Sleep is a fundamental property conserved across species. The homeostatic induction of sleep indicates the presence of a mechanism that is progressively activated by the awake state and that induces sleep. Several lines of evidence support that such function, namely, sleep need, lies in the neuronal assemblies rather than specific brain regions and circuits. However, the molecular mechanism underlying the dynamics of sleep need is still unclear. This review aims to summarize recent studies mainly in rodents indicating that protein (...) class='Hi'>phosphorylation, especially at the synapses, could be the molecular entity associated with sleep need. Genetic studies in rodents have identified a set of kinases that promote sleep. The activity of sleep-promoting kinases appears to be elevated during the awake phase and in sleep deprivation. Furthermore, the proteomic analysis demonstrated that the phosphorylation status of synaptic protein is controlled by the sleep-wake cycle. Therefore, a plausible scenario may be that the awake-dependent activation of kinases modifies the phosphorylation status of synaptic proteins to promote sleep. We also discuss the possible importance of multisite phosphorylation on macromolecular protein complexes to achieve the slow dynamics and physiological functions of sleep in mammals. (shrink)

Cadherin‐mediated cell‐cell adhesion is perturbed in protein tyrosine kinase (PTK)‐transformed cells. While cadherins themselves appear to be poor PTK substrates, their cytoplasmic binding partners, the Arm catenins, are excellent PTK substrates and therefore good candidates for mediating PTK‐induced changes in cadherin behavior. These proteins, p120ctn, β‐catenin and plakoglobin, bind to the cytoplasmic region of classical cadherins and function to modulate adhesion and/or bridge cadherins to the actin cytoskeleton. In addition, as demonstrated recently for β‐catenin, these proteins also have crucial signaling (...) roles that may or may not be related to their effects on cell‐cell adhesion. Tyrosine phosphorylation of cadherin complexes is well documented and widely believed to modulate cell adhesiveness. The data to date, however, is largely correlative and the mechanism of action remains unresolved. In this review, we discuss the current literature and suggest models whereby tyrosine phosphorylation of Arm catenins contribute to regulation or perturbation of cadherin function. (shrink)

The study of intrinsic phosphorylation dynamics and kinetics in the context of complex protein architecture in vivo has been challenging: Method limitations have prevented significant advances in the understanding of the highly variable turnover of phosphate groups, synergy, and cooperativity between P‐sites. However, over the last decade, powerful analytical technologies have been developed to determine the full catalog of the phosphoproteome for many species. The curated databases of phospho sites found by mass spectrometry analysis and the computationally predicted sites (...) based on the linear sequence of kinase motifs are valuable tools. They allow investigation of the complexity of phosphorylation in vivo, albeit with strong discrepancies between different methods. A series of hypothetical scenarios on combinatorial processive phosphorylation is proposed that are likely unverifiable with current methodologies. These proposed a priori postulates could be considered as possible extensions of the known schemes of the activation/inhibition signaling process in vivo. (shrink)

Phosphoinositide (PI) phosphatases such as the SH2 domain‐containing inositol 5‐phosphatases 1/2 (SHIP1 and 2) are important signalling enzymes in human physiopathology. SHIP1/2 interact with a large number of immune and growth factor receptors. Tyrosine phosphorylation of SHIP1/2 has been considered to be the determining regulatory modification. However, here we present a hypothesis, based on recent key publications, highlighting the determining role of Ser/Thr phosphorylation in regulating several key properties of SHIP1/2. Since a subunit of the Ser/Thr phosphatase PP2A (...) has been shown to interact with SHIP2, a putative mechanism for reversing SHIP2 Ser/Thr phosphorylation can be anticipated. PI phosphatases are potential target molecules in human diseases, particularly, but not exclusively, in cancer and diabetes. Therefore, this novel regulatory mechanism deserves further attention in the hunt for discovering novel or complementary therapeutic strategies. This mechanism may be more broadly involved in regulating PI signalling in the case of synaptojanin1 or the phosphatase, tensin homolog, deleted on chromosome TEN.Editor's suggested further reading in BioEssays: Pairing phosphoinositides with calcium ions in endolysosomal dynamics Abstract. (shrink)

The origins of oxidative phosphorylation, initially known as aerobic phosphorylation, grew out of three research areas of muscle metabolism, creatine phosphorylation, aerobic metabolism of lactic acid in muscle, and studies on the nature and role of adenosine triphosphate . Much of this work centred round the laboratory of Otto Meyerhof, and most of those contributing to the study of aerobic phosphorylation were influenced by that laboratory: particularly Lipmann and also Ochoa. The work of Engelhardt on ATP (...) levels in blood also appears to have been influenced by the studies of Meyerhof’s laboratory. However, with the work of Kalckar, influenced by Lipmann, biochemists began to realise the potential importance of the process. This was confirmed and extended by Belitzer and Ochoa and the theoretical contribution of Lipmann. Thus it is not easy to identify a single point that marked the initiation of studies in this field.The early work was based on the use of tissue homogenates and cells but ultimately this approach limited research possibilities. The development of techniques based on mitochondria opened up new possibilities and brought this first phase of the study of oxidative phorphorylation to a close. (shrink)

