Kinase cascades regulating entry into apoptosis.

All cells are constantly exposed to conflicting environment cues that signal cell survival or cell death. Survival signals are delivered by autocrine or paracrine factors that actively suppress a default death pathway. In addition to survival factor withdrawal, cell death can be triggered by environmental stresses such as heat, UV light, and hyperosmolarity or by dedicated death receptors (e.g., FAS/APO-1 and tumor necrosis factor [TNF] receptors) that are counterparts of growth factor or survival receptors at the cell surface. One of the ways that cells integrate conflicting exogenous stimuli is by phosphorylation (or dephosphorylation) of cellular constituents by interacting cascades of serine/threonine and tyrosine protein kinases (and phosphatases). Survival factors (e.g., growth factors and mitogens) activate receptor tyrosine kinases and selected mitogen-activated, cyclin-dependent, lipid-activated, nucleic acid-dependent, and cyclic AMP-dependent kinases to promote cell survival and proliferation, whereas environmental stress (or death factors such as FAS/APO-1 ligand and TNF-alpha) activates different members of these kinase families to inhibit cell growth and, under some circumstances, promote apoptotic cell death. Because individual kinase cascades can interact with one another, they are able to integrate conflicting exogenous stimuli and provide a link between cell surface receptors and the biochemical pathways leading to cell proliferation or cell death.     

MAP kinase phosphatases

Mitogen-activated protein MAP kinases are key signal-transducing enzymes that are activated by a wide range of extracellular stimuli. They are responsible for the induction of a number of cellular responses, such as changes in gene expression, proliferation, differentiation, cell cycle arrest and apoptosis. Although regulation of MAP kinases by a phosphorylation cascade has long been recognized as significant, their inactivation through the action of specific phosphatases has been less studied. An emerging family of structurally distinct dual-specificity serine, threonine and tyrosine phosphatases that act on MAP kinases consists of ten members in mammals, and members have been found in animals, plants and yeast. Three subgroups have been identified that differ in exon structure, sequence and substrate specificity.     

PTPs versus PTKs: the redox side of the coin

The phosphorylation of tyrosine, and to a lesser extent threonine and serine, plays a key role in the regulation of signal transduction during a plethora of eukaryotic cell functions, including cell activation, cell-cycle progression, cytoskeletal rearrangement and cell movement, differentiation, apoptosis and metabolic homeostasis. In vivo, tyrosine phosphorylation is reversible and dynamic; the phosphorylation states are governed by the opposing activities of protein tyrosine kinases (PTKs)2 and protein tyrosine phosphatases (PTPs). Reactive oxygen species (ROS) act as cellular messengers in cellular processes such as mitogenic signal transduction, gene expression, regulation of cell proliferation, senescence and apoptosis. Redox regulated proteins include PTPs and PTKs, although with opposite regulation of enzymatic activity. Transient oxidation of thiols in PTPs leads to their inactivation by the formation of either an intramolecular S-S bridge or a sulfenyl-amide bond. Conversely, oxidation of PTKs leads to their activation, either by direct SH modification or, indirectly, by concomitant inhibition of PTPs that guides to sustained activation of PTKs. This review focuses on the redox regulation of both PTPs and PTKs and the interplay of their specular regulation.     

Receptor type protein tyrosine phosphatases (RPTPs) – roles in signal transduction and human disease

Protein tyrosine phosphorylation is a fundamental regulatory mechanism controlling cell proliferation, differentiation, communication, and adhesion. Disruption of this key regulatory mechanism contributes to a variety of human diseases including cancer, diabetes, and auto-immune diseases. Net protein tyrosine phosphorylation is determined by the dynamic balance of the activity of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). Mammals express many distinct PTKs and PTPs. Both of these families can be sub-divided into non-receptor and receptor subtypes. Receptor protein tyrosine kinases (RPTKs) comprise a large family of cell surface proteins that initiate intracellular tyrosine phosphorylation-dependent signal transduction in response to binding of extracellular ligands, such as growth factors and cytokines. Receptor-type protein tyrosine phosphatases (RPTPs) are enzymatic and functional counterparts of RPTKs. RPTPs are a family of integral cell surface proteins that possess intracellular PTP activity, and extracellular domains that have sequence homology to cell adhesion molecules. In comparison to extensively studied RPTKs, much less is known about RPTPs, especially regarding their substrate specificities, regulatory mechanisms, biological functions, and their roles in human diseases. Based on the structure of their extracellular domains, the RPTP family can be grouped into eight sub-families. This article will review one representative member from each RPTP sub-family.     

