INTRODUCTION

TOP ABSTRACT INTRODUCTION OVERVIEW OF CURRENT PROTEOMIC... LINEAR PARADIGM OF PKC... SCAFFOLDING PARADIGM OF SIGNAL... THE SIGNALING MODULE HYPOTHESIS... USE OF PROTEOMIC TECHNOLOGIES... REFERENCES

The signaling system in cardioprotection is a production for which the cast of characters is ever growing, but the details of the script remain largely unknown. ...

THE PRECOCITY OF PROTEOMIC TECHNOLOGIES has transformed the manner in which proteins and their contributions to cellular function are viewed. In particular, proteomic technologies have the potential to impart a significant paradigm shift with respect to the way signal transduction systems are being studied. Prompted by the rapid development of these techniques, this review is intended to present an overview of proteomics, especially with respect to its ability to profoundly influence our understanding of the role of protein kinase C- (PKC) in the signaling infrastructure underlying cardioprotection. Accordingly, the classic linear paradigm of PKC signaling in cardioprotection, as well as the recently described scaffolding paradigm of signal transduction, are discussed. Furthermore, the signaling module hypothesis of PKC in cardioprotection is introduced as a view gained with the strategies of the proteomic era.

	OVERVIEW OF CURRENT PROTEOMIC TECHNOLOGY

TOP ABSTRACT INTRODUCTION OVERVIEW OF CURRENT PROTEOMIC... LINEAR PARADIGM OF PKC... SCAFFOLDING PARADIGM OF SIGNAL... THE SIGNALING MODULE HYPOTHESIS... USE OF PROTEOMIC TECHNOLOGIES... REFERENCES

The completed preliminary human genome map represents an invaluable tool that will undoubtedly assist in all studies of biology and human diseases. Although the map is a comprehensive account that details the structure and sequence of human genes, it offers limited insight regarding the characteristics of the final effectors of the encoded information, i.e., the proteins. Such pertinent knowledge as the identity of the protein products resultant from their corresponding genes, the expression levels and biochemical properties of these proteins, and the various modifications of these proteins that occur posttranslationally cannot be inferred from the DNA sequence. One approach to understanding the physiological significance of the genome is the systematic, large-scale analysis of the corresponding proteins. The characterization of all the proteins expressed by a genome in a specific cell type has acquired the name "proteomics." The very young age of this field is evidenced by the fact that the term was first used in 1994 at the Two-Dimensional Electrophoresis Meeting in Sienna, Italy, and initially appeared in print in 1995 (55).

The proteome is inherently more complex than the genome. Whereas four nucleotides are used to encode DNA, there are 20 unmodified amino acids that constitute proteins. Moreover, a single gene's mRNA can be differentially spliced to create more than one protein, and further variation can be introduced by posttranslational modifications (such as phosphorylation and glycosylation). Unlike the genome, the features of which are conserved throughout different organs and tissues, the visage of the proteome is dependent upon the cell type and cell condition. Consideration of these factors makes tenable the notion that the proteome is a major independent plexus in the central dogma that is responsible for the dynamic regulation of cellular function.