The ubiquitous nature of mitochondria, the dual genetic foundation of the respiratory chain in mitochondrial and nuclear genome, and the peculiar rules of mitochondrial genetics all contribute to the extraordinary heterogeneity of clinical disorders associated with defects of oxidative phosphorylation (mitochondrial encephalomyopathies). Here, we review recent findings about nuclear gene defects in isolated OXPHOS enzyme complex deficiency. This information should help in identifying patients with mitochondrial disease and defining a biochemical and molecular basis of the disorder found in each (...) patient. This knowledge is indispensable for accurate genetic counseling and prenatal diagnosis, and is a prerequisite for the development of rational therapies, which are still, at present, woefully inadequate. BioEssays 23:518–525, 2001. © 2001 John Wiley & Sons, Inc. (shrink)

The reversible phosphorylation of proteins on serine/threonine residues preceding proline (Ser/Thr‐Pro) is a major regulatory mechanism for the control of a series of cell cycle events. Although phosphorylation is thought to regulate protein function by inducing conformational changes, little is known about most of these conformational changes and their significance. Recent studies indicate that the conformation and function of a subset of these phosphorylated proteins are controlled by the prolyl isomerase Pin1 through isomerization of specific phosphorylated Ser/Thr‐Pro bonds. (...) Furthermore, compelling evidence supports the idea that proline‐directed phosphorylation and subsequent isomerization play a critical role not only in cell cycle control, but also in the development of Alzheimer's disease, where postmitotic neurons display various cell cycle markers, especially mitotic events, prior to degeneration. BioEssays 25:174–181, 2003. © 2003 Wiley Periodicals, Inc. (shrink)

One of the profound changes in cellular morphology during mitosis is a massive alteration in the organization of microfilament cytoskeleton. It has been recently discovered that nonmuscle caldesmon, an actin and calmodulin binding microfilament‐associated protein of relative molecular mass Mr = 83000, is dissociated from microfilaments during mitosis, apparently as a consequence of mitosis‐specific phosphorylation. cdc2 kinase, which is a catalytic subunit of MPF (maturation or mitosis promoting factor), is found to be responsible for the mitosis‐specific phosphorylation of (...) caldesmon. Because caldesmon is implicated in the regulation of actin myosin interactions and/or microfilament organization, these results suggest that cdc2 kinase directly affects microfilament re‐organization during mitosis. (shrink)

We investigate the context of discovery of two significant achievements of twentieth century biochemistry: the chemiosmotic mechanism of oxidative phosphorylation and the dark reaction of photosynthesis. The pursuit of these problems involved discovery strategies such as the transfer, recombination and reversal of previous causal and mechanistic knowledge in biochemistry. We study the operation and scope of these strategies by careful historical analysis, reaching a number of systematic conclusions: even basic strategies can illuminate “hard cases” of scientific discovery that go (...) far beyond simple extrapolation or analogy; the causal–mechanistic approach to discovery permits a middle course between the extremes of a completely substrate-neutral and a completely domain-specific view of scientific discovery; the existing literature on mechanism discovery underemphasizes the role of combinatorial approaches in defining and exploring search spaces of possible problem solutions; there is a subtle interplay between a fine-grained mechanistic and a more coarse-grained causal level of analysis, and both are needed to make discovery processes intelligible. (shrink)

We investigate the context of discovery of two significant achievements of twentieth century biochemistry: the chemiosmotic mechanism of oxidative phosphorylation and the dark reaction of photosynthesis. The pursuit of these problems involved discovery strategies such as the transfer, recombination and reversal of previous causal and mechanistic knowledge in biochemistry. We study the operation and scope of these strategies by careful historical analysis, reaching a number of systematic conclusions: even basic strategies can illuminate “hard cases” of scientific discovery that go (...) far beyond simple extrapolation or analogy; the causal–mechanistic approach to discovery permits a middle course between the extremes of a completely substrate-neutral and a completely domain-specific view of scientific discovery; the existing literature on mechanism discovery underemphasizes the role of combinatorial approaches in defining and exploring search spaces of possible problem solutions; there is a subtle interplay between a fine-grained mechanistic and a more coarse-grained causal level of analysis, and both are needed to make discovery processes intelligible. (shrink)

Phosphoinositide (PI) phosphatases such as the SH2 domain‐containing inositol 5‐phosphatases 1/2 (SHIP1 and 2) are important signalling enzymes in human physiopathology. SHIP1/2 interact with a large number of immune and growth factor receptors. Tyrosine phosphorylation of SHIP1/2 has been considered to be the determining regulatory modification. However, here we present a hypothesis, based on recent key publications, highlighting the determining role of Ser/Thr phosphorylation in regulating several key properties of SHIP1/2. Since a subunit of the Ser/Thr phosphatase PP2A (...) has been shown to interact with SHIP2, a putative mechanism for reversing SHIP2 Ser/Thr phosphorylation can be anticipated. PI phosphatases are potential target molecules in human diseases, particularly, but not exclusively, in cancer and diabetes. Therefore, this novel regulatory mechanism deserves further attention in the hunt for discovering novel or complementary therapeutic strategies. This mechanism may be more broadly involved in regulating PI signalling in the case of synaptojanin1 or the phosphatase, tensin homolog, deleted on chromosome TEN.Editor's suggested further reading in BioEssays: Pairing phosphoinositides with calcium ions in endolysosomal dynamics Abstract. (shrink)

