Organizing signal transduction through A-kinase anchoring proteins (AKAPs)

A fundamental role for protein-protein interactions in the organization of signal transduction pathways is evident. Anchoring, scaffolding and adapter proteins function to enhance the precision and directionality of these signaling events by bringing enzymes together. The cAMP signaling pathway is organized by A-kinase anchoring proteins. This family of proteins assembles enzyme complexes containing the cAMP-dependent protein kinase, phosphoprotein phosphatases, phosphodiesterases and other signaling effectors to optimize cellular responses to cAMP and other second messengers. Selected A-kinase anchoring protein signaling complexes are highlighted in this minireview.     

PP2A targeting by viral proteins: a widespread biological strategy from DNA/RNA tumor viruses to HIV-1

Protein phosphatase 2A (PP2A) is a large family of holoenzymes that comprises 1% of total cellular proteins and accounts for the majority of Ser/Thr phosphatase activity in eukaryotic cells. Although initially viewed as constitutive housekeeping enzymes, it is now well established that PP2A proteins represent a family of highly and sophistically regulated phosphatases. The past decade, multiple complementary studies have improved our knowledge about structural and functional regulation of PP2A holoenzymes. In this regard, after summarizing major cellular regulation, this review will mainly focus on discussing a particulate biological strategy, used by various viruses, which is based on the targeting of PP2A enzymes by viral proteins in order to specifically deregulate, for their own benefit, cellular pathways of their hosts. The impact of such PP2A targeting for research in human diseases, and in further therapeutic developments, is also discussed.     

Protein phosphatase 2C (PP2C) function in higher plants

In the past few years, molecular cloning studies have revealed the primary structure of plant protein serine/threonine phosphatases. Two structurally distinct families, the PP1/PP2A family and the PP2C family, are present in plants as well as in animals. This review will focus on the plant PP2C family of protein phosphatases. Biochemical and molecular genetic studies in Arabidopsis have identified PP2C enzymes as key players in plant signal transduction processes. For instance, the ABI1/ABI2 PP2Cs are central components in abscisic acid (ABA) signal transduction. Arabidopsis mutants containing a single amino acid exchange in ABI1 or ABI2 show a reduced response to ABA. Another member of the PP2C family, kinase-associated protein phosphatase (KAPP), appears to be an important element in some receptor-like kinase (RLK) signalling pathways. Finally, an alfalfa PP2C acts as a negative regulator of a plant mitogen-activated protein kinase (MAPK) pathway. Thus, the plant PP2Cs function as regulators of various signal transduction pathways.     

Signaling complexes: junctions on the intracellular information super highway.

An enigmatic yet fundamental principle of signal transduction is that parallel signaling pathways assembled from a common repertoire of enzymes are able to propagate diverse physiological responses. A key feature of such a mechanism is that separate signaling pathways are organized into localized transduction units, each tailored to respond optimally to a particular signal. Protein-protein interactions maintained by anchoring, adapter and scaffolding proteins provide the molecular glue that holds these signal transduction units together. A major objective of the signaling community is to ascertain how signals flow through compartmentalized transduction units that contain transmembrane receptors, protein kinases, phosphatases and their substrates.     

Phosphatases in apoptosis: to be or not to be,PP2A is in the heart of the question

Protein phosphatase type 2A (PP2A) is a major Ser/Thr phosphatase involved in several cellular signal transduction pathways. In this review, we will focus on recent progress concerning the role of PP2A in apoptotic signalling. Since PP2A activates pro-apoptotic and inhibits anti-apoptotic proteins of the Bcl-2 family, we conclude that PP2A has a positive regulatory function in apoptosis. However, in Drosophila, a specific subset of the PP2A holoenzyme family, containing B'/PR61 as third regulatory subunit, is inhibitory for apoptosis, suggesting different regulatory mechanisms and substrates in different species. Moreover, PP2A acts not only upstream as a regulator of the apoptotic signal transduction pathway but also downstream as a substrate of effector caspases. Hence, PP2A is involved in the regulation as well as in the cellular response of apoptosis. Probably, various PP2A holoenzymes with distinct regulatory subunits specifically target different apoptotic substrates. This could explain the implication of PP2A at several levels of the apoptotic signal transduction pathway. Finally, some viral proteins such as adenovirus E4orf4 and simian virus small t target PP2A to alter its activity, resulting in induction of apoptosis as a regulatory mechanism to enhance virus spread.     