The emergence of proteomic analysis can be dated from advances in protein separation first described in 1975. In that year, O'Farrell (34) and Klose and Spielman (25) autonomously described the use of two-dimensional gel electrophoresis to separate cellular proteins. During the first dimension, or isoelectric focusing stage, proteins were separated via an ampholyte pH gradient and allowed to migrate to their respective isoelectric points (pI) in the gel on the basis of their ionic character. The first-dimensional gel was then subjected to a second-dimensional SDS-PAGE, and the proteins were separated on the basis of mass (25, 34). O'Farrell's original two-dimensional gel technique resolved only about 1,000 proteins per gel. Since 1975, numerous advancements, including the introduction of high-sensitivity staining methods, refinements of ampholytic technology to accommodate extended pI ranges, and larger gel formats, have increased the resolution to approximately 10,000 proteins in a single gel. Contemporary two-dimensional gel electrophoretic separation (conceptually analogous to that described above) allows for the determination of protein charge, size, and relative quantity with significant reproducibility. As a complement to the analysis of these gels by Western immunoblotting, advancements in mass spectrometry that have occurred in parallel with the optimization of two-dimensional electrophoresis have markedly enhanced our ability to identify and characterize the proteins that are resolved in these gels. The advent of accurate, sensitive, and affordable matrix-assisted laser desorption ionization mass spectrometers (MALDI-MS) has made practical the use of peptide mass fingerprinting to identify proteins from two-dimensional gels. Significant barriers still exist that may limit the identification of proteins of low abundance, of high or low pI, or those that are bound to the hydrophobic region of the membrane. Despite this, the characterization of a protein can usually be achieved via the analysis of tryptic digests of the targeted protein. This is followed by peptide mass fingerprinting with automated MALDI-MS and/or via tandem mass spectrometry or postsource decay analysis to obtain primary amino acid sequence data (36, 37). The employment of tandem mass spectrometry to decode amino acid sequences also represents a powerful method to discern novel proteins. Of particular importance, a concerted effort to compile these findings into a database that will facilitate the integration of multiple proteomic investigations is incumbent with the accumulation of information pertaining to protein sequences.

Proteomic analysis, like genomic analysis, generates large data sets, and consequently relies heavily on bioinformatic technologies. Bioinformatic assessment plays at least three distinct roles in proteomics. First, computer software is utilized to faithfully recapitulate the image of two-dimensional gels, which enables the subsequent quantitative analysis of protein spots in-frame. Second, bioinformatic tools are applied to consolidate data from mass spectral analyses, which permits the comparison with existing proteomic or genomic databases. Finally, the Internet is used for data communication from multiple sites, which enhances the ability of investigators from different institutions to collectively characterize a cellular proteome. These applications of bioinformatic technologies will inevitably 1) expedite the acquisition, analysis, and communication of proteomic data from studies in all cell types; 2) transform and integrate information collected on individual subproteome levels; and 3) simultaneously impart a more refined understanding of cellular proteins and the manifestation of their biological functions.

	LINEAR PARADIGM OF PKC SIGNALING IN PRECONDITIONING

TOP ABSTRACT INTRODUCTION OVERVIEW OF CURRENT PROTEOMIC... LINEAR PARADIGM OF PKC... SCAFFOLDING PARADIGM OF SIGNAL... THE SIGNALING MODULE HYPOTHESIS... USE OF PROTEOMIC TECHNOLOGIES... REFERENCES

To date, most studies of signal transduction in preconditioning have focused on the role of individual kinases in contrast to the aforementioned proteomic approaches. Nevertheless, these studies yield important results pertaining to key signaling elements in the genesis of preconditioning (1, 2, 4, 7, 8, 17, 23, 27-30, 32, 39-46, 49, 52, 53, 57, 59), including the identification of the -isozyme of PKC as an essential protein kinase to mediate and induce cardioprotection (7, 11, 17, 28, 40-46). The PKC hypothesis of preconditioning was introduced by Downey and colleagues (59) and was tested in a rabbit model of myocardial infarction. They found that pharmacological inhibition of PKC blocked ischemic preconditioning and that activation of PKC via phorbol esters mimicked the infarct-sparing effects of ischemic preconditioning (59). However, this hypothesis was controversial because direct biochemical evidence supporting the role of PKC was absent, and because expression profiling of total PKC failed to detect a subcellular redistribution in response to ischemic preconditioning. Since then, our laboratory has reported that the rabbit myocardium expresses 11 isoforms of PKC and that ischemic preconditioning induces a selective activation of the - and -isoforms without a demonstrable perturbation of the total subcellular PKC pool (44). Subsequent studies by multiple investigators have identified that  is in fact the isoform that mediates various forms of cardioprotection, including ischemic preconditioning, nitric oxide-induced preconditioning, and other types of pharmacological preconditioning (2, 9, 17, 28, 40, 41, 46, 54). Furthermore, mounting evidence has corroborated a role for PKC as a central signaling hub during preconditioning in multiple experimental models (4, 9, 17, 28, 40, 41, 46, 54). Activation of PKC has been shown to be sufficient to induce preconditioning (4, 7, 41), whereas inhibition of this isozyme abrogates cardioprotection (11, 17, 28).