Chromosome segregation requires the ordered separation of the newly replicated chromosomes between the two daughter cells. In most cells, this requires nuclear envelope (NE) disassembly during mitotic entry and its reformation at mitotic exit. Nuclear envelope breakdown (NEB) results in the mixture of two cellular compartments. This process is controlled through phosphorylation of multiple targets by cyclin‐dependent kinase 1 (Cdk1)‐cyclin B complexes as well as other mitotic enzymes. Experimental evidence also suggests that nucleo‐cytoplasmic transport of critical cell cycle regulators (...) such as Cdk1‐cyclin B complexes or Greatwall, a kinase responsible for the inactivation of PP2A phosphatases, plays a major role in maintaining the boost of mitotic phosphorylation thus preventing the potential mitotic collapse derived from NEB. These data suggest the relevance of nucleo‐cytoplasmic transport not only to communicate cytoplasmic and nuclear compartments during interphase, but also to prepare cells for the mixture of these two compartments during mitosis. (shrink)

In the 1950s and 1960s, the search for the mechanism of oxidative phosphorylation by biochemists paralleled the description of mitochondrial form by George Palade and Fritiof Sjöstrand using electron microscopy. This paper explores the extent to which biochemists studying oxidative phosphorylation took mitochondrial form into account in the formulation of hypotheses, design of experiments, and interpretation of results. By examining experimental approaches employed by the biochemists studying oxidative phosphorylation, and their interactions with Palade, I suggest that use (...) of mitochondrial form as a guide to experimentation and interpretation varied considerably among investigators. Most notably, Peter Mitchell, whose chemiosmotic hypothesis was ultimately the basis of the correct mechanism of oxidative phosphorylation, incorporated crucial aspects of mitochondrial form into his model that others failed to recognize. I discuss these historical observations in terms of the background and training of the biochemists, as well as a proposed heuristic of form, whose use may increase the possibility that biologically meaningful molecular mechanisms will be discovered. (shrink)

Entry into mitosis is driven by the activity of kinases, which phosphorylate over 7000 proteins on multiple sites. For cells to exit mitosis and segregate their genome correctly, these phosphorylations must be removed in a specific temporal order. This raises a critical and important question: how are specific phosphorylation sites on an individual protein removed? Traditionally, the temporal order of dephosphorylation was attributed to decreasing kinase activity. However, recent evidence in human cells has identified unique patterns of dephosphorylation during (...) mammalian mitotic exit that cannot be fully explained by the loss of kinase activity. This suggests that specificity is determined in part by phosphatases. In this review, we explore how the physicochemical properties of an individual phosphosite and its surrounding amino acids can affect interactions with a phosphatase. These positive and negative interactions in turn help determine the specific pattern of dephosphorylation required for correct mitotic exit. (shrink)

Site‐specific phosphorylation of intermediate filament (IF) proteins on serine and threonine residues leads to alteration of the filament structure, in vitro and in vivo. Protein kinases involved in cell signaling and those activated in mitosis dynamically control spatial and temporal organization of intracellular IF phosphorylation. Thus, IF phosphorylation appears to be one of the most predominant strategies in coordinating intracellular organization of the IF network.

Hypoxia hampers ATP production and threatens cell survival. Since cellular energetics tightly controls cell responses and fate, ATP levels and dynamics are of utmost importance. An integrated mathematical model of ATP synthesis by the mitochondrial oxidative phosphorylation/electron transfer chain system has been recently published :e36, 2005). This model was validated under static conditions. To evaluate its performance under dynamical situations, we implemented and simulated it . Inner membrane potential and [NADH] were used as indicators of mitochondrial function. Root mean (...) squared error was used to compare simulations and experiments :39155–39165, 2003). Steady-state experimental data were reproduced within 2–6%. Model dynamics were evaluated under: baseline, activation of NADH production, addition of ADP, addition of inorganic phosphate, oxygen exhaustion. In all phases, except the last one, ΔΨ and [NADH] as well as oxygen consumption, were reproduced . Under anoxia, simulated ΔΨ markedly depolarized . In conclusion, the model reproduces dynamic data as long as oxygen is present. Anticipated improvement by the inclusion of ATP consumption and explicit Krebs cycle are under evaluation. (shrink)

The “Human Genome Project” was completed in 2003, shifting the focus to proteome and transcriptome research. One approach to proteomics involves the comprehensive visualization of the localization of proteins in all tissues and organs. We discuss in situ phospho‐protein atlases, which are systematized representations of the localization of proteins. Protein atlases provide important information about the identity and presence of proteins in specific organs, tissues and cells under physiological and pathological conditions. Antibody‐based immunohistochemical analysis is a powerful method for generating (...) a protein atlas. However, it is difficult to localize phosphorylated proteins under in vivo physiological conditions, even with immunohistochemistry, because these proteins tend to be dephosphorylated or phosphorylated due to the experimental manipulations. We also discuss an improved immunohistochemical method for precisely detecting phosphorylated protein, using the detection of phosphorylated ERK1/2 as an example. We consider that it is possible and useful to generate a phospho‐protein atlas. (shrink)

The interconversion of key regulatory proteins between phosphorylated and dephosphorylated forms is an extremely versatile mechanism for reversible altering their activities, and in mammalian cells may be almost as common as allosteric regulation. It is now evident that protein phosphorylation is the basis of a complex network of interlocking systems which allow a variety of hormones and other extracellular signals, acting through just a few second messengers to coordinate biochemical functions.

