Cyclosporin A, the cyclophilin class of peptidylprolyl isomerases, and blockade of T cell signal transduction.

Cyclosporin A, the major immunosuppressive drug in transplantation, and the more potent therapeutic drug candidate, FK506, have led to the discovery of two superfamilies of immunosuppressant binding proteins, the cyclophilins and the FK binding proteins. These proteins, enzymes with high kcat values for isomerization of X-Pro bonds in peptides and protein substrates, are distributed in all cell compartments where protein folding normally occurs. It is likely that they play major roles in the protein folding and protein trafficking in the cell. It is also likely that they have been suborned in T cells by the immunosuppressant drugs that are potent pseudosubstrate ligands that selectively block the signal transduction cascade. The discovery of the inhibition of protein phosphatase 2B (calcineurin) by the drug-immunophilin complex (CsA-CyP or FK506-FKBP) provides evidence for a specific downstream target of the drug-immunophilin complexes and may prompt a search for endogenous ligands of cyclophilin and FKBP that may effect signal transduction regulation. The molecular insights gained over a short time in this area have been remarkable; they promise to elucidate the steps in T cell activation and delineate new targets for immunosuppressive therapy.     

Scaffold State Switching Amplifies,Accelerates, and Insulates Protein Kinase C Signaling

Scaffold proteins localize two or more signaling enzymes in close proximity to their downstream effectors. A-kinase-anchoring proteins (AKAPs) are a canonical family of scaffold proteins known to bind protein kinase A (PKA) and other enzymes. Several AKAPs have been shown to accelerate, amplify, and specify signal transduction to dynamically regulate numerous cellular processes. However, there is little theory available to mechanistically explain how signaling on protein scaffolds differs from solution biochemistry. In our present study, we propose a novel kinetic mechanism for enzymatic reactions on protein scaffolds to explain these phenomena, wherein the enzyme-substrate-scaffold complex undergoes stochastic state switching to reach an active state. This model predicted anchored enzymatic reactions to be accelerated, amplified, and insulated from inhibition compared with those occurring in solution. We exploited a direct interaction between protein kinase C (PKC) and AKAP7α as a model to validate these predictions experimentally. Using a genetically encoded PKC activity reporter, we found that both the strength and speed of substrate phosphorylation were enhanced by AKAP7α. PKC tethered to AKAP7α was less susceptible to inhibition from the ATP-competitive inhibitor Gö6976 and the substrate-competitive inhibitor PKC 20-28, but not the activation-competitive inhibitor calphostin C. Model predictions and experimental validation demonstrated that insulation is a general property of scaffold tethering. Sensitivity analysis indicated that these findings may be applicable to many other scaffolds as well. Collectively, our findings provide theoretical and experimental evidence that scaffold proteins can amplify, accelerate, and insulate signal transduction.     

Protein tyrosine phosphatases: from genes, to function, to disease

The protein tyrosine phosphatase (PTP) superfamily of enzymes functions in a coordinated manner with protein tyrosine kinases to control signalling pathways that underlie a broad spectrum of fundamental physiological processes. In this review, I describe recent breakthroughs in our understanding of the role of the PTPs in the regulation of signal transduction and the aetiology of human disease.     

Proposed role of drug-metabolizing enzymes: regulation of steady state levels of the ligands that effect growth, homeostasis, differentiation, and neuroendocrine functions.

Every ligand known to bind to a receptor in the nuclear hormone receptor superfamily is involved in a variety of signal transduction pathways effecting growth, morphogenesis, homeostasis, proliferation, and neuroendocrine functions. Often these ligands are associated with increases in particular subsets of cytochromes P450 and other drug-metabolizing enzymes. Interestingly, certain of these enzymes participate in the metabolism (synthesis as well as degradation) of these ligands. It appears that genes coding for certain drug-metabolizing enzymes might have existed on this planet at least 1 billion years before the presence of plants, animals, and drugs. An early role for oxidative enzymes in prokaryotes most likely involved energy substrate utilization: insertion of oxygen into various inaccessible carbon and other food sources, thereby rendering them accessible to further metabolism. It is proposed that a later development of these "drug-metabolizing enzymes" in prokaryotes and early eukaryotes might be related to their metabolic ability to control the steady state levels of the ligands that modulate cell division, growth, morphogenesis, and mating, and that this role has diversified in numerous additional signal transduction pathways and exists today in all eukaryotes.     