The current paradigm of PKC signaling in cardioprotection has been primarily formulated on the basis of information acquired through the pharmacological inhibition of various signaling elements. This reductionist approach has yielded data that portrays a series of linear pathways that connect PKC to various other effectors. In support of this linear paradigm of PKC signaling, activation of multiple proteins in a PKC-dependent fashion has been demonstrated in response to ischemic preconditioning, indicating that these proteins may be signaling elements in the PKC pathway. These proteins include, but are not confined to, a number of G protein-coupled receptors (e.g., adenosine receptors, adrenergic receptors, and opioid receptors), mitogen-activated protein kinases (MAPK) [e.g., extracellular signal-regulated kinases (ERK), p38, c-Jun NH₂-terminal kinases (JNK), and MAPK-activated protein kinase-2 (MAPKAPK2)], tyrosine kinases (e.g., the Src family of tyrosine kinases), ion channels [e.g., mitochondrial and sarcolemmal ATP-sensitive K (K_ATP) channels], and transcriptional factors [e.g., activator protein-1 (AP-1) and nuclear factor-B (NFB)] (2, 4, 9, 18, 27, 30, 33, 41, 42, 45, 49, 50, 54, 57). Nevertheless, the linear model of PKC signaling in preconditioning is limited because it lacks the capacity to effectively assess the subcellular infrastructure utilized by PKC to integrate and transduce the cardioprotective signal. To further our understanding of the precise manner in which multiple protein kinases may interact with each other to facilitate the transmission of signals, characterization of the signaling architecture underlying PKC-mediated cardioprotection must be undertaken.

	SCAFFOLDING PARADIGM OF SIGNAL TRANSDUCTION

TOP ABSTRACT INTRODUCTION OVERVIEW OF CURRENT PROTEOMIC... LINEAR PARADIGM OF PKC... SCAFFOLDING PARADIGM OF SIGNAL... THE SIGNALING MODULE HYPOTHESIS... USE OF PROTEOMIC TECHNOLOGIES... REFERENCES

An essential feature that is missing from the linear paradigm of signal transduction is information regarding the cellular locations of multiple signaling molecules and the coordinated actions of these elements at a specific site. An emergent paradigm addressing positional coordination deals with scaffolding proteins (5, 6, 10, 13, 19, 22, 31, 56, 58). It is generally conceived that scaffolding proteins can perform at least two distinct tasks: they can serve to bring into close apposition different kinases in a signaling cascade, and they can elicit subcellular compartmentalization (5, 6, 10, 13, 19, 22, 31, 56, 58).

It has been suggested that scaffolding proteins can serve as linkers for pathways such as MAPK cascades, which are commonly thought to proceed in a unidirectional stepwise fashion (that is, a MAPKKK is first activated, which then proceeds to activate a MAPKK, which in turn acts upon the MAPK of interest). One such scaffolding protein, c-jun NH₂-terminal kinase-interacting protein (JIP) was identified by Davis and colleagues (58) in human brain tissue and was shown to bind all three members of the JNK cascade in vitro, including mixed-lineage kinase (MLK), MAPK kinase-7 (MKK7), and JNK. The JIP group scaffolding proteins has yet to be conclusively identified in the heart, suggesting that scaffolding mechanisms other than JIP may be utilized to accommodate the JNK cascades in the myocardium. Of other interest with respect to the regulation of MAPK messaging is the finding that the ₂-adrenergic receptor associated protein, -arrestin-2, in addition to its previously reported functions, can interact directly with JNK3 (as shown by reciprocal immunoprecipitation) and enhance apoptosis signal-regulating kinase 1 (ASK1)-mediated JNK3 activation in the rat brain (31). These findings suggest that -arrestin behaves as a scaffolding protein that directly links receptor stimulation to MAPK cascade activation, indicating a manner by which members of the MAPK super family may be differentially regulated in response to a specific agonist (31).