In the same year, 1961, Peter D. Mitchell and Robert R.J.P. Williams both put forward hypotheses for the mechanism of oxidative phosphorylation in mitochondria and photophosphorylation in chloroplasts. Mitchell's proposal was ultimately adopted and became known as the chemiosmotic theory. Both hypotheses were based on protons and differed markedly from the then prevailing chemical theory originally proposed by E.C. Slater in 1953, which by 1961 was failing to account for a number of experimental observations. Immediately following the publication of (...) Williams's hypothesis and before his own was published, Mitchell initiated a correspondence. Examination of the letters shows the development of a dispute based on the validity of the proposals, who should have priority and particularly whether Mitchell had drawn on Williams's work without acknowledgement. We have concluded that Mitchell's proposals were original although it is evident that prior to the correspondence Williams had considered and rejected a proposition similar to Mitchell's theory. However, a major cause of the dispute was the difference in disciplinary backgrounds of Mitchell, a microbial biochemist and Williams, a chemist. (shrink)

Saethre‐Chotzen syndrome (SCS), a human autosomal dominant condition with limb defects and craniosynostosis, is caused by haploinsufficiency of TWIST1, a basic helix–loop–helix (bHLH) transcription factor. Until recently, the molecular pathogenesis of the limb defects in SCS has not been well understood. Now, Firulli et al.1 show in mouse and chick that ectopic expression of a related bHLH protein, Hand2, results in phenocopies of the limb defects caused by Twist1 loss‐of‐function mutations. These two proteins interact in a dosage‐dependent antagonistic manner, and (...) both can be regulated through phosphorylation at conserved helix I amino acid residues. These findings provide an important link between the misregulation of Twist1 dimerization and the limb phenotypes observed in SCS. BioEssays 27:1102–1106, 2005. © 2005 Wiley Periodicals, Inc. (shrink)

The mathematical dynamic model of oxidative phosphorylation in muscle mitochondria developed previously was used to calculate the flux control coefficients of particular steps of this process in isolated mitochondria at different amounts of hexokinase and oxygen concentrations. The pattern of control was completely different under different conditions. For normoxic concentration, the main controlling steps in state 4, state 3.5 and state 3 were proton leak, ATP usage (hexokinase) and complex III, respectively. The pattern of control in state 4 was (...) not changed at hypoxic oxygen concentration, while in state 3.5 and state 3 much of the control was shifted from other steps to cytochrome oxidase. The implications of the theoretical results obtained for the regulation of oxidative phosphorylation in intact muscle are discussed. (shrink)

Engagement of integrins and other adhesion receptors can induce tyrosine phosphorylation of focal adhesion kinase (FAK), a tyrosine kinase present in focal adhesions. Furthermore, in addition to adhesion receptors, a surprising variety of stimuli, acting either on specific surface receptors or on intracellular molecules, such as PKC or Rho, can induce also tyrosine phosphorylation of FAK. I suggest that a potential mechanism by which such distinct factors may modulate the tyrosine phosphorylation of FAK is the promotion of (...) integrin or other adhesion receptor clustering at focal adhesions. BioEssays 21:1069–1075, 1999. © 1999 John Wiley & Sons, Inc. (shrink)

Osteopotin is a secreted glycosylated phosphoprotein found in bone and other normal and malignant tissues. Osteopontin can be autophosphorylated on tyrosine residues and can also be phosphorylated on serine and threonine residues by several protein kinases. Autophosphorylation of osteopontin may generate sites for specific interactions with other proteins on the cell surface and/or within the extracelluar matrix. These interactions of osteopontin are thought to be essential for bone mineralization and function. The polyaspartic acid motif of osteopontin, in combination with neighboring (...) sequences that include serine residues phosphorylated by protein kinases, could fold and assemble into a molecular structure that participates in the mineralization of the bone matrix. (shrink)

It has been known for 20 years that the ribosomal protein S6 is rapidly phosphorylated when cells are stimulated to grow or divide. Furthermore, numerous studies have documented that there is a strong correlation between increases in S6 phosphorylation and protein synthesis, leading to the idea that S6 phosphorylation is involved in up‐regulating translation. In an attempt to define a mechanism by which S6 phosphorylation exerts translational control, other studies have focused on characterizing the sites of (...) class='Hi'>phosphorylation of this protein and its location within the ribosome. Recent data show that S6 is a protein which may have diverse cellular functions and is essential for normal development, and that it may be involved in the translational regulation of a specific class of messages. (shrink)

The precise orchestration of two opposing protein complexes – one in the cytoplasm (β‐catenin destruction complex) and the other at the plasma membrane (LRP6 signaling complex) – is critical for controlling levels of the transcriptional co‐factor β‐catenin, and subsequent activation of the Wnt/β‐catenin signal transduction pathway. The Wnt pathway component Axin acts as an essential scaffold for the assembly of both complexes. How the β‐catenin destruction and LRP6 signaling complexes are modulated following Wnt stimulation remains controversial. A recent study in (...) Science by He and coworkers reveals an underlying logic for Wnt pathway control in which Axin phosphorylation toggles a switch between the active and inactive states. This mini‐review focuses on this and two other recent studies that provide insight into the initial signaling events triggered by Wnt exposure. We emphasize regulation of the β‐catenin destruction and LRP6 signaling complexes and propose a framework for future work in this area. (shrink)

Ca2+ transients in myocardial cells are modulated by cyclic AMP‐dependent phosphorylation of a protein in the sarcoplasmic reticulum. This protein, termed phospholamban, serves to regulate the Ca2+ pump ATPase of this membrane, thus altering the mode of Ca2+ transients and the myocardial contractile response. Elucidating the structure of phospholamban and its intimate interaction with the Ca2+ pump ATPase should provide the basis for understanding, at the molecular level, how the cAMP system contributes to excitation‐contraction coupling in muscle cells.

