Imidazolium trans-[tetrachloro (dimethyl sulfoxide)(imidazole)ruthenate(III)], NAMI-A, is an experimental metastasis inhibitor whose specific mechanism of activation and action remains to be elucidated. In the nucleophilic and reducing physiological environment; it is anticipated that the most relevant and available reductants upon introduction of NAMI-A as a therapeutic agent will be the biologically-relevant free thiols. The kinetics and mechanisms of interaction of NAMI-A with biologically-active thiols cysteamine, glutathione, cysteine and a popular chemoprotectant, 2-mercaptoethane sulfonate (MESNA) have been studied spectrophotometrically under physiologically-relevant conditions. The reactions are characterized by initial reduction of NAMI-A with simultaneous formation of dimeric thiol and subsequent ligand exchange with water to various degrees as evidenced by Electospray Ionization Mass Spectrometry. Stoichiometry of reactions shows that one molecule of NAMI-A reacted with one mole of thiol to form corresponding disulfide cystamine, dimeric MESNA, oxidized glutathione and cystine. Observed rate constants, ko, for the reaction of NAMI-A with cysteamine, MESNA, GSH and cysteine were deduced to be 6.85 + 0.3 x 10-1, 9.4 + 0.5 x 10-2 , 7.42 + 0.4 x 10-3 and 3.63 + 0.3 x 10-2 s-1 respectively. Activation parameters determined from Arrhenius plots are indicative of formation of associative intermediates prior to formation of products. A negative correlation was obtained from the Brønsted plot derived from observed rate constants and the pKa of the different thiols demonstrating significant contribution of thiolate species towards the rate. In conclusion, interactions of NAMI-A with biologically-active thiols are kinetically and thermodynamically favored and should play significant roles in in vivo metabolism of NAMI-A.

Indiana University-Purdue University Indianapolis (IUPUI) / The addition and removal of phosphate groups is a key regulatory mechanism for many cellular processes. The balance between phosphorylation and dephosphorylation is delicate and must be maintained in order for proper cell functions to be carried out. Protein kinases and phosphatases are the keepers of this balance with kinases adding phosphate groups and phosphatases removing them. As such, mutation and/or altered regulation of these proteins can be the driving factor in disease. Phosphatase of Regenerating Liver (PRL) is a family novel of three dual specificity phosphatases (DSPs) first discovered in the regenerating liver tissue of rats. PRLs have also been shown to act as oncogenes in cell culture and in animal models. However, the physiological substrate and mechanisms of the PRLs are not yet known. Recently, our lab has developed a PRL 2 knockout mouse and found several striking phenotypes all of which correspond to a significant increase in PTEN. We also found that PRL 2 is targetable by small molecular inhibitors that can potentially be used to disrupt tumor growth and spermatogenesis. Furthermore, a PTEN heterozygous mouse model crossed into our PRL 2 knockout line was generated to investigate the relevance of PRL interaction with PTEN in cancer.