Another scaffolding mechanism that has been described is the coordination of signal transduction via subcellular compartmentalization of signaling molecules. An example of such a mechanism is the colocalization of glutamate receptors and the membrane-associated guanylate kinases (MAGUK) (6). A-kinase anchoring proteins, or AKAPs, have recently been shown to direct the binding of these MAGUKs to the glutamate receptors in the rat brain (6). This information, when coupled with what is already known about the ability of AKAPs to bind other proteins such as PKC, PKA, and calcinuerin (56), portrays scaffolding proteins as junctions for signal coordination between receptors and signaling kinases. An investigation of PKA signaling in Swiss 3T3 fibroblasts found that WAVE-1 proteins (a member of the Wiskott-Aldrich syndrome Verprolin-homologous family of proteins) could behave as AKAPs by accommodating the localization of PKA and Abl (an Abelson tyrosine kinase, whose activity is known to be regulated by PKA) to a site of cytoskeletal remodeling (56). The functional role of scaffolding proteins to harbor signaling elements, and thereby facilitate signal transduction, is exemplified by the host of kinases that modulate IB activity and its dissociation from NFB. With the use of recombinant proteins, it has been shown that the IB kinases [IKK-, IKK-, and NFB-inducing kinase (NIK)] will colocalize only in the presence of the scaffolding protein IKK complex-associated protein (IKAP) (5). Inherent in this finding is the notion that activation of certain signaling kinases may require simultaneous association with and modification by more than one protein. These findings also support the notion that the role of a scaffolding protein can be assumed by various signaling molecules in response to a multitude of stimuli in a cell type-specific manner (31).

	THE SIGNALING MODULE HYPOTHESIS OF PKC IN CARDIOPROTECTION

TOP ABSTRACT INTRODUCTION OVERVIEW OF CURRENT PROTEOMIC... LINEAR PARADIGM OF PKC... SCAFFOLDING PARADIGM OF SIGNAL... THE SIGNALING MODULE HYPOTHESIS... USE OF PROTEOMIC TECHNOLOGIES... REFERENCES

The scaffolding paradigm of signaling significantly expands our understanding of signal transduction in that it highlights the colocalization of molecules at a specific subcellular compartment, a layer of information that has not been revealed with the linear paradigm of signaling. Nevertheless, using the linear paradigm approach, investigations concerning the role of PKC in preconditioning have thus far implicated a number of molecules as downstream effectors of the -isozyme of PKC (4, 9, 27, 30, 33, 41, 42, 45, 50, 54, 57). However, characterization of these candidate effectors was performed in isolation, and therefore insight concerning the manner in which these candidate effectors facilitate the integration and transmission of this PKC-mediated cardioprotective signal remains largely unknown. Predicated on these previous findings, our laboratory recently undertook a concerted effort to develop a holistic portrait of the moderators of PKC signaling in the murine myocardium (43). We found that PKC colocalizes with various signaling molecules at a number of subcellular locations (43). Furthermore, our data reveal a dynamic modulation of PKC-associated signaling molecules in the cardioprotected myocardium (43), suggesting a functional role of these molecules, and their association with PKC, during the genesis of cardioprotection. These data indicate that PKC may form signaling complexes to achieve the integration and transmission of a protective signal. It is highly intriguing that within these PKC complexes, we found not only other signaling molecules (e.g., the serine-threonine kinase, Akt, and the tyrosine kinase, Lck), but also a number of stress-activated proteins that have been previously implicated in preconditioning [e.g., endothelial nitric oxide synthase (eNOS) and inducible NOS (iNOS)] (43). Importantly, several studies (3, 15, 26, 35, 48) show that most of these stress-activated proteins can undergo kinase-dependent posttranslational modification. The colocalization of various kinases and stress-activated proteins provides physical evidence that these proteins may be elements of multi-tier signaling modules.

