Van Eyk, Dunn - Proteomic and Genomic Analysis of Cardiovascular Disease - 2003 (522919), страница 74
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Much of thefocus of proteomics has been on the advancement of mass spectrometry sequencing technology (i.e., automation, speed, and sensitivity) which has resulted in ade-emphasis on the design of proteomic experiments. Our general strategy hasbeen to devise techniques that enrich for low abundance proteins of significant relevance to the biological question at hand. The reduction in proteome complexitysaves us from identifying hundreds of irrelevant proteins which could consume amajority of our time and effort. We used c-linked ATP Sepharose affinity chroma-26326415 Investigations in Smooth Muscle Cell Physiology15.3 Defining Signal Transduction Modules in Smooth Muscletography to create a sub-proteome that was enriched with protein kinases.
This affinity resin was developed with ATP immobilized in the “protein kinase orientation” (via its c-phosphate); the entire SMC complement of ATP binding proteinscan be isolated following high stringency washes to remove non-specific proteins[43]. Using this methodology, we were successful in sequencing the previouslyunidentified MYPT1-kinase following affinity purification. Additional proof fromexpression and structure function studies has confirmed a role for the MYPT1kinase as a regulator of SMC contractile state [44, 45]. The kinase was shown toassociate with MYPT1, to phosphorylate MYPT1 at the inhibitory site in intactsmooth muscle, and to be regulated by a yet unidentified upstream-kinase.Several hormones and clinically administered nitrovasodilators exert their actionsto widen vessels and inhibit vasoconstriction through activation of smooth muscleguanylate cyclase to produce cGMP [46]. One of the primary effects of elevated intracellular cGMP is the reduction of [Ca2+]i.
The mechanisms by which cGMP lowersintracellular Ca2+ are well studied [47, 48]; they include stimulation of Ca2+ extrusionby activation of the calmodulin-stimulated Ca2+-ATPase, a decrease in Ca2+ influxdue to decreased Ca2+-channel activity, a decrease in Ca2+ influx through hyperpolerization as a result of increased K+-channel activity, stimulation of calcium uptakeinto the sarcoplasmic reticulum by phosphorylation of phospholamban, and antagonism of hormone triggered generation of second messengers. A second relevantphysiological effect of elevated intracellular [cGMP] (and cAMP) is the phenomenonof Ca2+-desensitization (reviewed in [49]).
Elevation of [cGMP] at fixed [Ca2+] causesmuscle relaxation in a manner that does not disrupt the LC20 phosphorylation/forcerelationship. The molecular mechanisms that bring about this phenomenon, presumably through the activation of cyclic-GMP dependent protein kinase (PKG)are unknown. We are using a functional proteomic approach to identify novel kinase substrates that are phosphorylated in response to cGMP administration. Theidentification of these physiologically-relevant, phosphorylated targets is a criticalstep to our understanding of the regulation of SMC tone. Several previously characterized targets were identified during cyclic nucleotide-evoked relaxation includingbut not limited to HSP20, HSP27 [50, 51], and telokin [49, 52]. Several additionalphosphorylation targets are detected after cGMP-dependent relaxation of ill longitudinal smooth muscle and vascular smooth muscle (MacDonald and Haystead, un3Fig. 15.3 Catching the adenine nucleotidebinding proteome on c-phosphate linked ATPSepharose.
Mouse extract was prepared andpassed over 50 ml of c-phosphate linked ATPSepharose containing 10 lmols/ml of linkedATP or N-6 linked ATP-Sepharose (SigmaChemical Co., St. Louis MO). Following washing, the column was eluted sequentially withthe indicated nucleotides and fractions collected (10 ml). Column fractions were separated by 1D SDS-PAGE and silver-stained.
Aportion of the ATP eluate was concentrated100 fold and 10 ll analyzed by 2DE. Proteinswere identified by ESI-MS/MS sequencing andwere matched against the entire publishedprotein or DNA data bases with the FASTS orTFASTS algorithms respectively. Expectationscores for the identified proteins ranged from2.6 e–7 for PKA to 1.2 e–54 for GAPDH. Expectation scores after each search for the nexthighest scoring non-related protein were generally < 2.3 e–4. (Redrawn from P. R. Graves, etal., 2002; submitted for publication.)26526615 Investigations in Smooth Muscle Cell PhysiologyFig.