WD40 repeat proteins striatin and S/G(2) nuclear autoantigen are members of a novel family of calmodulin-binding proteins that associate with protein phosphatase 2A

Protein phosphatase 2A (PP2A) is a multifunctional serine/threonine phosphatase that is critical to many cellular processes including development, neuronal signaling, cell cycle regulation, and viral transformation. PP2A has been implicated in Ca(2+)-dependent signaling pathways, but how PP2A is targeted to these pathways is not understood. We have identified two calmodulin (CaM)-binding proteins that form stable complexes with the PP2A A/C heterodimer and may represent a novel family of PP2A B-type subunits. These two proteins, striatin and S/G(2) nuclear autoantigen (SG2NA), are highly related WD40 repeat proteins of previously unknown function and distinct subcellular localizations. Striatin has been reported to associate with the post-synaptic densities of neurons, whereas SG2NA has been reported to be a nuclear protein expressed primarily during the S and G(2) phases of the cell cycle. We show that SG2NA, like striatin, binds to CaM in a Ca(2+)-dependent manner. In addition to CaM and PP2A, several unidentified proteins stably associate with the striatin-PP2A and SG2NA-PP2A complexes. Thus, one mechanism of targeting and organizing PP2A with components of Ca(2+)-dependent signaling pathways may be through the molecular scaffolding proteins striatin and SG2NA.     

Identification of proteins interacting with the catalytic subunit of PP2A by proteomics

The protein phosphatase 2A (PP2A) is a serine/threonine phosphatase involved in the regulation of multiple signaling pathways including the Wnt/beta-catenin and the ERK pathways. To understand the complex signaling networking associated with PP2A, we searched proteins interacting with the catalytic subunit of protein phosphatase 2A (PP2Ac) by a pull-down analysis followed by 2-D gel electrophoresis and proteomic analyses. The probability of identification of the proteins interacting with PP2Ac was increased by searching proteins differently interacting with PP2Ac according to stimulation of Wnt3a, which regulates both the Wnt/beta-catenin and the ERK pathways. Around 100 proteins, pulled-down by His-tagged PP2Ac, were identified in 2-D gels stained with CBB. By MALDI-TOF-MS analyses of 45 protein spots, we identified several proteins that were previously known to interact with PP2A, such as Axin and CaMK IV. In addition, we also identified many proteins that potentially interact with PP2Ac. The interactions of several candidate proteins, such as tuberous sclerosis complex 2, RhoB, R-Ras, and Nm23H2, with PP2Ac, were confirmed by in vitro binding analyses and/or coimmunoprecipitation experiments.     

Targeting of PP2A enzymes by viral proteins and cancer signalling

Protein phosphatase 2A (PP2A) is a large family of holoenzymes that comprises 1% of total cellular proteins and accounts for the majority of Ser/Thr phosphatase activity in eukaryotic cells. PP2A proteins are made of a core dimer, composed of a catalytic (C) subunit and a structural (A) subunit, in association with a third variable -regulatory (B) subunit. Although initially considered as a constitutive housekeeping enzyme, PP2A is indeed highly regulated by post-translational modifications of its catalytic subunit or by the identity of a regulatory type B subunit, which determines substrate specificity, subcellular localization and enzymatic activity of a defined holoenzyme. During the two last decades, multiple studies of structural and functional regulation of PP2A holoenzymes by viral proteins led to the identification of critical pathways for both viral biology and tumorigenesis. To date a dozen of different viruses (ADN/ARN or retrovirus) have been identified that encode viral proteins associated to PP2A. In this review, we analyze a biological strategy, used by various viruses based on the targeting of PP2A enzymes by viral proteins, in order to specifically deregulate cellular pathways of their hosts. The impact of such PP2A targeting for biomedical search, and in further therapeutic developments against cancer, will also be discussed.     

Small tumor antigen of polyomaviruses: role in viral life cycle and cell transformation

The regulatory proteins of polyomaviruses, including small and large T antigens, play important roles, not only in the viral life cycle but also in virus-induced cell transformation. Unlike many other tumor viruses, the transforming proteins of polyomaviruses have no cellular homologs but rather exert their effects mostly by interacting with cellular proteins that control fundamental processes in the regulation of cell proliferation and the cell cycle. Thus, they have proven to be valuable tools to identify specific signaling pathways involved in tumor progression. Elucidation of these pathways using polyomavirus transforming proteins as tools is critically important in understanding fundamental regulatory mechanisms and hence to develop effective therapeutic strategies against cancer. In this short review, we will focus on the structural and functional features of one polyomavirus transforming protein, that is, the small t-antigen of the human neurotropic JC virus (JCV) and the simian virus, SV40.     

