The role of phosphotyrosine phosphatases in haematopoietic cell signal transduction

Phosphotyrosine phosphatases (PTPases) are the enzymes which remove phosphate groups from protein tyrosine residues. An enormous number of phosphatases have been cloned and sequenced during the past decade, many of which are expressed in haematopoietic cells. This review focuses on the biochemistry and cell biology of three phosphatases, the transmembrane CD45 and the cytosolic SH2-domain-containing PTPases SHP-1 and SHP-2, to illustrate the diverse ways in which PTPases regulate receptor signal transduction. The involvement of these and other PTPases has been demonstrated in haematopoietic cell development, apoptosis, activation and non-responsiveness. A common theme in the actions of many haematopoietic cell PTPases is the way in which they modulate the thresholds for receptor signalling, thereby regulating critical events in the positive and negative selection of lymphocytes. There is growing interest in haematopoietic PTPases and their associated regulatory proteins as targets for pharmaceutical intervention and in the involvement of these enzymes in human disease.     

Coordinated action of protein tyrosine phosphatases in insulin signal transduction.

Insulin is the principal regulatory hormone involved in the tight regulation of fuel metabolism. In response to blood glucose levels, it is secreted by the beta cells of the pancreas and exerts its effects by binding to cell surface receptors that are present on virtually all cell types and tissues. In humans, perturbations in insulin function and/or secretion lead to diabetes mellitus, a severe disorder primarily characterized by an inability to maintain blood glucose homeostasis. Furthermore, it is estimated that 90-95% of diabetic patients exhibit resistance to insulin action. Thus an understanding of insulin signal transduction and insulin resistance at the molecular level is crucial to the understanding of the pathogenesis of this disease. The insulin receptor (IR) is a transmembrane tyrosine kinase that becomes activated upon ligand binding. Consequently, the receptor and its downstream substrates become tyrosine phosphorylated. This activates a series of intracellular signaling cascades which coordinately initiate the appropriate biological response. One important mechanism by which insulin signaling is regulated involves the protein tyrosine phosphatases (PTPs), which may either act on the IR itself and/or its substrates. Two well characterized examples include leuckocyte antigen related (LAR) and protein tyrosine phosphatase-1B (PTP-1B). The present review will discuss the current knowledge of these two and other potential PTPs involved in the insulin signaling pathway.     

The role of mitochondria in the oncogenic signal transduction

Mitochondria are intracellular organelles thought to have evolved from an alphaproteobacterium engulfed by the ancestor of the eukaryotic cell, an archeon, two billion years ago. Although mitochondria are frequently recognised as the “power plant” of the cell, the function of these organelles go beyond the simple generation of ATP. In fact, mounting evidence suggests that mitochondria are involved in several cellular processes, from regulation of cell death to signal transduction. Given this important role in cell physiology, mitochondrial dysfunction has been frequently associated with human diseases including cancer. Importantly, recent evidence suggests that mitochondrial function is directly regulated by oncogenes and tumour suppressors. However, the consequences of deregulation of mitochondrial function in tumour formation are still unclear. In this review, I propose that mitochondria play a pivotal role in shaping the oncogenic signalling cascade and that mitochondrial dysfunction, in some circumstances, is a required step for cancer transformation.     

Lipid phosphate phosphatases regulate signal transduction through glycerolipids and sphingolipids

