Phosphatase assay for multi-phosphorylated substrates using phosphatase specific-motif antibody

Protein phosphorylation plays central roles in a wide variety of signal transduction pathways and most phosphorylated proteins contain multi-phosphorylated sites. PPM1 type Ser/Thr protein phosphatase family is known to show rigid substrate specificity unlike other Ser/Thr phosphatase PPP family including PP1, PP2A and PP2B. PPM1 type phosphatases are reported to play important roles in growth regulation and in cellular stress signalling. In this study, we developed a phosphatase assay of PPM1D using phosphatase motif-specific antibody. PPM1D is a member of PPM1 type Ser/Thr phosphatase and known to dephosphorylate Ser(P)-Gln sequence. The gene amplification and overexpression of PPM1D were reported in many human cancers. We generated the monoclonal antibody specific for the Ser(P)-Gln sequence, named 3G9-H11. The specificity of this method using ELISA enables the convenient measurement of the dephosphorylation level of only PPM1D target residues of substrate peptides with multiple phosphorylated sites in the presence of multiple phosphatases. In addition, the antibody was applicable to immunoblotting assay for PPM1D function analysis. These results suggested that this method should be very useful for the PPM1D phosphatase assay, including high-throughput analysis and screening of specific inhibitors as anti-cancer drugs. The method using phosphatase motif-specific antibody can be applied to other PPM1 phosphatase family.     

The specificity of extracellular signal-regulated kinase 2 dephosphorylation by protein phosphatases

The extracellular signal-regulated protein kinase 2 (ERK2) is the founding member of a family of mitogen-activated protein kinases (MAPKs) that are central components of signal transduction pathways for cell proliferation, stress responses, and differentiation. The MAPKs are unique among the Ser/Thr protein kinases in that they require both Thr and Tyr phosphorylation for full activation. The dual phosphorylation of Thr-183 and Tyr-185 in ERK2 is catalyzed by MAPK/ERK kinase 1 (MEK1). However, the identity and relative activity of protein phosphatases that inactivate ERK2 are less well established. In this study, we performed a kinetic analysis of ERK2 dephosphorylation by protein phosphatases using a continuous spectrophotometric enzyme-coupled assay that measures the inorganic phosphate produced in the reaction. Eleven different protein phosphatases, many previously suggested to be involved in ERK2 regulation, were compared, including tyrosine-specific phosphatases (PTP1B, CD45, and HePTP), dual specificity MAPK phosphatases (VHR, MKP3, and MKP5), and Ser/Thr protein phosphatases (PP1, PP2A, PP2B, PP2C alpha, and lambda PP). The results provide biochemical evidence that protein phosphatases display exquisite specificity in their substrate recognition and implicate HePTP, MKP3, and PP2A as ERK2 phosphatases. The fact that ERK2 inactivation could be carried out by multiple specific phosphatases shows that signals can be integrated into the pathway at the phosphatase level to determine the cellular response to external stimuli. Important insights into the roles of various protein phosphatases in ERK2 kinase signaling are obtained, and further analysis of the mechanism by which different protein phosphatases recognize and inactivate MAPKs will increase our understanding of how this kinase family is regulated.     

Peptide microarrays for high-throughput studies of Ser/Thr phosphatases

Protein phosphorylation and dephosphorylation play an important role in regulation of intracellular signal transduction pathways in the biological system. A key step in the biological characterization of phosphatases and their use as drug targets is the identification of their cellular partners and suitable substrates for potential inhibitor development. Herein we describe a microarray-based protocol to map the substrate specificity of protein Ser/Thr phosphatases. This protocol uses Pro-Q dye to sensitively and quantitatively detect the amount of dephosphorylation that occurs from many putative peptide substrates in parallel, and therefore could be used to generate the so-called peptide substrate fingerprints as well as detailed kinetic information of a target phosphatase. Excluding the synthesis of the peptide substrates, the whole protocol takes a total of 11 h to complete and in future can be readily extended to the study of other classes of phosphatases, i.e., protein tyrosine phosphatases.     

Novel putative protein tyrosine phosphatases identified by the polymerase chain reaction

Protein tyrosine phosphatases (PTPases) are a family of enzymes that specifically dephosphorylate phosphotyrosyl residues in selected protein substrates. To more fully understand the regulatory role of protein tyrosine phosphorylation and dephosphorylation in cellular signal transduction, characterization of PTPases is essential. Using the polymerase chain reaction and degenerate oligonucleotide primers corresponding to conserved amino acid sequences within the catalytic domain of PTPases, we have identified 11 PTPase-related human liver cDNA sequences. Five of these have not been described previously. These results indicate that, like protein tyrosine kinases, PTPases may also comprise a gene family with a large number of members.     