Based upon these lines of evidence, we propose "the signaling module hypothesis of PKC in cardioprotection." The foundation of the signaling module hypothesis is the concept that the signaling system in preconditioning is characterized by a high degree of nonrandom arrangement and organization, which is necessary to accommodate its complexity in structure and function. This particular signaling paradigm of PKC cardioprotection would involve the formation of PKC signaling complexes. Specifically, we propose that PKC signaling complexes are organized collections of all those proteins that directly or indirectly interact with PKC to accomplish signaling tasks within the cell. Multiple PKC signaling complexes would exist at various subcellular locations. Signal transduction within a complex would be facilitated by the assembly of signaling modules that would be composed of at least one PKC binding partner, and a number of other proteins that have physical interactions with the binding partner(s). Within the PKC signaling complex, we found a number of multipurpose stress-activated proteins and various signaling kinases that participate in numerous different cellular functions, including, but not limited to, cardioprotection (43). Therefore, we envision that within the PKC signaling complex, multiple tiers of signaling elements (for instance, PKC may represent a tier, and it may interact with proteins from other tiers, such as a tier of stress-activated proteins or a tier of signaling kinases) form a signaling module, the latter of which is defined on the basis of the type of stimulus impinging on the cell, the subcellular location in question, and the desired biochemical function. The assembly of distinct, stimulus and subcellular location-specific signaling modules provides a means by which multipurpose stress-activated proteins and signaling kinases could function in accord to dynamically regulate a variety of cellular responses. The binding partners of PKC, i.e., those proteins that maintain a direct physical interaction with the kinase, simultaneously associate with other proteins in the PKC complex and facilitate the biochemical function of these proteins that maintain indirect interactions with PKC. Under the scenario presented in the signaling module hypothesis, PKC, via its direct interactions with a limited number of binding partners, can influence a significant number of proteins in different modules within the whole PKC signaling complex.

The logic driving the "module" nomenclature is grounded by data from our laboratory (47, 50) and others (8, 20), suggesting that the simultaneous physical interaction of two or more molecules may serve as a means for signal transduction. Essential to the experimental identification of a module must be the reconciling, in isolation from other proteins, of the ability of two or more proteins to physically interact. It has been previously reported that Akt can directly phosphorylate eNOS (15). Other studies have shown that ischemia-reperfusion and ischemic preconditioning are associated with increased phosphorylation of Akt (32, 54). In addition, our laboratory (43) has found that both Akt and eNOS reside in the PKC signaling complex in the murine myocardium, representing what may be the countenance of a putative signaling module that is specifically assembled for the regulation of NO production and the enactment of a protective response to ischemic stress. Further studies detailing the specific role of Akt in this model of cardioprotection, coupled with proteomic analyses of the temporal and spatial relationship among Akt, PKC, and eNOS in the generation of NO and cardioprotection, remain to be conducted and would be necessary to label absolutely these interactions as those of a signaling module. In addition, it has been shown that Lck tyrosine kinase is a binding partner of PKC (50) and that a tyrosine phosphorylation event is a primary mechanism for the regulation of iNOS activity (35). Thus the colocalization of Lck and iNOS into the PKC complex provides the molecular basis for the constitution of a signaling module involving these three proteins (43, 50).

It is possible that distinct types of modules that carry out specific protective tasks may exist within the same complex. The constellation of proteins in any one particular module within the signaling complex is regulated on the basis of the subcellular compartment where the complex is located and on the nature of the biochemical functions that the complex is designated to carry out. We hypothesize that these signaling modules are essential functional units of the signaling pathways during ischemic, and in various forms of pharmacological, preconditioning. Therefore, efforts to characterize the specific signaling modules that are utilized by these pathways are of critical importance in the development of pharmacological agents to protect the heart. The signaling module hypothesis, as presented here, is intended to represent the next stage in the evolution of our understanding of the signaling mechanisms in cardioprotection. Nevertheless, the concept of the signaling module hypothesis highlights what may be a conserved model that is employed in various types of cellular responses where multiple signaling elements collectively act to achieve a specific biological function.