In the same year, 1961, Peter D. Mitchell and Robert R.J.P. Williams both put forward hypotheses for the mechanism of oxidative phosphorylation in mitochondria and photophosphorylation in chloroplasts. Mitchell's proposal was ultimately adopted and became known as the chemiosmotic theory. Both hypotheses were based on protons and differed markedly from the then prevailing chemical theory originally proposed by E.C. Slater in 1953, which by 1961 was failing to account for a number of experimental observations. Immediately following the publication of (...) Williams 's hypothesis and before his own was published, Mitchell initiated a correspondence. Examination of the letters shows the development of a dispute based on the validity of the proposals, who should have priority and particularly whether Mitchell had drawn on Williams 's work without acknowledgement. We have concluded that Mitchell's proposals were original although it is evident that prior to the correspondence Williams had considered and rejected a proposition similar to Mitchell's theory. However, a major cause of the dispute was the difference in disciplinary backgrounds of Mitchell, a microbial biochemist and Williams, a chemist. (shrink)

In the current model of visual transduction, the lifetime of active cGMP phosphodiesterase depends upon the period of its interaction with GTP-bound transducin. If recoverin regulates the lifetime of light-activated cGMP phosphodiesterase through inhibition of rhodopsin phosphorylation, rhodopsin should directly interact with cGMP phosphodiesterase and/or GTP-bound transducin complexed with cGMP phosphodiesterase. Is this true?

Mitchell's formulation of the chemiosmotic theory of oxidative phosphorylation in 1961 lacked any experimental support for its three central postulates. The path by which Mitchell reached this theory is explored. A major factor was the role of Mitchell's philosophical system conceived in his student days at Cambridge. This system appears to have become a tacit influence on his work in the sense that Polanyi understood all knowledge to be generated by an interaction between tacit and explicit knowing. Early in (...) his life Mitchell had evolved a simple philosophy based on fluctoids, fluctids and statids which was developed in a thesis submitted for the Ph.D. at the University of Cambridge, England. This aspect of his work was rejected by the examiners and became a tacit element in his intellectual development. It is argued from his various publications that this philosophy can be traced as an underlying theme behind much of Mitchell's theoretical writing in the 50's leading, through his notion of vectorial metabolism, to the formulation and amplification of the chemiosmotic theory in the sixties. This philosophy formed the basis for Mitchell of his understanding of biological systems and gave him his unique approach to cell biology. (shrink)

Protein post‐translational modifications (PTMs) play a crucial role in all cellular functions by regulating protein activity, interactions and half‐life. Despite the enormous diversity of modifications, various PTM systems show parallels in their chemical and catalytic underpinnings. Here, focussing on modifications that involve the addition of new elements to amino‐acid sidechains, I describe historical milestones and fundamental concepts that support the current understanding of PTMs. The historical survey covers selected key research programmes, including the study of protein phosphorylation as a (...) regulatory switch, protein ubiquitylation as a degradation signal and histone modifications as a functional code. The contribution of crucial techniques for studying PTMs is also discussed. The central part of the essay explores shared chemical principles and catalytic strategies observed across diverse PTM systems, together with mechanisms of substrate selection, the reversibility of PTMs by erasers and the recognition of PTMs by reader domains. Similarities in the basic chemical mechanism are highlighted and their implications are discussed. The final part is dedicated to the evolutionary trajectories of PTM systems, beginning with their possible emergence in the context of rivalry in the prokaryotic world. Together, the essay provides a unified perspective on the diverse world of major protein modifications. (shrink)

Recoverin is a 23 kDa Ca2+binding protein that has been detected primarily in vertebrate photoreceptors. The role of recoverin in phototransduction has been investigated using a variety of biochemical methods. Initial reports suggesting that recoverin regulates photoreceptor guanylyl cyclase have not been confirmed. Instead, recoverin appears to determine the lifetime of lightstimulated phosphodiesterase activity, perhaps by regulating rhodopsin phosphorylation. Retinal recoverin is heterogeneously fatty acylated at its ammo-terminus. The amino-terminal fatty acid appears to be involved in the interaction of (...) recoverin with photoreceptor membranes. (shrink)