Indiana University-Purdue University Indianapolis (IUPUI) / Phosphatase of Regenerating Liver 3 (PRL3) is suspected to be a causative factor toward cellular metastasis when overexpressed. To date, the molecular basis for PRL3 function remains an enigma, justifying the use of 'shot-gun'-style phosphoproteomic strategies to define the PRL3-mediated signaling network. On the basis of aberrant Src tyrosine kinase activation following ectopic PRL3 expression, phosphoproteomic data reveal a signal transduction network downstream of a mitogenic and chemotactic PDGF (α and β), Eph (A2, B3, B4), and Integrin (β1 and β5) receptor array known to be utilized by migratory mesenchymal cells during development and acute wound healing in the adult animal. Tyrosine phosphorylation is present on a multitude of signaling effectors responsible for Rho-family GTPase, PI3K-Akt, Jak-STAT3, and Ras-ERK1/2 pathway 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 pro-metastatic molecular events through Src activation. The Src-homology 2 (SH2) domain-containing tyrosine phosphatase 2 (SHP2), encoded by the Ptpn11 gene, is a bona-fide proto-oncogene responsible for the activation of the Ras/ERK1/2 pathway following mitogen stimulation. The molecular basis for SHP2 function is pTyr-ligand-mediated alleviation of intramolecular autoinhibition by the N-terminal SH2 domain (N-SH2 domain) upon the PTP catalytic domain. Pathogenic mutations that reside within the interface region between the N-SH2 and PTP domains are postulated to weaken the autoinhibitory interaction leading to SHP2 catalytic activation in the open conformation. Conversely, a subset of mutations resides within the catalytic active site and cause catalytic impairment. These catalytically impaired SHP2 mutants potentiate the pathogenesis of LEOPARD-syndrome (LS), a neuro-cardio-facial-cutaneous (NCFC) syndrome with very similar clinical presentation to related Noonan syndrome (NS), which is known to be caused by gain-of-function (GOF) SHP2 mutants. Here we apply hydrogen-deuterium exchange mass spectrometry (H/DX-MS) to provide direct evidence that LS-associated SHP2 mutations which cause catalytic impairment also weaken the autoinhibitory interaction that the N-SH2 domain makes with the PTP domain. Our H/DX-MS study shows that LS-SHP2 mutants possess a biophysical property that is absolutely required for GOF-effects to be realized, in-vivo.

Indiana University-Purdue University Indianapolis (IUPUI) / Phosphatases of regenerating liver, consisting of PRL-1, PRL-2 and PRL-3, belong to a novel protein tyrosine phosphatases subfamily, whose overexpression promotes cell proliferation, migration and invasion and contributes to tumorigenesis and metastasis. However, although great efforts have been made to uncover the biological function of PRLs, limited knowledge is available on the underlying mechanism of PRLs’ actions, therapeutic value by targeting PRLs, as well as the physiological function of PRLs in vivo. To answer these questions, we first screened a phage display library and identified p115 RhoGAP as a novel PRL-1 binding partner. Mechanistically, we demonstrated that PRL-1 activates RhoA and ERK1/2 by decreasing the association between active RhoA with GAP domain of p115 RhoGAP, and displacing MEKK1 from the SH3 domain of p115 RhoGAP, respectively, leading to enhanced cell proliferation and migration. Secondly, structure-based virtual screening was employed to discover small molecule inhibitors blocking PRL-1 trimer formation which has been suggested to play an important role for PRL-1 mediated oncogenesis. We identified Cmpd-43 as a novel PRL-1 trimer disruptor. Structural study demonstrated the binding mode of PRL-1 with the trimer disruptor. Most importantly, cellular data revealed that Cmpd-43 inhibited PRL-1 induced cell proliferation and migration in breast cancer cell line MDA-MB-231 and lung cancer cell line H1299. Finally, in order to investigate the physiological function of PRLs, we generated mouse knockout models for Prl-1, Prl-2 and Prl-3. Although mice deficient for Prl-1 and Prl-3 were normally developed, Prl-2-null mice displayed growth retardation, impaired male reproductive ability and insufficient hematopoiesis. To further investigate the in vivo function of Prl-1, we generated Prl-1-/-/Prl-2+/- and Prl-1+/-/Prl-2-/- mice. Similar to Prl-2 deficient male mice, Prl-1-/-/Prl-2+/- males also have impaired spermatogenesis and reproductivity. More strikingly, Prl-1+/-/Prl-2-/- mice are completely infertile, suggesting that, in addition to PRL-2, PRL-1 also plays an important role in maintaining normal testis function. In summary, these studies demonstrated for the first time that PRL-1 activates ERK1/2 and RhoA through the novel interaction with p115 RhoGAP, targeting PRL-1 trimer interface is a novel anti-cancer therapeutic treatment and both PRL-1 and PRL-2 contribute to spermatogenesis and male mice reproductivity.