ReviewPTPs versus PTKs: The redox side of the coin

The phosphorylation of tyrosine, and to a lesser extent threonine and serine, plays a key role in the regulation of signal transduction during a plethora of eukaryotic cell functions, including cell activation, cell-cycle progression, cytoskeletal rearrangement and cell movement, differentiation, apoptosis and metabolic homeostasis. In vivo, tyrosine phosphorylation is reversible and dynamic; the phosphorylation states are governed by the opposing activities of protein tyrosine kinases (PTKs)² and protein tyrosine phosphatases (PTPs). Reactive oxygen species (ROS) act as cellular messengers in cellular processes such as mitogenic signal transduction, gene expression, regulation of cell proliferation, senescence and apoptosis. Redox regulated proteins include PTPs and PTKs, although with opposite regulation of enzymatic activity. Transient oxidation of thiols in PTPs leads to their inactivation by the formation of either an intramolecular S–S bridge or a sulfenyl–amide bond. Conversely, oxidation of PTKs leads to their activation, either by direct SH modification or, indirectly, by concomitant inhibition of PTPs that guides to sustained activation of PTKs. This review focuses on the redox regulation of both PTPs and PTKs and the interplay of their specular regulation.     

On the cross-regulation of protein tyrosine phosphatases and receptor tyrosine kinases in intracellular signaling

Intracellular signaling proteins are very often regulated by site-specific phosphorylation. For example, growth factor receptors in eukaryotic cells contain intrinsic tyrosine kinase activity and use inter- and intra-molecular interactions to recruit and orient potential protein substrates for phosphorylation. Equally important in determining the magnitude and kinetics of such a response is protein dephosphorylation, catalysed by phosphatase enzymes. A growing body of evidence indicates that certain protein tyrosine phosphatases (PTPs), like tyrosine kinases, are affected by intermolecular interactions that alter the specific activity or localization of their catalytic domains. Using a detailed kinetic modeling framework, we theoretically explore the regulation of PTPs through their association with receptor tyrosine kinases, as noted for the Src homology 2-domain-containing PTPs, SHP-1 and -2. Receptor-PTP binding, in turn, is expected to influence the phosphorylation pattern of those receptors and proteins they associate with, and we show how PTPs might serve to co- or counter-regulate parallel pathways in a signaling network.     

Reversible tyrosine phosphorylation and cell cycle control

In eukaryotic organisms, reversible tyrosine phosphorylation has been established as an important element in the regulation of cell growth and more recently as an essential element in the regulation of the cell division cycle. The activity of p34^cdc2, a protein kinase whose activity is required for the entry of cells into mitosis, is tightly controlled by reversible phosphorylation at tyrosine 15. A complex network of interacting protein kinases and protein phosphatases regulate the state of p34^cdc2 tyrosine phosphorylation and therefore the entry of cells into mitosis. In the fission yeast Schizosaccharomyces pombe, genes encoding several of these protein kinases and protein phosphatases have been obtained through genetic approaches. In this review, we will focus on the protein kinases encoded by wee1⁺, mik1⁺ and cdr1⁺/nim1⁺ and the protein phosphatases encoded by cdc25⁺ and pyp1⁺, pyp2⁺ and pyp3⁺. Homologs of many of these regulators have been identified and characterized in higher eukaryotes underscoring the importance of reversible tyrosine phosphorylation as a universal mechanism for the regulation of the cell division cycle.     

The sulfhydryl reagent N-ethylmaleimide induces hyperphosphorylation on tyrosine residues in the Jurkat T-cell line.

Tyrosine protein kinases have been shown to be functionally involved in regulation of cellular signalling, proliferation and transformation. The activity of tyrosine protein kinases is counterbalanced by phospho tyrosine phosphatases that maintain constitutively low levels of protein phosphotyrosine in most cells. In this study the effect of N-ethylmaleimide on the protein tyrosine phosphorylation was tested in Jurkat T-cells. Treatment of intact cells for 5-10 mins with 50-100 microM N-ethylmaleimide resulted in a dramatic increase in phosphorylation on tyrosine residues. Phosphoaminoacid analysis revealed an up to ten-fold increase in the content of phosphotyrosine. N-ethylmaleimide blocked the phospho tyrosine phosphatases activity of immunoprecipitated CD45 while in a kinase assay N-ethylmaleimide did not affect the 32P-gamma-ATP phosphorylation of substrates. The N-ethylmaleimide-induced hyperphosphorylation was reversed by treatment with 2 mM dithiotreitol. It is concluded that N-ethylmaleimide offers a novel useful tool for identification of substrates for tyrosine protein kinases and for studies on phosphotyrosine-dependent protein interactions.     

