Alistair T. R. Sim

Coordinated Control of Endothelial Nitric-oxide Synthase Phosphorylation by Protein Kinase C and the cAMP-dependent Protein Kinase

Endothelial nitric-oxide synthase (eNOS) is an important regulatory enzyme in the cardiovascular system catalyzing the production of NO from arginine. Multiple protein kinases including Akt/PKB, cAMP-dependent protein kinase (PKA), and the AMP-activated protein kinase (AMPK) activate eNOS by phosphorylating Ser-1177 in response to various stimuli. During VEGF signaling in endothelial cells, there is a transient increase in Ser-1177 phosphorylation coupled with a decrease in Thr-495 phosphorylation that reverses over 10 min. PKC signaling in endothelial cells inhibits eNOS activity by phosphorylating Thr-495 and dephosphorylating Ser-1177 whereas PKA signaling acts in reverse by increasing phosphorylation of Ser-1177 and dephosphorylation of Thr-495 to activate eNOS. Both phosphatases PP1 and PP2A are associated with eNOS. PP1 is responsible for dephosphorylation of Thr-495 based on its specificity for this site in both eNOS and the corresponding synthetic phosphopeptide whereas PP2A is responsible for dephosphorylation of Ser-1177. Treatment of endothelial cells with calyculin selectively blocks PKA-mediated dephosphorylation of Thr-495 whereas okadaic acid selectively blocks PKC-mediated dephosphorylation of Ser-1177. These results show that regulation of eNOS activity involves coordinated signaling through Ser-1177 and Thr-495 by multiple protein kinases and phosphatases.

[40] Use of okadaic acid to inhibit protein phosphatases in intact cells

Publisher Summary Okadaic acid is a marine toxin originally isolated from the black sponge, Halichondria okadaii . It is now known to be one of a family of related toxins, including dinophysistoxin 1 and acanthifolicin. These toxins are synthesized by dinoflagellates, especially of the genus Dinophysis , but the toxins accumulate in organisms further up the food chain, including sponges, shellfish, and, ultimately, humans. In humans, consumption of contaminated shellfish causes diarrhetic shellfish poisoning, and this is a particular problem at certain times of year when there is a proliferation of plankton containing the dinoflagellates. Okadaic acid is a complex fatty acid derivative containing numerous polyether linkages. It is an extremely valuable tool for testing the physiological role of protein phosphorylation in any physiological response in intact cells. It has been shown to act as a tumor promoter in the mouse skin bioassay, but unlike many other tumor promoters does not activate protein kinase C. The structure of okadaic acid is very hydrophobic, which makes it possible to be used it as a protein phosphatase inhibitor in intact cells.

Essential Requirement for PP2A Inhibition by the Oncogenic Receptor c-KIT Suggests PP2A Reactivation as a Strategy to Treat c-KIT+ Cancers

Oncogenic mutations of the receptor tyrosine kinase c-KIT play an important role in the pathogenesis of gastrointestinal stromal tumors, systemic mastocytosis, and some acute myeloid leukemias (AML). Although juxtamembrane mutations commonly detected in gastrointestinal stromal tumor are sensitive to tyrosine kinase inhibitors, the kinase domain mutations frequently encountered in systemic mastocytosis and AML confer resistance and are largely unresponsive to targeted inhibition by the existing agent imatinib. In this study, we show that myeloid cells expressing activated c-KIT mutants that are imatinib sensitive (V560G) or imatinib resistant (D816V) can inhibit the tumor suppressor activity of protein phosphatase 2A (PP2A). This effect was associated with the reduced expression of PP2A structural (A) and regulatory subunits (B55alpha, B56alpha, B56gamma, and B56delta). Overexpression of PP2A-Aalpha in D816V c-KIT cells induced apoptosis and inhibited proliferation. In addition, pharmacologic activation of PP2A by FTY720 reduced proliferation, inhibited clonogenic potential, and induced apoptosis of mutant c-KIT(+) cells, while having no effect on wild-type c-KIT cells or empty vector controls. FTY720 treatment caused the dephosphorylation of the D816V c-KIT receptor and its downstream signaling targets pAkt, pSTAT5, and pERK1/2. Additionally, in vivo administration of FTY720 delayed the growth of V560G and D816V c-KIT tumors, inhibited splenic and bone marrow infiltration, and prolonged survival. Our findings show that PP2A inhibition is essential for c-KIT-mediated tumorigenesis, and that reactivating PP2A may offer an attractive strategy to treat drug-resistant c-KIT(+) cancers.