	USE OF PROTEOMIC TECHNOLOGIES TO FACILITATE AN UNDERSTANDING OF THE SIGNALING ARCHITECTURE IN PRECONDITIONING

TOP ABSTRACT INTRODUCTION OVERVIEW OF CURRENT PROTEOMIC... LINEAR PARADIGM OF PKC... SCAFFOLDING PARADIGM OF SIGNAL... THE SIGNALING MODULE HYPOTHESIS... USE OF PROTEOMIC TECHNOLOGIES... REFERENCES

The classic application of proteomic technology is to identify novel protein targets in a nonhypothesis-driven fashion, whereas recent functional proteomic analyses have been specifically employed to interrogate signal transduction pathways (12, 14, 16, 24, 38, 43, 51). Inspired by these studies in signal transduction, the proteomic analyses described herein endeavor to characterize the signaling architecture of preconditioning from the signaling module hypothesis. There are at least three fundamental lines of information that one must acquire to achieve this goal: 1) the identification of the specific components that serve as binding partners, that constitute a signaling module, and that form a signaling complex; 2) the determination of the dynamic features of these components, including level of expression, enzymatic activity, and posttranslational modifications; and 3) the definition of the specific subcellular location (e.g., sarcolemmal membrane, mitochondria, or nuclei) where the signaling complex resides and which dictates the biological function of the signaling complex. This information will not only explain how the signaling architecture is assembled, but furthermore, it will provide insight as to how a signal is being transmitted and integrated in different subcellular arenas to achieve a biological function. Through the conscription of multiple techniques, including mass spectrometric analysis, two-dimensional gel electrophoresis, affinity chromatography, and Western immunoblotting, functional proteomic analysis enables the investigator to conduct a comprehensive assessment of the signaling architecture underlying a biological phenomenon, such as the preconditioning of the heart against ischemic insult.

The identification of specific components that serve as binding partners can be achieved by a number of biochemical assays including the overlay assay (8) and the affinity pull-down assay (19, 38, 43, 47). These assays will aid in the characterization of a direct physical association between two proteins in vitro and in vivo. These methods have been utilized for the description of the receptors for activated C kinase (RACK)-PKC and PKC-Lck interactions in PKC-mediated preconditioning (8, 11, 17, 39, 50). In contrast to the determination of binding partners, the characterization of the components in a signaling module or a signaling complex will require the simultaneous display of multiple molecules, which can be achieved by either one- or two-dimensional electrophoretic analyses. Subsequently, these electrophoretically separated molecules can be visualized by staining, identified through mass spectrometric analysis, and confirmed via Western immunoblotting. With the use of this approach, we and others (19, 43) found that signaling complexes are composed of proteins from distinct signaling tiers. In the normal myocardium, PKC forms signaling complexes with at least 36 different proteins that can be organized into structural elements, signaling molecules, and stress-responsive proteins (43). Furthermore, PKC-dependent cardioprotection induced dynamic modulation of these complexes (43), suggesting a functional role for these complexes in the genesis of protection against ischemic injury (a detailed representation of the protocols used by our laboratory in these studies is seen in Fig. 1). A similar signaling paradigm was also revealed for the NMDA receptor complexes in the mouse brain, which have been shown to include over 100 receptor, adaptor, signaling, and cytoskeletal proteins (19). Several other investigations (16, 38, 47, 51) also demonstrate the power of functional proteomic analysis for the characterization of multiple proteins in parallel.