Lipid phosphate esters including lysophosphatidate (LPA), phosphatidate (PA), sphingosine 1-phosphate (S1P) and ceramide 1-phosphate (C1P) are bioactive in mammalian cells and serve as mediators of signal transduction. LPA and S1P are present in biological fluids and activate cells through stimulation of their respective G-protein-coupled receptors, LPA(1-3) and S1P(1-5). LPA stimulates fibroblast division and is important in wound repair. It is also active in maintaining the growth of ovarian cancers. S1P stimulates chemotaxis, proliferation and differentiation of vascular endothelial and smooth muscle cells and is an important participant in the angiogenic response and neovessel maturation. PA and C1P are believed to act primarily inside the cell where they facilitate vesicle transport. The lipid phosphates are substrates for a family of lipid phosphate phosphatases (LPPs) that dramatically alter the signaling balance between the phosphate esters and their dephosphorylated products. In the case of PA, S1P and C1P, the products are diacylglycerol (DAG), sphingosine and ceramide, respectively. These latter lipids are also bioactive and, thus, the LPPs change signals that the cell receives. The LPPs are integral membrane proteins that act both inside and outside the cell. The "ecto-activity" of the LPPs regulates the circulating and locally effective concentrations of LPA and S1P. Conversely, the internal activity controls the relative accumulation of PA or C1P in response to stimulation by various agonists thereby affecting cell signaling downstream of EDG and other receptors. This article will review the various LPPs and discuss how these enzymes could regulate signal transduction by lipid mediators.     

The role of insulin receptor autophosphorylation in signal transduction.

We have examined the role of autophosphorylation in insulin signal transmission by oligonucleotide directed mutagenesis of seven potential tyrosine autophosphorylation sites in the human insulin receptor. Chinese hamster ovary cells transfected with these receptors were analyzed for insulin stimulated 2-deoxyglucose uptake, thymidine incorporation, endogenous substrate phosphorylation, and in vitro kinase activity. We found that phosphorylation on tyrosine residues 953, 1316, and 1322 were not necessary for receptor-mediated signal transduction. Mutation of tyrosine 960 reduced but did not abolish the signaling capabilities of the receptor. Finally, the simultaneous mutation of tyrosine residues 1146, 1150, and 1151 (the numbering system is that of Ullrich et al. (Ullrich, A., Bell, J. R., Chen, E. Y., Herrera, R., Petruzzelli, L. M., Dull, T. J., Gray, A., Coussens, L., Liao, Y. C., Tsubokawa, M., Mason, A., Seeburg, P.H., Grunfeld, C., Rosen, O. M., and Ramachandran, J. (1985) Nature 313, 756-761) resulted in a biologically inactive receptor, suggesting that the insulin receptor can be inactivated by removal of key autophosphorylation sites.     