Attempts to solve the puzzling problem of oxidative phosphorylation led to four very different hypotheses each of which suggested a different view of the ATP synthase, the phosphorylating enzyme. During the 1960s and 1970s evidence began to accumulate which rendered Peter Mitchell’s chemiosmotic hypothesis, the novel part of which was the proton translocating ATP synthase (ATPase), a plausible explanation. The conformational hypothesis of Paul Boyer implied an enzyme where ATP synthesis was driven by the energy of conformational changes in (...) the respiratory proteins. This was finally abandoned as an explanation of the overall process. Nevertheless the conformational understanding of the enzyme became an acceptable proposal during the early 1970s and eventually led Boyer to a view of the enzyme that incorporated both hypotheses. The correspondence between Mitchell and Boyer, both Nobel laureates, exposes their different approaches to both this enzyme and to the hypotheses of oxidative phosphorylation and illuminates a key step in the development of bioenergetics. In particular Boyer was suspicious of proton gradients, because he could not envisage a chemical mechanism for the synthesis of ATP, while Mitchell distrusted conformational arguments because he believed the proton must act vectorially at the active site of the enzyme. This resulted in two different views of the mechanisms operating in this enzyme. Ultimately while Boyer was able to marry the two approaches, Mitchell retained his insistence on the role of the proton at the active site and was thus unable to give significance to Boyer’s conformational ideas. The underlying issues in this debate are discussed particularly with reference to the differing styles of Boyer and Mitchell and the influence of molecular biology, especially the development of protein technology. (shrink)

It has long been recognized that cancer onset and progression represent a type of reversion to an ancestral quasi‐unicellular phenotype. This general concept has been refined into the atavistic model of cancer that attempts to provide a quantitative analysis and testable predictions based on genomic data. Over the past decade, support for the multicellular‐to‐unicellular reversion predicted by the atavism model has come from phylostratigraphy. Here, we propose that cancer onset and progression involve more than a one‐off multicellular‐to‐unicellular reversion, and are (...) better described as a series of reversionary transitions. We make new predictions based on the chronology of the unicellular‐eukaryote‐to‐multicellular‐eukaryote transition. We also make new predictions based on three other evolutionary transitions that occurred in our lineage: eukaryogenesis, oxidative phosphorylation and the transition to adaptive immunity. We propose several modifications to current phylostratigraphy to improve age resolution to test these predictions. Also see the video abstract here: https://youtu.be/3unEu5JYJrQ. (shrink)

Cell proliferation in response to growth factors is mediated by specific high affinity receptors. Ligand‐binding by receptors of the protein tyrosine kinase family results in the stimulation of several intracellular signal transduction pathways. Key signalling enzymes are recruited to the plasma membrane through the formation of stable complexes with activated receptors. These interactions are mediated by the conserved, non‐catalytic SH2 domains present in the signalling molecules, which bind with high affinity and specificity to tyrosine‐phosphorylated sequences on the receptors. The assembly (...) of enzyme complexes is emerging as a major mechanism of signal transduction and may regulate the pleiotropic effects of growth factors. (shrink)

Phosphatidylinositol‐3‐kinases (PI3Ks) are lipid kinases that produce 3‐phosphorylated derivatives of phosphatidylinositol upon activation by various cues. These 3‐phosphorylated lipids bind to various protein effectors to control many cellular functions. Lipid phosphatases such as phosphatase and tensin homolog (PTEN) terminate PI3K‐derived signals and are critical to ensure appropriate signaling outcomes. Many lines of evidence indicate that PI3Ks and PTEN, as well as some specific lipid effectors are highly compartmentalized, either in plasma membrane nanodomains or in endosomal compartments. We examine the evidence (...) for specific recruitment of PI3Ks, PTEN, and other related enzymes to membrane nanodomains and endocytic compartments. We then examine the hypothesis that scaffolding of the sources (PI3Ks), terminators (PTEN), and effectors of these lipid signals with a common plasma membrane nanodomain may achieve highly localized lipid signaling and ensure selective activation of specific effectors. This highlights the importance of spatial regulation of PI3K signaling in various physiological and disease contexts. (shrink)

It is a long-standing view that global translation varies during the cell cycle and is much lower in mitosis than in other cell-cycle phases. However, the central papers in the literature are not in agreement about the extent of downregulation in mitosis, ranging from a dramatic decrease to only a marginal reduction. Herein, it is argued that the discrepancy derives from technical challenges. Cell-cycle-dependent variations are most conveniently studied in synchronized cells, but the synchronization methods by themselves often evoke stress (...) responses that, in turn, affect translation rates. Further, it is argued that previously reported cell-cycle-dependent changes in the global translation rate to a large extent reflect responses to the synchronization methods. Recent findings strongly suggest that the global translation rate is not regulated in a cell-cycle-dependent manner. Novel techniques allowing a genome-wide analysis of translational profiles suggest that the extent and importance of selective translational regulation associated with cell-cycle transitions have been underestimated. Therefore, the main question is which messenger RNAs (mRNAs) are translated, rather than whether the global translation rate is decreased. (shrink)

Transcription factors provide nodes of information integration by serving as nuclear effectors of multiple signaling cascades, and thus elaborate layers of regulation, often involving post-translational modifications, modulating and coordinate activities. Such modifications can rapidly and reversibly regulate virtually all transcription factor functions, including subcellular localization, stability, interactions with cofactors, other post-translational modifications and transcriptional activities. Aside from analyses of the effects of serine/threonine phosphorylation, studies on post-translational modifications of transcription factors are only in the initial stages. In particular, the (...) regulatory possibilities afforded by combinatorial usage of and competition between distinct modifications on an individual protein are immense, and with respect to large families of closely related transcription factors, offer the potential of conferring critical specificity. Here we will review the post-translational modifications known to regulate ETS transcriptional effectors and will discuss specific examples of how such modifications influence their activities to highlight emerging paradigms in transcriptional regulation. BioEssays 27:285–298, 2005. © 2005 Wiley Periodicals, Inc. (shrink)

This paper considers papers on conceptual analysis by Laudan (1981) and Whitt (1989) and relates them to three biochemical episodes: (1) the modern 'biochemical explanation' of acupuncture; (2) the chemio-osmotic hypothesis of oxidative phosphorylation; (3) the theory of the complete digestion of proteins in the gut. The advantages of including philosophical debate in chemical/biochemical undergraduate courses is then discussed.