Erythropoiesis and molecular mechanisms for sexual determination in malaria parasites

Malaria parasites proliferate asexually within the vertebrate host but must undergo sexual reproduction for transmission to mosquitoes and hence infection of new hosts. The developmental pathways controlling gametocytogenesis are not known, but several protein kinases and other putative signal transduction elements possibly involved in this phenomenon have been found in Plasmodium. Recently, another developmental pathway, that of Plasmodium sex determination (male or female), has been shown to be triggered by erythropoiesis in the host. Rapid progress is being made in our understanding of the molecular basis of mammalian erythropoiesis, revealing kinase pathways that are essential to cellular responses triggered by the hormone erythropoietin. Although the molecular mechanisms whereby this hormone modulates the sex ratio of malaria parasites remain to be elucidated, it probably activates, within the parasite, transduction pathways similar to those found in other eukaryotes. Indeed, enzymes belonging to protein kinase families known to be involved in the response of mammalian cells to erythropoietin (such as the mitogen-activated protein kinases) have been identified in P. falciparum gametocytes. Some of these enzymes differ markedly from their mammalian homologs; therefore, identification of the transduction pathways of the parasite that are responsible for its developmental response to erythropoietin opens the way to the development of transmission-blocking drugs based on kinase inhibitors.     

Metalloenzymes and signal transduction: the protein serine/threonine phosphatases,a novel class of binuclear metal-containing enzymes

The serine/threonine protein phosphatases are important regulatory enzymes involved in signal transduction pathways in eukaryotic organisms. These enzymes include protein phosphatases 1, 2A, and 2B (also known as calcineurin). Recent structural data have indicated that the serine/threonine protein phosphatases are novel metalloenzymes containing a dinuclear metal ion cofactor at the active site. The dinuclear metal site is situated in a unique protein fold, a β-α-β-α-β motif which provides the majority of ligands to the metal ions. A similar fold is also seen in plant purple acid phosphatases, which also contain a dinuclear iron–zinc cofactor. In these enzymes, the two metal ions are bridged by a solvent molecule and a carboxylate group from an aspartic acid residue, juxtaposing the two metal ions to within 3.0–4.0?Å of each other. A similar motif has been identified in a number of other enzymes which exhibit phosphoesterase activity, implicating several of them as metalloenzymes which contain dinuclear metal ion cofactors.     

Structural determinants of phosphoinositide 3-kinase inhibition by wortmannin, LY294002, quercetin, myricetin, and staurosporine

The specific phosphoinositide 3-kinase (PI3K) inhibitors wortmannin and LY294002 have been invaluable tools for elucidating the roles of these enzymes in signal transduction pathways. The X-ray crystallographic structures of PI3Kgamma bound to these lipid kinase inhibitors and to the broad-spectrum protein kinase inhibitors quercetin, myricetin, and staurosporine reveal how these compounds fit into the ATP binding pocket. With a nanomolar IC50, wortmannin most closely fits and fills the active site and induces a conformational change in the catalytic domain. Surprisingly, LY294002 and the lead compound on which it was designed, quercetin, as well as the closely related flavonoid myricetin bind PI3K in remarkably different orientations that are related to each other by 180 degrees rotations. Staurosporine/PI3K interactions are reminiscent of low-affinity protein kinase/staurosporine complexes. These results provide a rich basis for development of isoform-specific PI3K inhibitors with therapeutic potential.     