The role of receptor interactions in regulating ethylene signal transduction

The phytohormone ethylene is perceived in Arabidopsis by a five-member receptor family. Earlier work has demonstrated that the basic functional unit for an ethylene receptor is a disulfide-linked homodimer. We recently reported in The Journal of Biological Chemistry that the ethylene-receptor ETR1 physically associates with other ethylene receptors through higher order interactions, suggesting the existence of receptor clusters. Here we consider the implications of such clusters upon the mechanism of ethylene signal transduction. In particular, we consider how such clustering provides a cooperative mechanism, akin to what has been found for the prokaryotic chemoreceptors, by which plant sensitivity to ethylene may be increased. In addition, we consider how the dominant ethylene insensitivity conferred by some receptor mutations, such as etr1-1, may also be propagated by interactions among members of the ethylene receptor family.Key words: ethylene, receptor, ETR1, cooperativity, ArabidopsisThe plant hormone ethylene regulates growth and development, and is perceived by a five-member family of receptors (ETR1, ERS1, ETR2, ERS2 and EIN4) in Arabidopsis.¹ Genetic analysis indicates that ethylene receptors are functionally redundant and negatively regulate ethylene responses through interactions with the Raf-like kinase CTR1.^2–5 The functional unit of an ethylene receptor in a disulfide-linked homodimer, with each homodimer capable of binding one ethylene molecule.^6,7 However, several observations suggest that propagation of the ethylene signal through the receptors is likely to involve more than just ethylene-induced changes within individual receptor homodimers. First, Arabidopsis is amazingly sensitive to ethylene and can respond to ethylene concentrations as low as 0.2 nl/L,⁸ 300-fold lower than the K_d of the receptors for ethylene, which suggests that some mechanism exists for amplifying the input signal.^7,9 Second, ethylene-insensitive mutations in the binding sites of the receptors exhibit greater dominance than would be predicted solely from a lesion within one member of the receptor family.¹⁰In our paper published in The Journal of Biological Chemistry,¹¹ we demonstrate that the Arabidopsis ethylene receptor ETR1 physically associates with other ethylene receptors through higher order interactions. Such physical interactions suggest that the receptors exist in plants as clusters, and that models for cooperative signaling previously applied to the histidine-kinaselinked chemoreceptors of bacteria may also be applicable to the evolutionarily related ethylene receptors of plants. In bacteria, the highly packed chemoreceptors are found in clusters at one or both poles of the cell.^12,13 Structural studies indicate that chemoreceptors can associate to form a ‘trimer of dimers’^14,15 and also support the possibility that domain swapping may occur to produce a large interconnected array of receptors. 16 Our studies indicate that ethylene receptors can interact through their cytosolic GAF domains, identifying one possible interface through which conformational changes could be propagated in an ethylene receptor cluster.A higher-order cooperative mechanism among the ethylene receptors may explain the high sensitivity of plants to ethylene. In this model, the ethylene receptors amplify ethylene signaling by lateral signal output. Binding of ethylene to one receptor induces the conformation change of the receptor from a tense state (T) to a relaxed state (R). This conformational change is then propagated to other empty receptors in the cluster due to their physical associations with the receptor in the R state. As a result empty receptors also adopt the relaxed state (R′), resulting in amplification of the initial signal. It should be noted here that mutational evidence supports the unbound state of the receptors (T state) as being the lower energy conformation of the receptors.¹⁷ Thus, according to this model, part of the energy from ligand binding would be used to transmit conformational changes to the neighboring receptors.An alternative model that may also explain the high sensitivity of ethylene responsiveness in plants, and one that is not necessarily incompatible with the previous model, is a conjugation model.¹⁸ Here it is hypothesized that, due to the physical proximity of the ethylene receptors, that ethylene released from one receptor then binds to another receptor rather than diffusing away. Through this conjugation mechanism, one ethylene molecule could amplify its signal by converting the conformations of multiple ethylene receptors from the ethylene-unbound state (T) to the ethylene-bound state (R). This model is based on several assumptions. One assumption is that a single ethylene molecule can bind ethylene receptors in the same cluster multiple times due to the dynamic binding of ethylene and ethylene receptor. A second assumption is that, after ethylene is released from one ethylene receptor, the recovery time for that receptor to resume the T state is longer than the time required for the released ethylene to bind to and convert another receptor from the T to the R state.Models for cooperativity need to also explain the dominant ethylene insensitivity of various mutant receptors such as etr1-1, in which a missense mutation results in a receptor incapable of binding ethylene. Several studies indicate that the etr1-1 mutant receptor acts cooperatively to affect the signal output from other wild-type receptors (i.e., the presence of the etr1-1 receptor in its T state increases the likelihood of other receptors adopting the T state).^10,11 This observation can be most readily explained if the dominant ethylene-insensitive mutations result in a receptor that requires more energy to undergo the T to R transition than do the wild-type receptors. For example, the etr1-1 mutation may increase the stability of the T form (a T′ state). There is evidence to support this possibility. The etr1-1 missense mutation results in a receptor unable to chelate a copper cofactor necessary for ethylene binding,¹⁹ but the effects of this mutation on signaling are different from wild-type receptors that lack their copper cofactor. The etr1-1 mutant receptor appears locked in its T state, whereas wild-type receptors lacking the copper cofactor appear to be in the R state.²⁰ Thus etr1-1 is truly a gain-of-function mutation that alters the conformation of the receptor in ways not necessarily predicted from just the loss of the copper cofactor.In conclusion, we have attempted here to provide models that can resolve an apparent contradiction in the cooperative signaling behavior exhibited by ethylene receptors. The high sensitivity of plants to ethylene suggest cooperative changes in which an R state can be propagated within a receptor cluster, but the dominance of the ethylene ethylene-insensitive mutant etr1-1 suggests that the T state can also be propagated within a receptor cluster. It should be born in mind, however, that ethylene signaling is mediated by multiple signaling components. The ethylene receptors regulate ethylene responses through interaction with and modulation of CTR1 kinase activity. Thus, the total kinase activity of CTR1 represents the signal output from the receptors. This situation is very similar to that of the bacterial chemoreceptors, which regulate the activity of an associated histidine kinase, and, as with the chemoreceptors, the stoichiometry of CTR1 interactions with the ethylene receptors and the means by which its kinase activity is regulated are important for the elucidation of the mechanism of ethylene signal transduction.     