The term ‘cell’, in addition to designating fundamental units of life, has also been applied since the nineteenth century to technical apparatuses such as fuel and galvanic cells. This paper shows that such technologies, based on the electrical effects of chemical reactions taking place in containers, had a far-reaching impact on the concept of the biological cell. My argument revolves around the controversy over oxidative phosphorylation in bioenergetics between 1961 and 1977. In this scientific conflict, a two-level mingling of (...) technological culture, physical chemistry and biological research can be observed. First, Peter Mitchell explained the chemiosmotic hypothesis of energy generation by representing cellular membrane processes via an analogy to fuel cells. Second, in the associated experimental scrutiny of membranes, material cell models were devised that reassembled spatialized molecular processes in vitro. Cells were thus modelled both on paper and in the test tube not as morphological structures but as compartments able to perform physicochemical work. The story of cells and membranes in bioenergetics points out the role that theories and practices in physical chemistry had in the molecularization of life. These approaches model the cell as a ‘topology of molecular action’, as I will call it, and it involves concepts of spaces, surfaces and movements. They epitomize an engineer’s vision of the organism that has influenced diverse fields in today’s life sciences. (shrink)

Protons are the most common coupling ions in bacterial energy conversions. However, while many organisms, such as the alkaliphilic Bacilli, employ H+‐bioenergetics for electron transport phosphorylation, they use Na+ as the coupling ion for transport and flagellar movement. The Na+ gradient required for these bioenergetic functions is established by the secondary Na+/H+ antiporter. In contrast, Vibrio alginolyticus and methanogenic bacteria have primary pumps for both H+ and Na+. They use the proton gradient for ATP synthesis while other, less energy‐consuming (...) membrane reactions are powered by the Na+ gradient. In a third mode, some anaerobic bacteria possess decarboxylases acting as primary Na+ pumps. For instance, in Klebsiella pneumoniae, the Na+ gradient established by oxaloacetate decarboxylase is used for the uptake of the growth substrate citrate, and Propionigenium modestum consumes the energy of the Na+ gradient formed by methylmalonyl‐CoA decarboxylase directly for ATP synthesis. (shrink)

The autophagy initiation complex is brought about via a highly ordered and stepwise assembly process. Two crucial signaling molecules, mTORC1 and AMPK, orchestrate this assembly by phosphorylating/dephosphorylating autophagy‐related proteins. Activation of Atg1 followed by recruitment of both Atg9 vesicles and the PI3K complex I to the PAS (phagophore assembly site) are particularly crucial steps in its formation. Ypt1, a small Rab GTPase in yeast cells, also plays an essential role in the formation of the autophagy initiation complex through multiple regulatory (...) pathways. In this review, our primary focus is to discuss how signaling molecules initiate the assembly of the autophagy initiation complex, and highlight the significant roles of Ypt1 in this process. We end by addressing issues that need future clarification. (shrink)

Unraveling the molecular detail of insulin receptor activation has proved challenging, but a major advance is the recent determination of crystallographic structures of insulin in complex with its primary binding site on the receptor. The current model for insulin receptor activation is that two distinct surfaces of insulin monomer engage sequentially with two distinct binding sites on the extracellular surface of the insulin receptor, which is itself a disulfide‐linked (αβ)2 homodimer. In the process, conformational changes occur both within the hormone (...) and the receptor, the latter resulting in the disruption of the intracellular interactions that hold the kinase domains in their basal state and in the initiation of the phosphorylation events that drive insulin signaling. The purpose of this paper is to summarize the extant structural data relating to hormone binding and how it effects receptor activation, as well as to discuss the issues that remain unresolved. (shrink)

Zygotic gene activation (ZGA) is the critical event that governs the transition from maternal to embryonic control of development. In the mouse, ZGA occurs during the 2‐cell stage and appears to be regulated by the time following fertilization, i.e. a zygotic clock, rather than by progression through the first cell cycle. The onset of ZGA must depend on maternally inherited proteins, and post‐translational modification of these maternally derived proteins is likely to play a role in ZGA. Consistent with this prediction (...) is that protein phosphorylation catalyzed by the cAMP‐dependent protein kinase is involved in ZGA and that protein synthesis is not required for ZGA. Recent results suggest that ZGA may occur earlier than previously thought, i.e. not during the 2‐cell stage, but rather in G2 of the 1‐cell embryo. Thus ZGA may comprise a period of minor gene activation in the 1‐cell embryo that is followed by a period of major gene activation in the 2‐cell embryo. Following ZGA, the expression of constitutively activated genes may require an enhancer. (shrink)

