Van Eyk, Dunn - Proteomic and Genomic Analysis of Cardiovascular Disease - 2003 (522919), страница 77
Текст из файла (страница 77)
Protein compositionof the human heart: the construction ofa myocardial two-dimensional electrophoresis database. Electrophoresis. 1994,15, 685–707.Macri, J., Rapundalo, S. T. Trends Cardiovasc. Med. Application of proteomics tothe study of cardiovascular biology. 2001,11, 66–75.Evans, G. W., Wheeler, C. H., Corbett,J. M., Dunn, M. J. Construction of HSC2DPAGE: a two-dimensional gel electrophoresis database of heart proteins. Electrophoresis. 1997, 18, 471–479.Pleissner, K. P., Sander, S., Oswald, H.,Regitz-Zagrosek, V., Fleck, E. Towardsdesign and comparison of World WideWeb-accessible myocardial two-dimensional gel electrophoresis protein databases. Electrophoresis.
1997, 18, 480–483.Service, R. F. High speed biologistssearch for gold in proteins. Science. 2001,294, 2074–2078.Somlyo, A. P., Somlyo A. V., in TheHeart and Cardiovascular System, H.A.Fozzard (Ed), Raven Press, New York,1991, pp. 1–30.Hartshorne, D. J., in Physiology of Gastrointestinal tract, L.R. Johnson (Ed.), Raven Press, New York, 1987, pp. 423–482.Sellers, J. R., Adelstein, R. S. Themechanism of regulation of smoothmuscle myosin by phosphorylation. Curr.Top.
Cell. Regul. 1985, 27, 51–62.Somlyo, A. P., Somlyo, A. V. Signal transduction and regulation in smooth muscle. Nature. 1994, 372, 231–236.Hartshorne, D. J., Ito, M., Erdodi, F.Myosin light chain phosphatase: subunitcomposition, interactions and regulation.J. Muscle Res. Cell Motil. 1998, 19, 325–341.Kimura, K., Ito, M., Amano, M., Chihara, K., Fukata, Y., Nakafuku, M., Yamamori, B. Feng, J., Nakano, T., Okawa, K., Iwamatsu, A., Kaibuchi, K. Reg-27527615 Investigations in Smooth Muscle Cell Physiology4041424344454647ulation of myosin phosphatase by Rhoand Rho-associated kinase (Rho-kinase)Science.
1996, 273, 245–248.Somlyo, A. P., Kitawawa, T., Himpens,B., Matthijs, G., Horiuti, K., Kowyashi, S., Goldman, Y. E., Somlyo, A. V.Modulation of Ca2+-sensitivity and of thetime course of contraction in smoothmuscle: a major role of protein phosphatase? Adv. Protein Phosphatases. 1989, 5,181–195.Shirazi, A., Izuka, K., Fadden, P.,Mosse, C., Somlyo, A. P., Somlyo, A. V.,Haystead, T. A. Purification and characterization of the mammalian myosinlight chain phosphatase holoenzyme.The differential effects of the holoenzyme and its subunits on smooth muscle.
J. Biol. Chem. 1994, 269, 31598–31606.Ichikawa, K., Ito, M., Hartshorne,D. J. Phosphorylation of the large subunitof myosin phosphatase and inhibition ofphosphatase activity. J. Biol. Chem. 1996,271, 4733–4470.Haystead, C. M., Gregory, P., Sturgill,T. W., Haystead, T. A. J. Gamma-phosphate-linked ATP-sepharose for the affinity purification of protein kinases. Rapidpurification to homogeneity of skeletalmuscle mitogen-activated protein kinasekinase. Eur.
J. Biochem. 1993, 214, 459–467.MacDonald, J. A., Borman, M. A., Muranyi, A., Somlyo, A. V., Hartshorne,D. J., Haystead, T. A. Identification of theendogenous smooth muscle myosinphosphatase-associated kinase. Proc. Natl.Acad. Sci. USA. 2001, 98, 2419–2424.MacDonald, J. A., Eto, M., Borman,M. A., Brautigan, D. L., Haystead,T. A. J. Dual Ser and Thr phosphorylationof CPI-17, an inhibitor of myosin phosphatase, by MYPT-associated kinase.F.E.B.S Lett. 2001, 493, 91–94.Moncada, S., Palmer, R. M. J., Higgs,E.
A. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol. Rev.1991, 43, 109–142.Cornwell, T. L., Pryzwansky, K. B.,Wyatt, T. A., Lincoln, T. M. Regulationof sarcoplasmic reticulum protein phosphorylation by localized cyclic GMP-de-48495051525354555657pendent protein kinase in vascularsmooth muscle cells. Mol. Pharmacol.1991, 40, 923–931.Lincoln, T. M., Komalavilas, P., Cornwell, T.L.
Hypertension. Pleiotropic regulation of vascular smooth muscle tone bycyclic GMP-dependent protein kinase.1994, 23, 1141–1147.Somlyo, A. P., Wu, X., Walker, L. A.,Somlyo, A. V. Pharmacomechanical coupling: the role of calcium, G-proteins, kinases and phosphatases. Rev. Physiol. Biochem.
Pharmacol. 1999, 440, 201–234.Beall, A., Bagwell, D., Woodrum, D.,Stoming, T. A., Kato, K., Suzuki, A.,Rasmussen, H., Brophy, C. M. The smallheat shock-related protein, HSP20, isphosphorylated on serine 16 during cyclic nucleotide-dependent relaxation.J. Biol. Chem. 1999, 274, 11344–11351.Woodrum, D. A., Brophy, C. M., Wingard, C. J., Beall, A., Rasmussen, H.Am. J.
Physiol. Phosphorylation events associated with cyclic nucleotide-dependentinhibition of smooth muscle contraction.1999, 277, H931–939.Wu, X., Haystead, T. A. J., Nakamoto,R. K., Somlyo, A. V., Somlyo, A. P. Acceleration of myosin light chain dephosphorylation and relaxation of smoothmuscle by telokin.
Synergism with cyclicnucleotide-activated kinase. J. Biol. Chem.1998, 273, 11362–11369.Pinna, L. A., Ruzzene, M. How do proteinkinases recognize their substrates? Biochim. Biophys. Acta. 1996, 1314, 191–225.Shenolikar, S., Nairn, A. C. Proteinphosphatases: recent progress. Adv. Second Messenger Phosphoprotein Res. 1991,23, 1–121.Fadden, P., Haystead, T. A. Quantitativeand selective fluorophore labeling ofphosphoserine on peptides and proteins:characterization at the attomole level bycapillary electrophoresis and laser-induced fluorescence. Anal. Biochem. 1995,10, 81–88.Oda, Y., Nagasu, T., Chait, B.
T. Enrichment analysis of phosphorylated proteinsas a tool for probing the phosphoproteome. Nat. Biotech. 2001, 19, 379–382.Zhou, H., Watts, J. D., Aebersold, R.A systematic approach to the analysis of15.6 Referencesprotein phosphorylation. Nat. Biotech.2001, 19, 375–378.58 Pawson, T., Scott., J. D. Signalingthrough scaffold, anchoring, and adaptorproteins. Science. 1997, 278, 2075–2080.59 Corcoran, E.
E., Means, A. R. DefiningCa2+/calmodulin-dependent protein kinase cascades in transcriptional regulation. J. Biol. Chem. 2001, 276, 2975–2978.60 Alms, G. R., Sanz, P., Carlson, M.,Haystead, T. A. J. Reg1p targets proteinphosphatase 1 to dephosphorylate hexoki-nase II in Saccharomyces cerevisiae:characterizing the effects of a phosphatase subunit on the yeast proteome.EMBO J. 1999, 18, 4157–4168.61 Cunningham, M. J. Genomics and proteomics: the new millennium of drugdiscovery and development.
J. Pharm.Tox. Meth. 2000, 44, 291–300.62 Haystead, T. A. J. Proteome mining: exploiting serendipity in drug discovery.Curr. Drug Discovery. 2001, 1, 22–24.27727916Identification of Targets of Phosphorylationin Heart MitochondriaRoberta A. Gottlieb and Huaping HeIn this chapter, we will describe the approach that led to the identification of EFTu as a mitochondrial target of phosphorylation in the ischemic myocardium,which we published in Circulation Research [1]. In addition to discussing the methods and difficulties, we will comment on the broader applications of this experimental approach.16.1Experimental Approach and PitfallsAbundant evidence has shown immediate activation of cytosolic MAP kinases,protein kinase C isoforms, and tyrosine kinases during ischemia or ischemic preconditioning; some of these signals converge upon the mitochondria [2].
We hypothesized that we would be able to detect mitochondrial phosphorylation due tocytosolic protein kinases in a reconstituted system, using isolated mitochondriaand cytosol from treated hearts. We assessed differential phosphorylation by com-Fig. 16.1 Experimental approach utilizedfor the identification of differentially phosphorylated proteins in mitochondria.Proteomic and Genomic Analysis of Cardiovascular Disease.Edited by Jennifer E. van Eyk, Michael J.