View larger version (34K): [in this window] [in a new window]   Fig. 1.   Illustration of the three-step protocol utilized for the identification of proteins in the protein kinase C- (PKC) signaling complexes. This protocol has been previously used to identify 36 proteins residing in PKC complexes (43). In step 1, cardiac tissue samples from either PKC transgenic positive (Tg+) or their transgenic negative littermates (Tg) were processed (43). To accumulate sufficient protein samples for subsequent proteomic analysis, 10 hearts of Tg+ or Tg were pooled to yield one independent sample. PKC monoclonal antibodies were then used to immunoprecipitate PKC signaling complexes (43). In step 2, putative candidate proteins that associate with PKC in the complex were identified through either one-dimensional electrophoresis coupled with Western immunoblotting, or two-dimensional electrophoresis in conjunction with matrix-assisted laser desorption ionization mass spectrometer (MALDI) analysis and confirmed with Western immunoblotting. In step 3, the colocalization of the candidate proteins (those that were identified in step 2) with the PKC complex was verified using PKC-GST affinity pull-down assays. Furthermore, the expression of these proteins in cardiac cells was confirmed using isolated mouse cardiac myocytes. This protocol can readily be extrapolated in order to characterize the signaling complexes of other proteins in any cell type.

Identification of the binding partners and other components in the signaling complex provides specific targets for the subsequent determination of the dynamic features of these proteins. These features of a signaling molecule can usually be assessed through the molecule's level of expression, enzymatic activity, and posttranslational modifications. One- and two-dimensional gel electrophoresis, coupled with immunoblotting, can be used to determine the level of expression, whereas the gel electrophoresis, in conjunction with mass spectrometry, can yield sufficient data for a definitive assessment of the posttranslational modifications of multiple proteins (see Ref. 43 and Fig. 1). Once a protein has been identified, its enzymatic activity can then be determined by standard kinase assays or protein chips that are specifically designed for the given protein and that are intended for the discernment of its enzymatic activity with multiple substrates (60). The latter method accommodates a rapid, yet comprehensive, analysis of multiple substrates en masse. The specific subcellular compartment where the complexes are located inevitably dictates the biological functions that the complex is designated to achieve. Subcellular compartments, such as nuclei, mitochondria, caveolae, and sarcolemmal membranes, can be isolated by well-established techniques (21, 41, 48). Subsequently, signaling complexes residing at these different compartments can be characterized using the methodology detailed above.

The signaling architecture in cardiac cells is highly intricate and is dynamically regulated to adapt to various cellular stresses, such as ischemic injury. We envision that the composition of the PKC signaling complex may change with respect to the subcellular location under consideration, and that this change is a direct reflection of the manner in which these distinct compartments modify and transduce intracellular signals. The fact that PKC complexes contain multiple signaling molecules and stress-responsive proteins presents the opportunity for a great level of plasticity in the formation of various signaling modules. It is conceivable that the signaling architecture may possess an intrinsic ability to organize proteins into various task-specific modules and that the same stress-activated proteins may reside in different signaling modules and perform distinct functions. Indeed, this ability to organize modules in response to distinct cellular stimuli may be the impetus for the differential responses of multipurpose stress proteins that are exhibited in a variety of adaptive biological processes.

In summary, proteomic studies are poised to enable our conception of proteins and their biological functions to transcend to a level that has not been feasible with previous approaches. Proteomic technologies not only allow for the nonbiased large-scale analysis of proteins that participate in a biological phenomenon, but also empower the investigator to conduct hypothesis-driven research and to gain definitive answers concerning the specific roles played by individual proteins of interest in the manifestation of a cellular phenotype. Most importantly, proteomic analyses present the opportunity to understand the elegant architecture of signal transduction with an unprecedented degree of sophistication. Insofar as novel players in the cardioprotection score will continue to be discerned, the implementation of proteomic technologies will allow for the much anticipated revealing of the choreography of the production. As reviewed here, these techniques have been employed to study protein-protein interactions in numerous cell types, and in particular, to characterize the PKC signaling complex in cardioprotection. The signaling module hypothesis of PKC in cardioprotection has been offered as a discussion of the insight into cellular signaling that has been garnered through the utilization of experimental approaches embraced by the proteomic era.