Protein tyrosine kinases and phosphatases in the nervous system.

Evidence in the past year has provided support for a prominent role of tyrosine phosphorylation in the regulation of neuronal function. The discovery that many novel forms of protein tyrosine kinases and phosphatases are expressed in the brain has revealed that the regulation of tyrosine phosphorylation is highly complex. The recent identification of substrate proteins in the brain for the protein tyrosine kinases and phosphatases has begun to clarify the functional role of tyrosine phosphorylation in the development and modulation of the nervous system.     

蛋白酪氨酸磷酸酶-PEST在生理调节及相关疾病中的作用

蛋白质分子中酪氨酸残基可逆性的磷酸化是细胞内信号分子传导的基本方式。两类作用相反的酶参与磷酸化的调节:蛋白酪氨酸激酶(protein tyrosinekinase,PTK)和蛋白酪氨酸磷酸酶(protein tyrosine phosphatase,PTP)。含脯氨酸-谷氨酸-丝氨酸-苏氨酸(P-E-S-T)结构域的蛋白酪氨酸磷酸酶(PTP-PEST)属于非受体型酪氨酸磷酸酶类,其本身能与多种蛋白质相互作用,并在细胞迁移、免疫细胞活化和胚胎发育等生理过程中发挥重要作用。本文对PTP-PEST的结构特点、生理功效、介导的信号传导途径和近年来PTP-PEST在疾病中的作用作一综述。     

Regulation of cell adhesion by protein-tyrosine phosphatases. I. Cell-matrix adhesion

Protein-tyrosine phosphatases are key regulators of protein tyrosine phosphorylation. More than merely terminating the pathways initiated by protein-tyrosine kinases, phosphatases are active participants in many signaling pathways. Signals involving tyrosine phosphorylation are frequently generated in response to cell-matrix adhesion. In addition, high levels of protein tyrosine phosphorylation generally promote disassembly or turnover of adhesions. In this brief review, we will discuss the role of protein-tyrosine phosphatases in cell-matrix adhesions.     

Activation of ErbB2 by 2-methyl-1,4-naphthoquinone (menadione) in human keratinocytes: role of EGFR and protein tyrosine phosphatases

Activation of ErbB receptor tyrosine kinases triggers multiple signaling pathways that regulate cellular proliferation and survival. We here demonstrate that ErbB2 is activated via the epidermal growth factor receptor (EGFR) upon exposure of cultured human keratinocytes to 2-methyl-1,4-naphthoquinone (menadione). Both ErbB2 and EGFR are shown to be regulated by protein tyrosine phosphatases that are inhibited by menadione, giving rise to the hypothesis that phosphatase inhibition by menadione may result in a net activation of EGFR and an enhanced ErbB2 phosphorylation. Isolated PTP-1B, a protein tyrosine phosphatase known to be associated with ErbB receptors, is demonstrated to be inhibited by menadione.     

RAFTK/Pyk2-mediated cellular signalling

Intracellular signal transduction following extracellular ligation by a wide variety of surface molecules involves the activation and tyrosine phosphorylation of protein tyrosine kinases (PTKs). Tyrosine phosphorylation, controlled by the coordinated actions of protein tyrosine phosphatases (PTPs) and tyrosine kinases, is a critical regulatory mechanism for various physiological processes, including cell growth, differentiation, metabolism, cell cycle regulation and cytoskeleton function. The focal adhesion PTK family consists of the focal adhesion kinase (FAK) and the RAFTK/Pyk2 kinase (also known as CAK-beta and CADTK). RAFTK/Pyk2 can be activated by a variety of extracellular signals that elevate intracellular calcium concentration, and by stress signals. RAFTK/Pyk2 is expressed mainly in the central nervous system and in cells derived from hematopoietic lineages, while FAK is widely expressed in various tissues and links transmembrane integrin receptors to intracellular pathways. This review describes the role of RAFTK/Pyk2 in various signalling cascades and details the differential signalling by FAK and RAFTK/Pyk2.     