The YAP/TAZ family of transcriptional co‐activators drives cell proliferation in epithelial tissues and cancers. Yet, how YAP and TAZ are physiologically regulated remains unclear. Here we review recent reports that YAP and TAZ act primarily as sensors of epithelial cell polarity, being inhibited when cells differentiate an apical membrane domain, and being activated when cells contact the extracellular matrix via their basal membrane domain. Apical signalling occurs via the canonical Crumbs/CRB‐Hippo/MST‐Warts/LATS kinase cascade to phosphorylate and inhibit YAP/TAZ. Basal signalling occurs (...) via Integrins and Src family kinases to phosphorylate and activate YAP/TAZ. Thus, YAP/TAZ is localised to the nucleus in basal stem/progenitor cells and cytoplasm in differentiated squamous cells or columnar cells. In addition, other signals such as mechanical forces, tissue damage and possibly receptor tyrosine kinases (RTKs) can influence MST‐LATS or Src family kinase activity to modulate YAP/TAZ activity. (shrink)

Here we discuss a “chromosome separation checkpoint” that might regulate the anaphase‐telophase transition. The concept of cell cycle checkpoints was originally proposed to account for extrinsic control mechanisms that ensure the order of cell cycle events. Several checkpoints have been shown to regulate major cell cycle transitions, namely at G1‐S and G2‐M. At the onset of mitosis, the prophase‐prometaphase transition is controlled by several potential checkpoints, including the antephase checkpoint, while the spindle assembly checkpoint guards the metaphase‐anaphase transition. Our hypothesis (...) is based on the recently uncovered feedback control mechanism that delays chromosome decondensation and nuclear envelope reassembly until effective separation of sister chromatids during anaphase is achieved. A central player in this potential checkpoint is the establishment of a constitutive, midzone‐based Aurora B phosphorylation gradient that monitors the position of chromosomes along the spindle axis. We propose that this surveillance mechanism represents an additional step towards ensuring mitotic fidelity. (shrink)

Lysine acetylation has been shown to occur in many protein targets, including core histones, about 40 transcription factors and over 30 other proteins. This modification is reversible in vivo, with its specificity and level being largely controlled by signal‐dependent association of substrates with acetyltransferases and deacetylases. Like other covalent modifications, lysine acetylation exerts its effects through “loss‐of‐function” and “gain‐of‐function” mechanisms. Among the latter, lysine acetylation generates specific docking sites for bromodomain proteins. For example, bromodomains of Gcn5, PCAF, TAF1 and CBP (...) are able to recognize acetyllysine residues in histones, HIV Tat, p53, c‐Myb or MyoD. In addition to the acetyllysine moiety, the flanking sequences also contribute to efficient recognition. The relationship between acetyllysine and bromodomains is reminiscent of the specific recognition of phosphorylated residues by phospho‐specific binding modules such as SH2 domains and 14‐3‐3 proteins. Therefore, lysine acetylation forges a novel signaling partnership with bromodomains to govern the temporal and spatial regulation of protein functions in vivo. BioEssays 26:1076–1087, 2004. © 2004 Wiley Periodicals, Inc. (shrink)

Previous studies have shown that zinc deficiency leads to apoptosis of neuronal precursor cells in vivo and in vitro. In addition to the role of p53 as a nuclear transcription factor in zinc deficient cultured human neuronal precursors (NT-2), we have now identified the translocation of phosphorylated p53 to the mitochondria and p53-dependent increases in the pro-apoptotic mitochondrial protein BAX leading to a loss of mitochondrial membrane potential as demonstrated by a 25% decrease in JC-1 red:green fluorescence ratio. Disruption of (...) mitochondrial membrane integrity was accompanied by efflux of the apoptosis inducing factor (AIF) from the mitochondria and translocation to the nucleus with a significant increase in reactive oxygen species (ROS) after 24 h of zinc deficiency. Measurement of caspase cleavage, mRNA, and treatment with caspase inhibitors revealed the involvement of caspases 2, 3, 6, and 7 in zinc deficiency-mediated apoptosis. Down-stream targets of caspase activation, including the nuclear structure protein lamin and polyADP ribose polymerase (PARP), which participates in DNA repair, were also cleaved. Transfection with a dominant-negative p53 construct and use of the p53 inhibitor, pifithrin- ␮, established that these alterations were largely dependent on p53. Together these data identify a cascade of events involving mitochondrial p53 as well as p53-dependent caspase-mediated mechanisms leading to apoptosis during zinc deficiency. (shrink)

D‐amino acids are being recognized as functionally important molecules in mammals. We recently identified endogenous D‐cysteine in mammalian brain. D‐cysteine is present in neonatal brain in substantial amounts (mM) and decreases with postnatal development. D‐cysteine binds to MARCKS and a host of proteins implicated in cell division and neurodevelopmental disorders. D‐cysteine decreases phosphorylation of MARCKS in neural progenitor cells (NPCs) affecting its translocation. D‐cysteine controls NPC proliferation by inhibiting AKT signaling. Exogenous D‐cysteine inhibits AKT phosphorylation at Thr 308 (...) and Ser 473 in NPCs. D‐cysteine treatment of NPCs led to 50% reduction in phosphorylation of Foxo1 at Ser 256 and Foxo3a at Ser 253. We hypothesize that in the developing brain endogenous D‐cysteine is as a physiologic regulator of NPC proliferation by inhibiting AKT signaling mediated by Foxo1 and Foxo3a. Endogenous D‐cysteine may regulate mammalian neurodevelopment with roles in schizophrenia and Alzheimer's disease (AD). (shrink)