The role of ras proteins in insulin signal transduction.

Ras-proteins are guanine nucleotide binding proteins, which, in the GTP bound state emit a strong mitogenic signal. In the GDP bound state, the protein appears inactive. We have found that stimulation by insulin of cells expressing elevated levels of insulin receptors results in a rapid conversion of Ras-GDP into Ras-GTP. This process is part of the signalling pathway leading to immediate-early gene expression and a mitogenic response. There seems to be no involvement of Ras-GTP formation in the process of insulin stimulated glucose transport. Though the precise mechanism by which Ras is converted to the GTP bound state remains to be established, a tight correlation exists between receptor autophosphorylation and Ras-GTP formation.     

Potential role for triglycerides in signal transduction

We previously reported that endothelin-1 or platelet-derived growth factor promoted in aortic smooth muscle cells a rapid hydrolysis of 1-O-alkyl-2-acyl-sn-glycero-3-phosphoethanolamine (alkyl-PE) which was immediately converted into 1-O-alkyl-2,3-diacyl-sn-glycerol (alkyl-TG) within 5 s or 60 s respectively [C. Comminges et al. (1996) Biochem. Biophys. Res. Commun. 220, 1008-1013 and C. Comminges et al. (1997) Biochim. Biophys. Acta 1355, 69-80]. In this study, we show that this alkyl-PE hydrolysis is triggered by a transient activation of a specific phospholipase C (PLC) regulated by pertussis toxin-sensitive heterotrimeric G-proteins. Moreover, this PLC can be triggered through a Ca2+ influx depending on L-type Ca2+ channel activation, as suggested by the use of a specific 'activator' S(-)-BayK 8644 and of selective inhibitors such as nimodipine. Interestingly, low concentrations (10(-8)-10(-7)M) of alkyl-TG block the opening of L-type Ca2+ channels, whereas identical concentrations of DG do not alter L-type Ca2+ channels. This study thus unravels a hitherto unrecognized signaling pathway generating alkyl-TG as a novel lipid second messenger, potentially acting as a negative feedback regulator of L-type Ca2+ channels.     

The role of calcium in signal transduction processes in Sertoli cells

Sertoli cells play a pivotal role in regulation and maintenance of spermatogenesis. They are hormonally regulated predominantly by follicle-stimulating hormone (FSH) and testosterone (T). Although FSH and T have distinct mechanisms of action they act synergistically in promoting spermatogenesis. Stimulation of freshly isolated Sertoli cells with FSH evokes a prompt rise in cytosolic calcium which is quantitatively reproduced by cAMP. The cytosolic calcium response to FSH in Sertoli cells is predominantly attributable to serial signaling after the generation of endogenous cAMP. Calcium homeostasis of Sertoli cells may also be regulated by cAMP-independent metabolism. Vasoactive testicular paracrine hormones such as angiotensin II (AII) and vasopressin acting via inositol triphosphate generation induce cytosolic calcium rise predominantly derived from the thapsigargin-sensitive endoplasmic reticulum. Investigations involving androgens action on cytosolic calcium reveal a common mechanism of action between the peptide and steroid regulators of Sertoli cell function, indicating that cytosolic calcium ions may represent a unifying biochemical mechanism that could explain the synergism of FSH and T. Androgens rapidly and specifically increase cytosolic calcium, consistent with a plasma membrane site of action. This argues for the possible existence of a short term non-genomic signaling pathway in hormonal regulation of Sertoli cell function in addition to the classical longer term, slower genomic response.     