The role of A-kinase anchoring proteins in cAMP-mediated signal transduction pathways

Compartmentalization of signal transduction enzymes is an important mechanism of cellular signaling specificity. This occurs through the interaction of enzymes with scaffolding or anchoring proteins. To date, one of the best-studied examples of kinase anchoring is the targeting of protein kinase A to cellular locations through its association with A-kinase anchoring proteins (AKAPs). AKAPs mediate a high-affinity interaction with the type II regulatory subunit of protein kinase A for the purpose of localizing the kinase to pools of cyclic adenosine monophosphate and within proximity of preferred substrates. Furthermore, AKAPs can organize entire signaling complexes made up of kinases, phosphatases, signaling enzymes, and additional regulatory proteins.     

Phosphoproteins involved in the signal transduction of cryptogein, an elicitor of defense reactions in tobacco

We previously reported that the signal transduction of cryptogein, an elicitor of defense reactions in Nicotiana tabacum cells, involves upstream protein phosphorylation. In the present study, induction of these early physiological events was further investigated with inhibitors of protein phosphatase (PP), okadaic acid, and calyculin A. Calyculin A mimicked the effects of cryptogein, inducing an influx of calcium, an extracellular alkalinization, and the production of active oxygen species (AOS), suggesting that during cryptogein signal transduction the balance between specific protein kinase (PK) and PP activities was modified. To identify the phosphorylated proteins that could be involved early in the elicitor signaling pathway, we analyzed by 2-D electrophoresis the in vivo phosphorylation status of proteins after cryptogein, staurosporine, and calyculin A treatments of tobacco cells (5 min). Of about 100 phospho-labeled polypeptides, 19 showed increased 32P incorporation after 5 min of cryptogein treatment. Phosphorylation of 12 of the 19 polypeptides depended upon calcium influx. Staurosporine inhibited the phosphorylations induced by cryptogein whereas calyculin A activated the phosphorylation of 18 of these polypeptides. This study highlighted the role of PKs and/or constitutive active PPs whose activation and inhibition, respectively, resulted in an increased phosphorylation of proteins that may be involved in cryptogein signal transduction. Identification of the phosphoproteins is in progress and will increase our knowledge of signal transduction pathways implicated in plant defense responses.     

Metal dependent protein phosphatase PPM family in cardiac health and diseases

Protein phosphorylation and dephosphorylation is central to signal transduction in nearly every aspect of cellular function, including cardiovascular regulation and diseases. While protein kinases are often regarded as the molecular drivers in cellular signaling with high specificity and tight regulation, dephosphorylation mediated by protein phosphatases is also gaining increasing appreciation as an important part of the signal transduction network essential for the robustness, specificity and homeostasis of cell signaling. Metal dependent protein phosphatases (PPM, also known as protein phosphatases type 2C, PP2C) belong to a highly conserved family of protein phosphatases with unique biochemical and molecular features. Accumulating evidence also indicates important and specific functions of individual PPM isoform in signaling and cellular processes, including proliferation, senescence, apoptosis and metabolism. At the physiological level, abnormal PPM expression and activity have been implicated in major human diseases, including cancer, neurological and cardiovascular disorders. Finally, inhibitors for some of the PPM members have been developed as a potential therapeutic strategy for human diseases. In this review, we will focus on the background information about the biochemical and molecular features of major PPM family members, with emphasis on their demonstrated or potential roles in cardiac pathophysiology. The current challenge and potential directions for future investigations will also be highlighted.     

S100 proteins modulate protein phosphatase 5 function: a link between CA2+ signal transduction and protein dephosphorylation

PP5 is a unique member of serine/threonine phosphatases comprising a regulatory tetratricopeptide repeat (TPR) domain and functions in signaling pathways that control many cellular responses. We reported previously that Ca(2+)/S100 proteins directly associate with several TPR-containing proteins and lead to dissociate the interactions of TPR proteins with their client proteins. Here, we identified protein phosphatase 5 (PP5) as a novel target of S100 proteins. In vitro binding studies demonstrated that S100A1, S100A2, S100A6, and S100B proteins specifically interact with PP5-TPR and inhibited the PP5-Hsp90 interaction. In addition, the S100 proteins activate PP5 by using a synthetic phosphopeptide and a physiological protein substrate, Tau. Overexpression of S100A1 in COS-7 cells induced dephosphorylation of Tau. However, S100A1 and permanently active S100P inhibited the apoptosis signal-regulating kinase 1 (ASK1) and PP5 interaction, resulting the inhibition of dephosphorylation of phospho-ASK1 by PP5. The association of the S100 proteins with PP5 provides a Ca(2+)-dependent regulatory mechanism for the phosphorylation status of intracellular proteins through the regulation of PP5 enzymatic activity or PP5-client protein interaction.     