Kinetics, substrate specificity, and stereospecificity of two new protein tyrosine phosphatase-like inositol polyphosphatases from Selenomonas lacticifex

Inositol polyphosphatases (IPPases) play an important role in the metabolism of inositol polyphosphates, a class of molecules involved in signal transduction. Here we characterize 2 new protein tyrosine phosphatase-like IPPases (PhyAsl and PhyBsl) cloned from Selenomonas lacticifex that can hydrolyze myo-inositol hexakisphosphate (InsP6) in vitro. To determine their preferred substrates and stereospecificity of InsP6 dephosphorylation, a combination of kinetic and high-performance ion pair chromatography studies were conducted. Despite only 33% amino acid sequence identity between them, both enzymes display strict specificity for IPP substrates and cleave InsP6 primarily at the d-3-phosphate position (>90%). Furthermore, both enzymes predominantly degrade InsP6 to Ins(2)P via identical and very specific routes of dephosphorylation (3,4,5,6,1). Despite these similarities, PhylAsl is shown to have a slight kinetic preference for the major inositol pentakisphosphate intermediate in its InsP6 hydrolysis pathway, whereas PhyBsl displays a unique and substantial preference for an inositol tetrakisphosphate intermediate.     

Effects of sequestration on signal transduction cascades

The building blocks of most signal transduction pathways are pairs of enzymes, such as kinases and phosphatases, that control the activity of protein targets by covalent modification. It has previously been shown [Goldbeter A & Koshland DE (1981) Proc Natl Acad Sci USA 78, 6840-6844] that these systems can be highly sensitive to changes in stimuli if their catalysing enzymes are saturated with their target protein substrates. This mechanism, termed zero-order ultrasensitivity, may set thresholds that filter out subthreshold stimuli. Experimental data on protein abundance suggest that the enzymes and their target proteins are present in comparable concentrations. Under these conditions a large fraction of the target protein may be sequestrated by the enzymes. This causes a reduction in ultrasensitivity so that the proposed mechanism is unlikely to account for ultrasensitivity under the conditions present in most in vivo signalling cascades. Furthermore, we show that sequestration changes the dynamics of a covalent modification cycle and may account for signal termination and a sign-sensitive delay. Finally, we analyse the effect of sequestration on the dynamics of a complex signal transduction cascade: the mitogen-activated protein kinase (MAPK) cascade with negative feedback. We show that sequestration limits ultrasensitivity in this cascade and may thereby abolish the potential for oscillations induced by negative feedback.     

9-polylysine protein transduction domain: enhanced penetration efficiency of superoxide dismutase into mammalian cells and skin

Antioxidant enzymes, such as superoxide dismutase (SOD) and catalase (CAT), have been considered to have a beneficial effect against various diseases that are mediated by the reactive oxygen species (ROS). Although a variety of modified recombinant antioxidant enzymes have been generated to protect against oxidative stresses, the lack of their transduction ability into cells resulted in a limited ability to detoxify intracellular ROS. To render the SOD enzyme capable of detoxifying intracellular ROS when added extracellularly, cell-permeable recombinant SOD proteins were generated. A human Cu,Zn-superoxide dismutase (Cu,Zn-SOD) gene was fused with a gene fragment that encodes the 9 amino acids Tat protein transduction domain (RKKRRQRRR) of HIV-1 and lysine rich peptide (KKKKKKKKK) in a bacterial expression vector in order to produce a genetic in-frame Tat-SOD and 9Lys-SOD fusion protein, respectively. The expressed and purified Tat-SOD and 9Lys-SOD fusion proteins can transduce into human fibroblast cells, and they were enzymatically active and stable for 24 h. The cell viability of the fibroblast cells that were treated with paraquat, an intracellular superoxide anion generator, was increased by the transduced Tat-SOD or 9Lys-SOD. The transduction efficacy of 9Lys-SOD was more efficient than that of Tat-SOD. We evaluated the ability of the SOD fusion pmteins to transduce into animal skin. This analysis showed that Tat-SOD and 9Lys-SOD fusion proteins efficiently penetrated into the epidermis as well as the dermis of the subcutaneous layer, when sprayed on mice skin (judged by the immunohistochemistry and specific enzyme activities). The enzymatic activity of the transduced 9Lys-SOD was higher than that of Tat-SOD, indicating that the penetration of 9Lys-SOD was more efficient when put into the skin. These results suggest Tat-SOD and 9Lys-SOD fusion proteins can be used as anti-aging cosmetics, or in protein therapy, for various disorders that are related to this antioxidant enzyme and ROS.     

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.     