Cytoplasmic protein tyrosine phosphatases, regulation and function: the roles of PTP1B and TC-PTP

PTP1B and TC-PTP are closely related protein tyrosine phosphatases, sharing 74% homology in their catalytic domain. However, their cellular localization, function, and regulation are found to be different. Their substrate specificity has implicated these enzymes in various signaling pathways, regulating metabolism, proliferation and cytokine signaling. For instance, PTP1B has been shown to regulate the activation of cytokine receptors through the dephosphorylation of specific members of the JAK family, namely JAK2 and TYK2, whereas TC-PTP is involved in the modulation of cytokine signaling via JAK1 and JAK3 molecules. Gene-targeting approaches will help us to unravel the physiological functions of these enzymes.     

Regulation of DNA fragmentation: the role of caspases and phosphorylation

DNA fragmentation is a hallmark of apoptosis that is induced by apoptotic stimuli in various cell types. Apoptotic signal pathways, which eventually cause DNA fragmentation, are largely mediated by the family of cysteinyl aspartate-specific protease caspases. Caspases mediate apoptotic signal transduction by cleavage of apoptosis-implicated proteins and the caspases themselves. In the process of caspase activation, reversible protein phosphorylation plays an important role. The activation of various proteins is regulated by phosphorylation and dephosphorylation, both upstream and downstream of caspase activation. Many kinases/phosphatases are involved in the control of cell survival and death, including the mitogen-activated protein kinase signal transduction pathways. Reversible protein phosphorylation is involved in the widespread regulation of cellular signal transduction and apoptotic processes. Therefore, phosphatase/kinase inhibitors are commonly used as apoptosis inducers/inhibitors. Whether protein phosphorylation induces apoptosis depends on many factors, such as the type of phosphorylated protein, the degree of activation and the influence of other proteins. Phosphorylation signaling pathways are intricately interrelated; it was previously shown that either induction or inhibition of phosphorylation causes cell death. Determination of the relationship between protein and phosphorylation helps to reveal how apoptosis is regulated. Here we discuss DNA fragmentation and protein phosphorylation, focusing on caspase and serine/threonine protein phosphatase activation.     

How regulators of G protein signaling achieve selective regulation

The regulators of G protein signaling (RGS) are a family of cellular proteins that play an essential regulatory role in G protein-mediated signal transduction. There are multiple RGS subfamilies consisting of over 20 different RGS proteins. They are basically the guanosine triphosphatase (GTPase)-accelerating proteins that specifically interact with G protein alpha subunits. RGS proteins display remarkable selectivity and specificity in their regulation of receptors, ion channels, and other G protein-mediated physiological events. The molecular and cellular mechanisms underlying such selectivity are complex and cooperate at many different levels. Recent research data have provided strong evidence that the spatiotemporal-specific expression of RGS proteins and their target components, as well as the specific protein-protein recognition and interaction through their characteristic structural domains and functional motifs, are determinants for RGS selectivity and specificity. Other molecular mechanisms, such as alternative splicing and scaffold proteins, also significantly contribute to RGS selectivity. To pursue a thorough understanding of the mechanisms of RGS selective regulation will be of great significance for the advancement of our knowledge of molecular and cellular signal transduction.     

Fluctuations of cellular, available zinc modulate insulin signaling via inhibition of protein tyrosine phosphatases.

Extracellular zinc ions are effectors of many signaling pathways in mammalian cells, including the insulin/IGF-1 pathway. Molecular targets of zinc are intracellular, however, because otherwise ineffective zinc concentrations alter the extent of protein phosphorylation only in the presence of the ionophore pyrithione. The tight inhibition of protein tyrosine phosphatases by zinc (nanomolar inhibition constants) is likely responsible for the known insulinomimetic effects of zinc ions, which increase net phosphorylation of the insulin/IGF-1-receptors and activate their signaling cascades. More importantly, not only do extracellular zinc ions affect signal transduction, but growth factors induce cellular zinc fluctuations that are of sufficient magnitude to inhibit protein tyrosine phosphatases. In conclusion, a pool of cellular, available zinc participates in phosphorylation/dephosphorylation cascades, suggesting the existence of a cellular signaling system based on zinc as a second messenger.     

生长分化因子15及其特异性受体GFRAL信号通路

生长分化因子 15(growth differentiation factor 15,GDF15)属于转化生长因子 β(transforming growth factor β,TGF-β)超家族的成员之一,是与转化生长因子β家族成员同源性很低的新一类二聚体多肽.GDF15最初发现于活化的巨噬细胞中,可通过2种不同的细...     