S-glutathionylation in protein redox regulation

Protein S-glutathionylation, the reversible formation of mixed disulfides between glutathione and low-pKa cysteinyl residues, not only is a cellular response to mild oxidative/nitrosative stress, but also occurs under basal (physiological) conditions. S-glutathionylation has now emerged as a potential mechanism for dynamic, posttranslational regulation of a variety of regulatory, structural, and metabolic proteins. Moreover, substantial recent studies have implicated S-glutathionylation in the regulation of signaling and metabolic pathways in intact cellular systems. The growing list of S-glutathionylated proteins, in both animal and plant cells, attests to the occurrence of S-glutathionylation in cellular response pathways. The existence of antioxidant enzymes that specifically regulate S-glutathionylation would emphasize its importance in modulating protein function, suggesting that this protein modification too might have a role in cell signaling. The continued development of proteomic and analytical methods for disulfide analysis will help us better understand the full extent of the roles these modifications play in the regulation of cell function. In this review, we describe recent breakthroughs in our understanding of the potential role of protein S-glutathionylation in the redox regulation of signal transduction.     

The PP4R1 subunit of protein phosphatase PP4 targets TRAF2 and TRAF6 to mediate inhibition of NF-κB activation

TRAFs constitute a family of proteins that have been implicated in signal transduction by immunomodulatory cellular receptors and viral proteins. TRAF2 and TRAF6 have an E3-ubiquitin ligase activity, which is dependent on the integrity of their RING finger domain and it has been associated with their ability to activate the NF-κB and AP1 signaling pathways. A yeast two-hybrid screen with TRAF2 as bait, identified the regulatory subunit PP4R1 of protein phosphatase PP4 as a TRAF2-interacting protein. The interaction of TRAF2 with PP4R1 depended on the integrity of the RING finger domain of TRAF2. PP4R1 could interact also with the TRAF2-related factor TRAF6 in a RING domain-dependent manner. Exogenous expression of PP4R1 inhibited NF-κB activation by TRAF2, TRAF6, TNF and the Epstein–Barr virus oncoprotein LMP1. In addition, expression of PP4R1 downregulated IL8 induction by LMP1, whereas downregulation of PP4R1 by RNA interference enhanced the induction of IL8 by LMP1 and TNF. PP4R1 could mediate the dephosphorylation of TRAF2 Ser11, which has been previously implicated in TRAF2-mediated activation of NF-κB. Finally, PP4R1 could inhibit TRAF6 polyubiquitination, suggesting an interference with the E3 ubiquitin ligase activity of TRAF6. Taken together, our data identify a novel mechanism of NF-κB pathway inhibition which is mediated by PP4R1-dependent targeting of specific TRAF molecules.     

Global strategies to integrate the proteome and metabolome

A fundamental goal of proteomics is to assign physiological functions to all proteins encoded by eukaryotic and prokaryotic genomes. Of the many activities performed by proteins, the chemical transformations catalyzed by enzymes form the basis for most, if not all, metabolic and signaling pathways. Elucidation of these pathways and their integration into larger cellular networks require new strategies to rapidly and systematically identify physiological substrates of enzymes. Here, we review emerging technologies that aim to assign endogenous biochemical functions to enzymes by profiling the metabolome.     

Pertussis Toxin Modification of PC12 Cells Inhibits a Protein Phosphatase 2A-Like Phosphatase

Abstract: We have found that modification of rat PC12 cells with pertussis toxin resulted in an ∼50% inhibition of a protein phosphatase 2A-like phosphatase. Protein phosphatase 2A (PP2A) is a major cellular serine/threonine-specific protein phosphatase. Treatment of extracts from pertussis toxin-modified PC12 cells with either immobilized alkaline phosphatase or Ca²⁺ reversed this inhibition. Reactivation of the PP2A-like phosphatase in Ca²⁺ appears to result from the dephosphorylation of a protein by the Ca²⁺/calmodulin-dependent protein phosphatase calcineurin. The PP2A-like phosphatase in extracts from pertussis toxin-modified PC12 cells eluted from a Mono Q column at a higher ionic strength than did the PP2A-like phosphatase in extracts from control cells. After incubation in Ca²⁺, the PP2A-like phosphatase in extracts from pertussis toxin-modified cells eluted from a Mono Q column at the same ionic strength as did the PP2A-like phosphatase in extracts from control cells. These results indicate that the effect of pertussis toxin on this PP2A-like activity results from the phosphorylation of either one of the subunits of the PP2A-like phosphatase or a protein that when phosphorylated binds to and inhibits this phosphatase. Pertussis toxin modification did not result in the phosphorylation of the catalytic subunit of PP2A. Because phosphorylation regulates the activities of many enzymes and cell surface receptors, a pertussis toxin-induced decrease in PP2A activity could alter signaling pathways and other cellular processes in which G proteins are not directly involved.