Anhydride modified cantharidin analogues: synthesis, inhibition of protein phosphatases 1 and 2A and anticancer activity

Two series of anhydride modified cantharidin analogues were synthesised and screened for their phosphatase inhibition (PP1 and PP2A) and cytotoxicity in various cancer cell lines (Ovarian A2780, ADDP; Osteosarcoma 143B; and Colon HCT116 and HT29). One series was synthesised by a novel, high yielding one-pot hydrogenation-ring-opening-esterification procedure, the other by acid catalysed acetal formation. Analogues 5-7 and 9 displayed moderate PP2A selectivity (ca. 5- to 20-fold) and inhibition typically in the low microM range (comparable, in some cases to cantharidin). The anticancer activity of these analogues varied with the cell line under study; however, many of them showed selective cytotoxicity for the colon tumour cell lines.

Identification of Novel Diagnostic Biomarkers for Asthma and Chronic Obstructive Pulmonary Disease

RATIONALE Proteomics may identify a useful panel of biomarkers for identification of asthma and chronic obstructive pulmonary disease (COPD). OBJECTIVES To conduct an unsupervised analysis of peripheral blood proteins in well-characterized subjects with asthma and COPD, and identify and validate a biomarker panel for disease discrimination. METHODS Two-dimensional difference gel electrophoresis was used to separate plasma proteins from healthy control subjects, stable patients with asthma, and individuals with COPD. Candidate protein markers were identified by matrix assisted laser desorption ionization time of flight mass spectrometry and subsequently validated in two populations via immunoassay. A panel of four biomarkers was selected and their ability to distinguish between groups was assessed in isolation and in combination in two separate validation populations. MEASUREMENTS AND MAIN RESULTS Seventy-two protein spots displayed significantly different expression levels between the three subject groupings (P < 0.05). Fifty-eight were positively identified, representing 20 unique proteins. A panel of four biomarkers (α(2)-macroglobulin, haptoglobin, ceruloplasmin, and hemopexin) was able to discriminate with statistical significance between the clinical groups of patients with asthma, patients with COPD, and control subjects, and these results were confirmed in a second clinical population of older adults with airflow obstruction. CONCLUSIONS Proteomics has identified novel biomarkers for asthma and COPD, and shown that the iron metabolism pathways and acute-phase response may be involved in the pathogenesis of airway disease. The panel of peripheral blood biomarkers has the potential to become an extremely useful addition to the clinical diagnosis and management of respiratory disease.

Modulation of the phosphorylation and activity of calcium/calmodulin- dependent protein kinase II by zinc

Calcium/calmodulin‐dependent protein kinase II (CaMPK‐II) is a key regulatory enzyme in living cells. Modulation of its activity, therefore, could have a major impact on many cellular processes. We found that Zn2+ has multiple functional effects on CaMPK‐II. Zn2+ generated a Ca2+/CaM‐independent activity that correlated with the autophosphorylation of Thr286, inhibited Ca2+/CaM binding that correlated with the autophosphorylation of Thr306, and inhibited CaMPK‐II activity at high concentrations that correlated with the autophosphorylation of Ser279. The relative level of autophosphorylation of these three sites was dependent on the concentration of zinc used. The autophosphorylation of at least these three sites, together with Zn2+ binding, generated an increased mobility form of CaMPK‐II on sodium dodecyl sulfate gels. Overall, autophosphorylation induced by Zn2+ converts CaMPK‐II into a different form than the binding of Ca2+/CaM. In certain nerve terminals, where Zn2+ has been shown to play a neuromodulatory role and is present in high concentrations, Zn2+ may turn CaMPK‐II into a form that would be unable to respond to calcium signals.

