human CAMP phosphodiesterase pde46 (HSPDE4A4) is altered upon the binding of SH3 domains of src family tyrosyl kinases to the PDE4A LR2 region.
Biochemical Society Transactions (1999) 27 Lyn tyrosyl b a s e binds to the human CAMP-specific phosphodiesterase pde46 (HSPDUA4B) and causes a conformational change in its catalytic unit 9. G. Scotland, M. Beard, A.H. Ross and M.D. Houslay Mol. Pharmacol. Group, Divn of Biochem. & Mol. Biol., University of Glasgow, Glasgow G I 2 8QQ. Specific PDE isoenzymes can be regulated through the action of a variety of intracellular signalling systems which suggests that CAMP signalling may be regulated in distinct fashions in different cells (1). Type 4 CAMPspecific phosphodiesterases (PDE4s) are encoded by four genes each of which produce a series of isoenzymes through alternative mRNA splicing (2). In this study we show that the PDE4 isoenzyme, pde46 (HSPDE4A4B) exhibited a selectivity for interaction with the SH3 domains of src family tyrosyl kinases expressed as GST fusion proteins. This interaction led to a profound change in the inhibition of pde46 by the anti-inflammatory and anti-depressant agent, rolipram. Such a change mimicked that seen when pde46 was bound to the particulate fraction of COS7 cells. Co-immunoprecipitation and co-localisation studies indicated that pde46 was associated with lyn kinase in COS7 cells. SH3 domain interaction with pde46 occurred at sites within both its Linker Region 2 (LR2) and also within its N-terminal alternatively spliced region. SH3 domain interaction with the LR2 region caused an increase in its susceptibility to rolipram inhibition, as demonstrated using deletion mutants in both pde46 and the N-terminal truncate h6.1. It is suggested that the conformation of the catalytic unit of the human CAMP phosphodiesterase pde46 (HSPDE4A4) is altered upon the binding of SH3 domains of src family tyrosyl kinases to the PDE4A LR2 region. This change can be detected by an altered susceptibility of pde46 to inhibition by rolipram. Such an interaction between PDE4A enzymes and src family tyrosyl kinases highlights a potentially novel regulatory system and point of cross-talk between these signalling systems.
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I.Houslay, M. D., and Milligan, G. (1997) TiBS 22(6), 217-224 Z.Houslay, M. D., et a1 (1997) Adv. Pharmacology 44, 225-342
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PTEN,A Tumour Suppresser Is A 3 Phosphorylated Phosphoinositide 3 Phosphatase
Ian Pass, Ian H Batty, C Peter Downes Department of Biochemistry, University of Dundee, MSI/WTB Complex, Dow Street, Dundee, DDI 5EH Deletions or mutations of the tumour suppresser, phosphatase and tensin homologue deleted on chromosome 10 (PTEN) also known as mutated in multiple advanced cancers 1 (MMACI) and TGF-Pregulated and epithelial cell - enriched phosphatase (TEPI) are associated with an array of human cancers. Also germ line mutations of the PTEN gene have been identified in several related neoplastic inherited disorders, such as Cowden disease, an autosomal dominant cancer predisposition syndrome that is associated with an elevated risk of tumours. An important step in understanding how PTEN functions as a tumour suppresser is the identification of it’s physiological substrates. PTEN has been shown to share homology to protein phosphatases and will dephosphorylate the synthetic peptide polyGluTyr, here we show in detail and previously (Myers, Pass et al. 1998) that PTEN is able to remove with total positional selectively the phosphate from the D3 position of the inositol ring. Further we show that both missense mutation and small deletions of FTEN observed in both Cowden disease kindred and tumour samples are able specifically to ablate the ability of PTEN to recognise inositol phospholipids as a substrate while retaining protein phosphatase activity. Myers, M. P., I. Pass, et al. (1998). “The lipid phosphatase activity of PTEN is critical for its tumor supressor function.” Proc Natl Acad Sci U S A 95(23): 135 13-8.
Kinetics ofvitamin K 2,3 epoxide reductase
L.A. Begent, S.T. Chan, G.B. Steventon Department of Pharmacy, King’s College London, London SW3 6LX. The vitamin K cycle is essential for the formation of active zymogens of the vitamin K-dependent blood-clotting factors. Dietary vitamin quinone is converted to the hydroquinone by vitamin K quinone reductase and the active vitamin K metabolites is an essential cofactor for the vitamin K-dependent carboxylase which y-carboxylates glutamic acid side chains in the inactive precursor proteins. The vitamin K hydroquinone is oxidised to vitamin K 2,3 epoxide which must be reduced to vitamin K quinone for the cycle to begin again. The enzyme vitamin K 2,3 epoxide reductase is responsible for this and is the main target of inhibition in oral anticoagulation therapy with (WS) warfarin a potent inhibitor ofthe enzyme. Vitamin K 2,3 epoxide reductase has yet to be purified from any species and the enzyme is known to thiol dependent since the in vitro cofactor for the enzyme, dithiothreitol (DTT)will reduce disulphide bonds. The inhibitor, (WS) warfarin is known to only bind to the protein in its oxidised (disulphide) form. The in vitro enzyme kinetics of rat hepatic microsomal vitamin K 2,3 epoxide reductase show biphasic kinetics with the enzyme having a high affinity and low affinity form for the substrate vitamin K 2,3 epoxide but monophasic kinetics for the cofactor DTT. This may possibly indicate that two enzymes are involved in the metabolism of vitamin K 2,3 epoxide to vitamin K quinone in the rat
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Purification of vitamin K 2,3 epoxide reductase
Anthony Hill*, C. Pallister*, D. Cowell*, G. Steventon’ *DepaNnent of Biological Sciences, University of the West of England, Bristol, BS 16 1 QY and the ‘Department of Pharmacy, King’s College London, London SW3 6LX. Vitamin K is an essential co-factor in hepatocytes for the endoplasmic reticulum enzyme, vitamin Kdependent carboxylase. The active vitamin K metabolite, vitamin K hydroquinone is converted by the carboxylase into vitamin K 2,3 epoxide. The conversion of the 2,3 epoxide metabolite to vitamin K quinone and then the hydroquinone is carried out by the enzyme vitamin K 2,3 epoxide reductase. This protein has proved difficult to purify and thus the study of the kinetics of vitamin K 2,3 epoxide turnover and the interactionsof the protein with the anti-coagulant drug (WS)warfarin have been carried out in only the crude microsomal fraction of liver from a number of species. The purification of rat liver vitamin K 2,3 epoxide reductase was attempted. Solubilisation of the microsomal fraction and subsequent ion exchange chromatography stages resulted in a 100 fold purification from the microsomal fraction Subsequentsize exclusion chromatography gave a 327 fold increase in purification over the microsomal fraction. Polyacrylamide gel electrophoresisof the size exclusion fraction resulted in 573 fold increase in purification of the enzyme. The size exclusion stage however resulted in the isolation of vitamin K 2,3 epoxide reductase fraction that was now insensitive to the action of (WS) warfarin. Thus possibly indicating that vitamin K 2,3 epoxide reductase is a complex of at least two proteins with one protein binding the substrate and the other protein(s) being responsible for inhibitor binding.