高等植物中的蛋白磷酸酶与信号传递途径

蛋白激酶与蛋白磷酸酶在细胞信号传递中起着重要作用。有关高等植物中蛋白激酶的研究工作已经较多,但关于蛋白磷酸酶的研究在以前却未受到足够的重视。本文主要介绍最近有关蛋白磷酸酶在高等植物的信号传递中有重要作用的研究工作。这些与蛋白磷酸酶有关的信号传递途径包括气孔运动调节与脱落酸的信号转导、植物对病原及逆境的响应以及植物发育的调控。这些研究工作清楚地证明,蛋白磷酸酶的功能不仅表现为蛋白激酶功能的逆向平衡机制,而且在许多信号传递过程中蛋白磷酸酶起着主导作用。     

Protein Phosphatases and Signaling Cascades in Higher Plants

Protein kinases and phosphatases play a central role in cellular signaling. Although protein kinases have been widely studied in higher plants, protein phosphatases have been largely neglected until recently. The article focuses on the most recent studies that have placed protein phosphatases in the context of several signal cascades in higher plants. These pathways include stomatal regulation and abscisic acid signal transduction, pathogen and stress responses, and developmental control. Studies clearly have demonstrated that protein phosphatases function not only to counterbalance the protein kinases but also take a leading role in many signaling processes.     

Regulation of MAP kinases by MAP kinase phosphatases

MAP kinase phosphatases (MKPs) catalyze dephosphorylation of activated MAP kinase (MAPK) molecules and deactivate them. Therefore, MKPs play an important role in determining the magnitude and duration of MAPK activities. MKPs constitute a structurally distinct family of dual-specificity phosphatases. The MKP family members share the sequence homology and the preference for MAPK molecules, but they are different in substrate specificity among MAPK molecules, tissue distribution, subcellular localization and inducibility by extracellular stimuli. Our understanding of their protein structure, substrate recognition mechanisms, and regulatory mechanisms of the enzymatic activity has greatly increased over the past few years. Furthermore, although there are a number of MKPs, that have similar substrate specificities, non-redundant roles of MKPs have begun to be identified. Here we focus on recent findings regarding regulation and function of the MKP family members as physiological regulators of MAPK signaling.     

Structural fingerprints,interactions, and signaling networks of RAS family proteins beyond RAS isoforms

Among the signaling molecules indirectly linked to many different cell surface receptors, RAS proteins essentially respond to a diverse range of extracellular cues. They control activities of multiple signaling pathways and consequently a wide array of cellular processes, including survival, growth, adhesion, migration, and differentiation. Any dysregulation of these pathway leads, thus, to cancer, developmental disorders, metabolic, and cardiovascular diseases. The biochemistry of RAS family proteins has become multifaceted since the discovery of the first members, more than 40 years ago. Substantial knowledge has been attained about molecular mechanisms underlying post-translational modification, membrane localization, regulation, and signal transduction through diverse effector molecules. However, the increasing complexity of the underlying signaling mechanisms is considerable, in part due to multiple effector pathways, crosstalks between them and eventually feedback mechanisms. Here, we take a broad view of regulatory and signaling networks of all RAS family proteins that extends beyond RAS paralogs. As described in this review, a lot is known but a lot has to be discovered yet.     

Two ancient bacterial-like PPP family phosphatases from Arabidopsis are highly conserved plant proteins that possess unique properties

Protein phosphorylation, catalyzed by the opposing actions of protein kinases and phosphatases, is a cornerstone of cellular signaling and regulation. Since their discovery, protein phosphatases have emerged as highly regulated enzymes with specificity that rivals their counteracting kinase partners. However, despite years of focused characterization in mammalian and yeast systems, many protein phosphatases in plants remain poorly or incompletely characterized. Here, we describe a bioinformatic, biochemical, and cellular examination of an ancient, Bacterial-like subclass of the phosphoprotein phosphatase (PPP) family designated the Shewanella-like protein phosphatases (SLP phosphatases). The SLP phosphatase subcluster is highly conserved in all plants, mosses, and green algae, with members also found in select fungi, protists, and bacteria. As in other plant species, the nucleus-encoded Arabidopsis (Arabidopsis thaliana) SLP phosphatases (AtSLP1 and AtSLP2) lack genetic redundancy and phylogenetically cluster into two distinct groups that maintain different subcellular localizations, with SLP1 being chloroplastic and SLP2 being cytosolic. Using heterologously expressed and purified protein, the enzymatic properties of both AtSLP1 and AtSLP2 were examined, revealing unique metal cation preferences in addition to a complete insensitivity to the classic serine/threonine PPP protein phosphatase inhibitors okadaic acid and microcystin. The unique properties and high conservation of the plant SLP phosphatases, coupled to their exclusion from animals, red algae, cyanobacteria, archaea, and most bacteria, render understanding the function(s) of this new subclass of PPP family protein phosphatases of particular interest.     

Pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP): a new player in cell signaling

Precise balance between phosphorylation, catalyzed by protein kinases, and dephosphorylation, catalyzed by protein phosphatases, is essential for cellular homeostasis. Deregulation of this balance leads to pathophysiological states that drive diseases such as cancer, heart disease, and diabetes. The recent discovery of the PHLPP (pleckstrin homology domain leucine-rich repeat protein phosphatase) family of Ser/Thr phosphatases adds a new player to the cast of phosphate-controlling enzymes in cell signaling. PHLPP isozymes catalyze the dephosphorylation of a conserved regulatory motif, the hydrophobic motif, on the AGC kinases Akt, PKC, and S6 kinase, as well as an inhibitory site on the kinase Mst1, to inhibit cellular proliferation and induce apoptosis. The frequent deletion of PHLPP in cancer, coupled with the development of prostate tumors in mice lacking PHLPP1, identifies PHLPP as a novel tumor suppressor. This minireview discusses the structure, function, and regulation of PHLPP, with particular focus on its role in disease.     

Coordinated control of endothelial nitric-oxide synthase phosphorylation by protein kinase C and the cAMP-dependent protein kinase

Endothelial nitric-oxide synthase (eNOS) is an important regulatory enzyme in the cardiovascular system catalyzing the production of NO from arginine. Multiple protein kinases including Akt/PKB, cAMP-dependent protein kinase (PKA), and the AMP-activated protein kinase (AMPK) activate eNOS by phosphorylating Ser-1177 in response to various stimuli. During VEGF signaling in endothelial cells, there is a transient increase in Ser-1177 phosphorylation coupled with a decrease in Thr-495 phosphorylation that reverses over 10 min. PKC signaling in endothelial cells inhibits eNOS activity by phosphorylating Thr-495 and dephosphorylating Ser-1177 whereas PKA signaling acts in reverse by increasing phosphorylation of Ser-1177 and dephosphorylation of Thr-495 to activate eNOS. Both phosphatases PP1 and PP2A are associated with eNOS. PP1 is responsible for dephosphorylation of Thr-495 based on its specificity for this site in both eNOS and the corresponding synthetic phosphopeptide whereas PP2A is responsible for dephosphorylation of Ser-1177. Treatment of endothelial cells with calyculin selectively blocks PKA-mediated dephosphorylation of Thr-495 whereas okadaic acid selectively blocks PKC-mediated dephosphorylation of Ser-1177. These results show that regulation of eNOS activity involves coordinated signaling through Ser-1177 and Thr-495 by multiple protein kinases and phosphatases.     

Protein-tyrosine phosphatases and the regulation of insulin action.

Protein-tyrosine phosphatases (PTPases) play an important role in the regulation of insulin action by dephosphorylating the active (autophosphorylated) form of the insulin receptor and attenuating its tyrosine kinase activity. PTPases can also modulate post-receptor signalling by catalyzing the dephosphorylation of cellular substrates of the insulin receptor kinase. Dramatic advances have recently been made in our understanding of PTPases as an extensive family of transmembrane and intracellular proteins that are involved in a number of pathways of cellular signal transduction. Identification of the PTPase(s) which act on various components of the insulin action cascade will not only enhance our understanding of insulin signalling but will also clarify the potential involvement of PTPases in the pathophysiology of insulin-resistant disease states. This brief review provides a summary of reversible tyrosine phosphorylation events in insulin action and available data on candidate PTPases in liver and skeletal muscle that may be involved in the regulation of insulin action.     

Involvement of protein tyrosine phosphatases in adipogenesis: New anti-obesity targets?

Obesity is a worldwide epidemic as well as being a major risk factor for diabetes, cardiovascular diseases and several types of cancers. Obesity is mainly due to the overgrowth of adipose tissue arising from an imbalance between energy intake and energy expenditure. Adipose tissue, primarily composed of adipocytes, plays a key role in maintaining whole body energy homeostasis. In view of the treatment of obesity and obesity-related diseases, it is critical to understand the detailed signal transduction mechanisms of adipogenic differentiation. Adipogenic differentiation is tightly regulated by many key signal cascades, including insulin signaling. These signal cascades generally transfer or amplify the signal by using serial tyrosine phosphorylations. Thus, protein tyrosine kinases and protein tyrosine phosphatases are closely related to adipogenic differentiation. Compared to protein tyrosine kinases, protein tyrosine phosphatases have received little attention in adipogenic differentiation. This review aims to highlight the involvement of protein tyrosine phosphatases in adipogenic differentiation and the possibility of protein tyrosine phosphatases as drugs to target obesity. [BMB Reports 2012; 45(12): 700-706]