The regulation and function of protein phosphatases in the brain

Data emerging from a number of different systems indicate that protein phosphatases are highly regulated and potentially responsive to changes in the levels of intracellular second messengers produced by extracellular stimulation. They may therefore be involved in the regulation of many cell functions. The protein phosphatases in the nervous system have not been well studied. However, a number of neuronal-specific regulators (such as DARPP-32 and G-substrate) exist, and brain protein phosphatases appear to have particularly low specific activity, suggesting that neuronal protein phosphatases possess considerable and unique potential for regulation. Several early events following depolarization or receptor activation appear to involve specific dephosphorylations, indicating that regulation of protein phosphatase activity is important for the control of many neuronal functions. This article reviews the current literature concerning the identification, regulation, and function of serine/threonine protein phosphatases in the brain, with particular emphasis on the regulation of the major protein phosphatases, PP1 and PP2A, and their potential roles in modulating neurotransmitter release and postsynaptic responses.

Multiple Interactions within the AKAP220 Signaling Complex Contribute to Protein Phosphatase 1 Regulation

The phosphorylation status of cellular proteins is controlled by the opposing actions of protein kinases and phosphatases. Compartmentalization of these enzymes is critical for spatial and temporal control of these phosphorylation/dephosphorylation events. We previously reported that a 220-kDa A-kinase anchoring protein (AKAP220) coordinates the location of the cAMP-dependent protein kinase (PKA) and the type 1 protein phosphatase catalytic subunit (PP1c) (Schillace, R. V., and Scott, J. D. (1999) Curr. Biol. 9, 321–324). We now demonstrate that an AKAP220 fragment is a competitive inhibitor of PP1c activity (K i = 2.9 ± 0.7 μm). Mapping studies and activity measurements indicate that several protein-protein interactions act synergistically to inhibit PP1. A consensus targeting motif, between residues 1195 and 1198 (Lys-Val-Gln-Phe), binds but does not affect enzyme activity, whereas determinants between residues 1711 and 1901 inhibit the phosphatase. Analysis of truncated PP1c and chimeric PP1/2A catalytic subunits suggests that AKAP220 inhibits the phosphatase in a manner distinct from all known PP1 inhibitors and toxins. Intermolecular interactions within the AKAP220 signaling complex further contribute to PP1 inhibition as addition of the PKA regulatory subunit (RII) enhances phosphatase inhibition. These experiments indicate that regulation of PP1 activity by AKAP220 involves a complex network of intra- and intermolecular interactions.

The role of serine/threonine protein phosphatases in exocytosis.

Modulation of exocytosis is integral to the regulation of cellular signalling, and a variety of disorders (such as epilepsy, hypertension, diabetes and asthma) are closely associated with pathological modulation of exocytosis. Emerging evidence points to protein phosphatases as key regulators of exocytosis in many cells and, therefore, as potential targets for the design of novel therapies to treat these diseases. Diverse yet exquisite regulatory mechanisms have evolved to direct the specificity of these enzymes in controlling particular cell processes, and functionally driven studies have demonstrated differential regulation of exocytosis by individual protein phosphatases. This Review discusses the evidence for the regulation of exocytosis by protein phosphatases in three major secretory systems, (1) mast cells, in which the regulation of exocytosis of inflammatory mediators plays a major role in the respiratory response to antigens, (2) insulin-secreting cells in which regulation of exocytosis is essential for metabolic control, and (3) neurons, in which regulation of exocytosis is perhaps the most complex and is essential for effective neurotransmission.

The First Two Cantharidin Analogues Displaying PP1 Selectivity

High pressure Diels-Alder reactions of furan and dimethylmaleate, and thiophene and maleimide resulted in two cantharidin analogues, 3 and 6 possessing PP1 selectivity (>40- and >30-fold selectivity) over PP2A. Both compounds exhibited moderate PP1 activity, 3 IC(50) 50 microM and 6 IC(50) 12.5 microM. Interestingly, the corresponding mono-ester derivatives of 3 showed no such selectivity.