DunnCopyright © 2003 WILEY-VCH Verlag GmbH & Co. KGaA, WeinheimISBN: 3-527-30596-328016 Phosphoproteins in Heart Mitochondriaparing continuously perfused control hearts with hearts that had been subjectedto ischemia and reperfusion. A flow chart of the experimental approach is shownin Fig.
16.1.We prepared a reconstituted system by incubating cytosol from control or ischemic/reperfused hearts with mitochondria from control hearts in labeling bufferwith 32P-c-ATP. After the 10 min kinase reaction, the mitochondria were recovered by centrifugation and washed three times to eliminate cytosolic contamination. This gave us some differences in phosphorylation that could be detected onparallel lanes of a 1-D gel (Fig. 16.2 A). Most notably, there was increased phosphorylation of a 46 kDa protein in mitochondria that had been incubated with cytosol from ischemic/reperfused hearts.One concern was that the heart cytosol was prepared from a mixed populationof cells including cardiomyocytes, endothelial cells, and fibroblasts, while the mitochondria would be predominantly obtained from the cardiomyocytes.
It was theoretically possible we were creating an artifact if the phosphorylation of cardiomyocyte mitochondria was in fact due to kinases that were activated in endothelial cells or fibroblasts. A second concern related to the fact that the mitochondriawere prepared by Polytron homogenization; it was therefore possible that theirFig. 16.2 Comparison of phosphoproteins inmitochondria subjected to various incubations. Panel A: Mitochondria from a normalheart (CTRL MITOS) or from cardiomyocytesdisrupted by nitrogen cavitation (CAVITATEDMITOS) were incubated with c32P-ATP and cytosol from a control heart (CON) or from aheart subjected to ischemia/reperfusion (I/R).Panel B: A similar reconstitution using heartmitochondria or mitochondria prepared fromJurkat cells disrupted by nitrogen cavitation.Panel C: Mitochondria were isolated fromcontrol hearts or from hearts subjected toischemia/reperfusion, then incubated withc32P-ATP without added cytosol.
Reproducedwith permission from Circulation Research89:461–467, 2001.16.1 Experimental Approach and Pitfallsouter membranes were damaged, resulting in artifactual phosphorylation of targetproteins in a compartment that would not ordinarily be accessible to a kinase. Toaddress these concerns, we prepared cytosol from isolated cardiomyocytes thatwere subjected to simulated ischemia by metabolic inhibition with cyanide and 2deoxyglucose, followed by recovery. We prepared mitochondria from isolated cardiomyocytes, using nitrogen cavitation. This method of preparing mitochondria preserves outer membrane integrity [3]. This preparation yielded the same pattern ofdifferential phosphorylation of the 46 kDa protein, although some minor differences in other bands were noted (Fig.
16.2 A). We also confirmed that the samepattern of phosphorylation was obtained when heart cytosols were incubated withmitochondria prepared from Jurkat T-lymphoblast cells after disruption by nitrogen cavitation (Fig. 16.2 B).A third concern was that the 46 kDa protein might represent a cytosolic contaminant, despite the fact that the mitochondria had been washed extensivelyafter the kinase reaction. We reasoned that in ischemic hearts, some kinaseswould have already translocated to mitochondria (or become activated within themitochondria), and could be demonstrated merely by incubating isolated mitochondria from ischemic (vs control) hearts in labeling buffer.
This was indeed thecase, as seen in Fig. 16.2 C.Interestingly, the results seemed to suggest that the phosphorylation of p46 wasnot directly due to a cytosolic protein kinase, because even isolated mitochondriaFig. 16.3 Effect of varying the amount of cytosol on mitochondrial phosphorylation. Mitochondria from a control (CTRL) or ischemic(ISCH) heart (100 lg) were incubated with in-creasing amounts of cytosol from control (C)or ischemic hearts (I) in the presence of c32PATP.28128216 Phosphoproteins in Heart Mitochondria16.1 Experimental Approach and Pitfallsfrom a control heart exhibited a certain degree of phosphorylation of p46, whichwas suppressed by the addition of cytosol from control hearts and enhanced by cytosol from ischemic hearts.
![](https://s.studizba.com/z.php?f=/templates/si/i/noavatar.png&w=100&h=100&t=1"){kind=avatar}