Les protéines phosphatases et protéines kinases des plantes supérieures

The recent gain in knowledge concerning enzymes involved in signal transduction pathways is a direct consequence of the considerable advances made in molecular biology. Protein kinases and protein phosphatases, the two major enzymes implicated in post-translational modifications, have been studied in particular. The number of characterized plant genes and/or cDNAs encoding these enzymes is increasing everyday. Since 1991, 26 genes and cDNAs coding for plant protein phosphatases have been isolated and characterized. The huge number of protein kinases (estimated at several thousands) makes it impossible to give an exhaustive list of the genes already identified, but a classification of these enzymes, based on phylogenetic criteria, allows us to appreciate the range of functions this protein family may play in plants.     

PESI--an intelligent system for prediction of enzyme-substrate interactions based on experimental constraints

We present a system for predicting protein-protein modifications, and demonstrate its usefulness in the field of signal transduction research. Signal transduction is one of the most important areas of investigation in biological research. One of the major mechanisms frequently employed by cells to regulate signal transduction processes involves protein phosphorylation by various kinases. As many as 1,000 protein kinases and 500 protein phosphatases in the human genome are thought to be involved in phosphorylation processes which regulate all aspects of cell function. The complexity of such interactions stems from the enormous number of factors and interactions, which makes the identification of putative substrates for any given enzyme by straightforward experimentation increasingly difficult. We present here a data mining algorithm, based on the similarity between the modifier proteins and between the modified proteins, and on experimental constraints. The application presented here (PESI) focuses on substrate phosphorylation by various enzymes. This algorithm reduces the number of substrate candidates for experimental study by about two orders of magnitude. Moreover, this algorithm has already yielded predictions for previously unknown substrates of the enzymes PKCdelta and PKCeta, which we have confirmed experimentally.     

The complete pathway for catalytic activation of the mitogen-activated protein kinase, ERK2

The mitogen-activated protein (MAP) kinase ERK2 is an essential signal transduction molecule that mediates extracellular signaling by all polypeptide growth factors. Full activation of ERK2 requires phosphorylation at both a threonine residue (Thr(183)) conserved in most protein kinases as well as a tyrosine residue (Tyr(185)) unique to members of the mitogen-activated protein kinase family. We have characterized the kinetic role of phosphorylation at each site with respect to the overall activation mechanism, providing a complete picture of the reaction steps involved. Phosphorylation at Tyr(185) serves to configure the ATP binding site, while phosphorylation at both residues is required to stabilize binding of the protein substrate, myelin basic protein. Similar control mechanisms are employed to stabilize ATP and myelin basic protein in the phosphoryl group transfer reaction, accounting for the enormous increase in turnover rate. The mechanism of ERK2 activation is kinetically similar to that of the cell cycle control protein, cdk2/cyclinA. Phosphorylation of Tyr(185) in ERK2 and association of cyclinA with cdk2 both serve to stabilize ATP binding. Subsequent phosphorylation of both enzymes on threonine serves to stabilize binding of the phosphoacceptor substrate.     

The mAKAP signaling complex: integration of cAMP, calcium, and MAP kinase signaling pathways

Following its production by adenylyl cyclases, the second messenger cAMP is in involved in pleiotrophic signal transduction. The effectors of cAMP include the cAMP-dependent protein kinase (PKA), the guanine nucleotide exchange factor Epac (exchange protein activated by cAMP), and cAMP-dependent ion channels. In turn, cAMP signaling is attenuated by phosphodiesterase-catalyzed degradation. The association of cAMP effectors and the enzymes that regulate cAMP concentration into signaling complexes helps to explain the differential signaling initiated by members of the G(s)-protein coupled receptor family. The signal transduction complex formed by the scaffold protein mAKAP (muscle A kinase-anchoring protein) at the nuclear envelope of both striated myocytes and neurons contains three cAMP-binding proteins, PKA, Epac1, and the phosphodiesterase PDE4D3. In addition, the mAKAP complex also contains components of the ERK5 MAP kinase signaling pathway, the calcium release channel ryanodine receptor and the phosphatases PP2A as well as calcineurin. Analysis of the mAKAP complex illustrates how a macromolecular complex can serve as a node in the intracellular signaling network of cardiac myocytes to integrate multiple cAMP signals with those of calcium and MAP kinases to regulate the hypertrophic actions of several hormones.