Redox signalling in anchorage-dependent cell growth

Current data have provided new perspectives concerning the regulation of non-transformed cell proliferation in response to both soluble growth factors and to adhesive cues. Non-transformed cells are anchorage dependent for the execution of the mitotic program and cannot avoid the concomitant signals starting from mitogenic molecules, as growth factors, and adhesive agents belonging to extracellular matrix. Reactive oxygen species play a key role during both growth factor and integrin receptor signalling and these second messengers are recognised to have a synergistic function for anchorage-dependent growth signalling. Redox regulated proteins include protein tyrosine phosphatases and protein tyrosine kinases, although with opposite regulation of their enzymatic activity, and cytoskeletal proteins as beta-actin. In this review we support a role of ROS as key second messengers granting a proper executed mitosis for anchorage-dependent cells, through redox regulation of several downstream targets. Deregulation of these redox pathways may help to guide transformed cells to elude the native apoptotic response to abolishment of signals started by cell/ECM contact, sustaining ectopic anchorage-independent cancer cell growth.     

Regulation of mitochondrial oxidative phosphorylation through cell signaling

The mitochondrial oxidative phosphorylation (OxPhos) system plays a key role in energy production, the generation of free radicals, and apoptosis. A lack of cellular energy, excessive radical production, and dysregulated apoptosis are found alone or in combination in most human diseases, including neurodegenerative diseases, stroke, cardiovascular disorders, ischemia/reperfusion, and cancer. In the context of its relevance to human disease, this article reviews current knowledge about the regulation of OxPhos with a focus on cell signaling and discusses identified phosphorylation sites with the aid of crystal structures of OxPhos complexes. Several recent studies have shown that all OxPhos components can be phosphorylated; even the small electron carrier cytochrome c is tyrosine phosphorylated in vivo. We propose that in higher organisms, in contrast to bacteria, cell signaling pathways are the main regulator of energy production, triggered for example by hormones. Pathways that have been identified to act on OxPhos include protein kinases A and C and growth factor activated receptor tyrosine kinase signaling. Present knowledge about kinases and phosphatases that execute signals at the level of the mitochondrial OxPhos system, and newly emerging concepts, such as the translocation of kinases to the mitochondria upon stimulation of a signaling pathway, are discussed.     

Functional regulation of gamma-aminobutyric acid transporters by direct tyrosine phosphorylation

Tyrosine phosphorylation regulates multiple cell signaling pathways and functionally modulates a number of ion channels and receptors. Neurotransmitter transporters, which act to clear transmitter from the synaptic cleft, are regulated by multiple second messenger pathways that exert their effects, at least in part, by causing a redistribution of the transporter protein to or from the cell surface. To test the hypothesis that tyrosine phosphorylation affects transporter function and to determine its mechanism of action, we examined the regulation of the rat brain gamma-aminobutyric acid (GABA) transporter GAT1 expressed endogenously in hippocampal neurons and expressed heterologously in Chinese hamster ovary cells. Inhibitors of tyrosine kinases decreased GABA uptake; inhibitors of tyrosine phosphatases increased GABA uptake. The decrease in uptake seen with tyrosine kinase inhibitors was correlated with a decrease in tyrosine phosphorylation of GAT1 and resulted in a redistribution of the transporter from the cell surface to intracellular locations. A mutant GAT1 construct that was refractory to tyrosine phosphorylation could not be regulated by tyrosine kinase inhibitors. Activators of protein kinase C, which are known to cause a redistribution of GAT1 from the cell surface, were additive to the effects of tyrosine kinase inhibitors suggesting that multiple signaling pathways control transporter redistribution. Application of brain-derived neurotrophic factor, which activates receptor tyrosine kinases, up-regulated GAT1 function suggesting one potential trigger for the cellular regulation of GAT1 signaling by tyrosine phosphorylation. These data support the hypothesis that transporter expression and function is controlled by the interplay of multiple cell signaling cascades.     

Role of the SHP-2 tyrosine phosphatase in cytokine-induced signaling and cellular response

Cytokines and growth factors are important extracellular regulatory proteins. They exert their biological functions through binding to their cognate receptors on the cell surface and triggering intracellular signaling cascades. However, the intracellular signaling mechanisms of cytokines and growth factors are not well understood. Accumulating evidence has shown that protein phosphorylation and dephosphorylation carried out by protein kinases and protein phosphatases are fundamental biochemical events in intracellular signal transduction. SHP-2, a Src homology (SH) 2 domain-containing protein tyrosine phosphatase (PTP), is widely involved in a variety of signaling pathways triggered by cytokines and growth factors, including the MAP kinase, Jak-Stat, and PI3 kinase pathways. Recent studies have clearly demonstrated that this phosphatase plays an important role in transducing signals relayed from the cell surface to the nucleus, and is a critical intracellular regulator in cytokine and growth factor-induced cell survival, proliferation, and differentiation.