for 4 mg baricitinib), or congestive heart failure (CHF) broad term (IRs of 4.3 per 100 patient-years for placebo and 2.4 per 100 patient-years for 4 mg baricitinib). Deep vein thrombosis (DVT)/pulmonary embolism (PE) were reported in 0 of 1,070 patients treated with placebo and 6 of 997 patients treated with 4 mg baricitinib during the placebo-controlled period; these events were serious in 2 of 6 patients, while all 6 had risk factors and 1 patient developed DVT/PE after discontinuation of the study drug. In the 2 mg-4 mg-extended data analysis set, IRs of DVT/PE were comparable between the doses across event types (IRs of 0.5 per 100 patient-years in those receiving 2 mg baricitinib and 0.6 per 100 patient-years in those receiving 4 mg baricitinib). In the All-bari-RA data analysis set, the rates were stable over time, with an IR of DVT/PE of 0.5 per 100 patient-years. CONCLUSION: In RA clinical trials, no association was found between baricitinib treatment and the incidence of MACE, ATE, or CHF. With regard to incidence of DVT/PE, 6 events occurred in patients treated with 4 mg baricitinib, but no cases of DVT/PE were reported in the placebo group. During longer-term evaluation, the incidence of DVT/PE was similar between the baricitinib dose groups, with consistent IR values over time, and this was similar to the rates previously reported in patients with RA

Phosphatase of regenerating liver 3 (PRL3) provokes a tyrosine phosphoproteome to drive prometastatic signal transduction

Phosphatase of regenerating liver 3 (PRL3) is suspected to be a causative factor toward cellular metastasis when in excess. To date, the molecular basis for PRL3 function remains an enigma, making efforts at distilling a concerted mechanism for PRL3-mediated metastatic dissemination very difficult. We previously discovered that PRL3 expressing cells exhibit a pronounced increase in protein tyrosine phosphorylation. Here we take an unbiased mass spectrometry-based approach toward identifying the phosphoproteins exhibiting enhanced levels of tyrosine phosphorylation with a goal to define the "PRL3-mediated signaling network." Phosphoproteomic data support intracellular activation of an extensive signaling network normally governed by extracellular ligand-activated transmembrane growth factor, cytokine, and integrin receptors in the PRL3 cells. Additionally, data implicate the Src tyrosine kinase as the major intracellular kinase responsible for "hijacking" this network and provide strong evidence that aberrant Src activation is a major consequence of PRL3 overexpression. Importantly, the data support a PDGF(α/β)-, Eph (A2/B3/B4)-, and Integrin (β1/β5)-receptor array as being the predominant network coordinator in the PRL3 cells, corroborating a PRL3-induced mesenchymal-state. Within this network, we find that tyrosine phosphorylation is increased on a multitude of signaling effectors responsible for Rho-family GTPase, PI3K-Akt, STAT, and ERK activation, linking observations made by the field as a whole under Src as a primary signal transducer. Our phosphoproteomic data paint the most comprehensive picture to date of how PRL3 drives prometastatic molecular events through Src activation

Integrated Analysis of Global mRNA and Protein Expression Data in HEK293 Cells Overexpressing PRL-1

<div><p>Background</p><p>The protein tyrosine phosphatase PRL-1 represents a putative oncogene with wide-ranging cellular effects. Overexpression of PRL-1 can promote cell proliferation, survival, migration, invasion, and metastasis, but the underlying mechanisms by which it influences these processes remain poorly understood.</p><p>Methodology</p><p>To increase our comprehension of PRL-1 mediated signaling events, we employed transcriptional profiling (DNA microarray) and proteomics (mass spectrometry) to perform a thorough characterization of the global molecular changes in gene expression that occur in response to stable PRL-1 overexpression in a relevant model system (HEK293).</p><p>Principal Findings</p><p>Overexpression of PRL-1 led to several significant changes in the mRNA and protein expression profiles of HEK293 cells. The differentially expressed gene set was highly enriched in genes involved in cytoskeletal remodeling, integrin-mediated cell-matrix adhesion, and RNA recognition and splicing. In particular, members of the Rho signaling pathway and molecules that converge on this pathway were heavily influenced by PRL-1 overexpression, supporting observations from previous studies that link PRL-1 to the Rho GTPase signaling network. In addition, several genes not previously associated with PRL-1 were found to be significantly altered by its expression. Most notable among these were Filamin A, RhoGDIα, SPARC, hnRNPH2, and PRDX2.</p><p>Conclusions and Significance</p><p>This systems-level approach sheds new light on the molecular networks underlying PRL-1 action and presents several novel directions for future, hypothesis-based studies.</p></div

Protein changes in the Rho-signaling canonical pathway resulting from PRL-1 overexpression in HEK293 cells.

<p>Selected proteins that conduct signals to remodel the cytoskeleton through RhoA, Rac1, and CDC42 are represented by their coding gene names in a canonical pathway diagram adapted from Ingenuity Pathway Analysis (IPA). The symbols of proteins that were detected in the mass-spectrometry experiment at Tier-1 or Tier-2 levels are colored according to the direction of their fold change (FC) in expression in the PRL-1-transfectants as compared to the empty vector group, with yellow hues indicating an increased quantity of protein and blue hues indicating a decrease. An asterisk (*) indicates that a protein expression change is significant at a level of p≤0.05. Tier-1 proteins are noted with bold font labels. Groups of related or complex-forming proteins are illustrated with double-outlined symbols. Connecting lines with arrowheads indicate an activating, de-activating, or translocating influence, and the absence of an arrowhead indicates a protein-protein binding interaction or group membership. Solid connecting lines show direct interactions while dashed lines show indirect interactions. The known direct interaction of PRL-1 (PTP4A1) with ARHGAP4 is represented here, but indirect connections between PRL-1 and the components of this pathway are not shown.</p

Significant (q≤0.05) differentially expressing mRNA transcripts from qRT-PCR analysis of PRL-1 overexpressing HEK293 cells.

<p>Abbreviations: ID  =  identification; Overex.  =  overexpressing; FDR  =  false discovery rate.</p

Volcano plot of significant (q≤0.10) differentially-expressed proteins integrated with changes in corresponding mRNA signals

<p>. The dot (•) symbols represent the Tier-1 proteins that were observed to be differentially expressed under PRL-1-overexpressing conditions in HEK293 cells. These protein data are plotted along the X- and Y-axes according to the log of the protein expression ratio and FDR-corrected significance respectively. A positive log2(protein ratio) value indicates an up-regulation of protein expression under PRL-1-overexpressing conditions as compared to controls, while a negative value indicates down-regulation of protein expression. Each protein's corresponding mRNA data is represented by a colored circle around that protein's dot symbol. Each probeset in the microarray experiment that was 1) mapped to a plotted protein's coding gene and was 2) differentially expressed with a significance of p≤0.20 is represented by a colored region. An asterisk (*) indicates an mRNA signal with a significance of p≤0.05. In cases where multiple detected probesets were mapped to the same protein's coding gene, the colored circle is divided into sectors according to the relative contribution that each probeset had to the total mRNA signal. Yellow colors represent an up-regulation of mRNA expression and blue colors indicate a down-regulation at the mRNA level. FC  =  fold change; FDR  =  false discovery rate.</p

Significant (q≤0.10) differentially expressing mRNA signals from microarray analysis of PRL-1 overexpressing HEK293 cells.

<p>Abbreviations: ID  =  identification; Overex.  =  overexpressing; FDR  =  false discovery rate.</p