Redox regulation of protein tyrosine phosphatases during receptor tyrosine kinase signal transduction

In addition to protein phosphorylation, redox-dependent post-translational modification of proteins is emerging as a key signaling system that has been conserved throughout evolution and that influences many aspects of cellular homeostasis. Both systems exemplify dynamic regulation of protein function by reversible modification, which, in turn, regulates many cellular processes such as cell proliferation, differentiation and apoptosis. In this article we focus on the interplay between phosphorylation- and redox-dependent signaling at the level of phosphotyrosine phosphatase-mediated regulation of receptor tyrosine kinases (RTKs). We propose that signal transduction by oxygen species through reversible phosphotyrosine phosphatase inhibition, represents a widespread and conserved component of the biochemical machinery that is triggered by RTKs.     

The role of trans-membrane signal transduction in turing-type cellular pattern formation

The Turing mechanism (Phil. Trans. R. Soc. B 237 (1952) 37) for the production of a broken spatial symmetry in an initially homogeneous system of reacting and diffusing substances has attracted much interest as a potential model for certain aspects of morphogenesis (Models of Biological Pattern Formation, Academic Press, London, 1982; Nature 376 (1995) 765) such as pre-patterning in the embryo. The two features necessary for the formation of Turing patterns are short-range autocatalysis and long-range inhibition (Kybernetik 12 (1972) 30) which usually only occur when the diffusion rate of the inhibitor is significantly greater than that of the activator. This observation has sometimes been used to cast doubt on applicability of the Turing mechanism to cellular patterning since many messenger molecules that diffuse between cells do so at more-or-less similar rates. Here we show that Turing-type patterns will be able to robustly form under a wide variety of realistic physiological conditions though plausible mechanisms of intra-cellular chemical communication without relying on differences in diffusion rates. In the mechanism we propose, reactions occur within cells. Signal transduction leads to the production of messenger molecules, which diffuse between cells at approximately equal rates, coupling the reactions occurring in different cells. These mechanisms also suggest how this process can be controlled in a rather precise way by the genetic machinery of the cell.     

Structural organization of mammalian lipid phosphate phosphatases: implications for signal transduction

This article describes the regulation of cell signaling by lipid phosphate phosphatases (LPPs) that control the conversion of bioactive lipid phosphates to their dephosphorylated counterparts. A structural model of the LPPs, that were previously called Type 2 phosphatidate phosphatases, is described. LPPs are characterized by having no Mg²⁺ requirement and their insensitivity to inhibition by N-ethylmaleimide. The LPPs have six putative transmembrane domains and three highly conserved domains that define a phosphatase superfamily. The conserved domains are juxtaposed to the proposed membrane spanning domains such that they probably form the active sites of the phosphatases. It is predicted that the active sites of the LPPs are exposed at the cell surface or on the luminal surface of intracellular organelles, such as Golgi or the endoplasmic reticulum, depending where various LPPs are expressed. LPPs could attenuate cell activation by dephosphorylating bioactive lipid phosphate esters such as phosphatidate, lysophosphatidate, sphingosine 1-phosphate and ceramide 1-phosphate. In so doing, the LPPs could generate alternative signals from diacylglycerol, sphingosine and ceramide. The LPPs might help to modulate cell signaling by the phospholipase D pathway. For example, phosphatidate generated within the cell by phospholipase D could be converted by an LPP to diacylglycerol. This should change the relative balance of signaling by these two lipids. Another possible function of the LPPs relates to the secretion of lysophosphatidate and sphingosine 1-phosphate by activated platelets and other cells. These exogenous lipids activate phospholipid growth factor receptors on the surface of cells. LPP activities could attenuate cell activation by lysophosphatidate and sphingosine 1-phosphate through their respective receptors.     

The role of GRAS proteins in plant signal transduction and development

GRAS proteins are a recently discovered family of plant-specific proteins named after GAI, RGA and SCR, the first three of its members isolated. Although the Arabidopsis genome encodes at least 33 GRAS protein family members only a few GRAS proteins have been characterized so far. However, it is becoming clear that GRAS proteins exert important roles in very diverse processes such as signal transduction, meristem maintenance and development. Here we present a survey of the different GRAS proteins and review the current knowledge of the function of individual members of this protein family.