15.4 2DE profile of phosphoprotein targets in ileum and femoral artery SMC following cGMP-treatment. Smooth muscle stripswere permeabilized with a-toxin, contractedwith exposure to pCa6.3 solution with 1 lMcalmodulin, and treated with vehicle (control)or 100 lM 8-bromo-cGMP solution (8BrcGMP) in the presence of [c-32P]ATP. Cellularextracts were applied to 2DE, and proteins ofinterest were identified by ESI-MS/MS se-quencing with FASTS or TFASTS searching ofprotein and DNA databases. 1 a, b, c) HSP27;2 a, b) p20; 3) ubiquitin-conjugating enzyme;4) below detection limit; 5) actin depolymerizing factor; 6) dbEST putative gene product, 7)unknown, 8) unknown; 9) telokin; 10) RIKENcDNA putative gene product; and 11) unknown.
(Redrawn from J.A. MacDonald et al.,2002; submitted for publication.)published observations). The proteins have been identified in the dbEST and RIKENcDNA libraries as novel putative gene products. Our initial observations suggest thatthere are different molecular mechanisms that mediate cGMP-dependent relaxationin tonic versus phasic SMC.15.3 Defining Signal Transduction Modules in Smooth MuscleProteomic Approaches to Substrate Profiling. A significant hindrance to the signaltransduction field has been the difficulty in identifying true in vivo substrates toindividual protein kinases and phosphatases.
This problem has been perpetuatedby the in vitro promiscuity of kinases [53] and phosphatases [54] toward proteinsubstrates. Two significant problems plague kinase substrate preference characterization. First, there are no methods available for the systematic, unbiased characterization of substrate preferences; existing techniques for substrate profiling (i.e.site prediction from phosphorylation consensus site mapping) must start fromknown determinants. Even peptide libraries cannot generate all possible aminoacid combinations for testing due to the shear number of combinatorial possibilities and the technological limitations those numbers impose.
Second, there is nosimple way to compare the general pattern of kinase substrate preferences. Bothof these issues can be partially addressed through proteomics by taking advantageof the fact that only a small fraction of all possible amino acid combinations areactually produced as proteins. By defining a substrate proteome as a set of proteins in a cell which could be in vitro substrates for a particular kinase, we canuse a cellular protein extract to explore differences in that set between kinases.This limits substrate possibilities to sequences which are available for phosphorylation, at least in one cell type, and increases the complexity relative to a combinatorial library, since each sequence is independent of the next. Moreover, becausean extract contains proteins not peptides, we further restrict substrate specificityto secondary and tertiary structures, and have added the complexity of post-translational modifications.
These differences may actually reduce the number of possible sequences tested, but do so in a way that corresponds to biological possibilities. As a screen, it is unbiased in any assumption of substrate sequence,although choices of source material for the extract and of biochemical techniquesto solubilize the proteins will affect the results.Test conditions were designed to provide the best possible definition of the substrate proteome. This was primarily achieved by using high concentrations of exogenous kinase relative to the substrate proteome concentration, so that the kinase tested could overwhelm kinases present in the extract.
By fractionating the extract prior to the kinase reaction, we are able to significantly increase the sensitivity toward less abundant substrates. Recently developed methodology for the selective enhancement of phosphoproteins from the cellular proteome [55–57] couldalso be used for the selective isolation of phosphoproteins from a highly complexmixture.High kinase concentrations, short reactions times that approximate initial rateconditions, and high specific radioactivity all increase sensitivity by increasingradioactive incorporation into any give substrate.
Importantly, these assays werenot designed to identify solely physiological substrates. While the proteins phosphorylated should reflect the best targets for phosphorylation among the proteinspresented to the kinase, this methodology, without further studies to present invivo validation, cannot attribute physiological relevance to any particular phosphorylation in the assay. Local concentrations of kinase and substrate in a cell areregulated and vary widely from the total cellular concentrations [58], further em-26726815 Investigations in Smooth Muscle Cell PhysiologyFig. 15.5 Protein kinase substrate screeningstrategy. Proteomes (1) are obtained from a cellline, organ, or animal source and fractionated(2) with an appropriate chromatography methodi.e.
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