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Institute of Interdisciplinary Research, Free University of Brussels, Campus Erasme, B-1070 Brussels, Belgium
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ABSTRACT |
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 ABSTRACT
 INTRODUCTION
MANY RELATIONS DESCRIBED IN...
HOW IS CELL SPECIFICITY...
LOSS OF SPECIFICITY MAY...
UTILITY OF NETWORKS
GENERAL CONCLUSIONS
REFERENCES
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The literature on intracellular signal transduction presents a confusing picture: every regulatory factor appears to be regulated by all signal transduction cascades and to regulate all cell processes. This contrasts with the known exquisite specificity of action of extracellular signals in different cell types in vivo. The confusion of the in vitro literature is shown to arise from several causes: the inevitable artifacts inherent in reductionism, the arguments used to establish causal effect relationships, the use of less than adequate models (cell lines, transfections, acellular systems, etc.), and the implicit assumption that networks of regulations are universal whereas they are in fact cell and stage specific. Cell specificity results from the existence in any cell type of a unique set of proteins and their isoforms at each level of signal transduction cascades, from the space structure of their components, from their combinatorial logic at each level, from the presence of modulators of signal transduction proteins and of modulators of modulators, from the time structure of extracellular signals and of their transduction, and from quantitative differences of expression of similar sets of factors.
signal transduction; effect of hormone modulations; isoforms; combinatorial logic
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MANY RELATIONS DESCRIBED IN...
HOW IS CELL SPECIFICITY...
LOSS OF SPECIFICITY MAY...
UTILITY OF NETWORKS
GENERAL CONCLUSIONS
REFERENCES
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CELL SIGNAL TRANSDUCTION ENCOMPASSES
all the biological and biochemical phenomena that lead from the
perception of a signal by a cell to the response of the cell. The
signal transduction machinery of a cell integrates all the signals it
recognizes and translates them in a coordinated behavior. A signal for
a cell is whatever is recognized as such by a receptor that itself
initiates a response to this signal. A receptor is the structure that
recognizes and reacts to the signal and interprets the specificity of
the signal. These are circular definitions. Our physiology, which integrates the requirements of a living organism (for metabolism, growth, reproduction) and its responses to the outside world, uses
thousands of signals. For each cell, these include hormones and
neurotransmitters, signals from neighboring cells, soluble such as
paracrine factors, or membrane bound such as ephrins, and signals from
the inert substratum such as fibronectin. Because each signal may be
recognized by different receptors (e.g., for norepinephrine or
serotonin), the number of receptors is a multiple of the number of
signals; hence the tremendous complexity and specificity of signals and
their receptors. One category of receptors, the seven transmembrane
receptors, are coded by ~700 genes. i.e., ~2% of the number of
genes in the human genome. This contrasts with the rather limited
repertoire of known signal transduction pathways that are modulated by
such receptors, with only a few ubiquitous intracellular signal
molecules (cAMP, cGMP, Ca2+, etc.), phosphorylation
cascades, and other pathways [nuclear factor (NF)-
B, etc.].
The cross signalings (anthropomorphically called cross talks) between
the various cascades further simplify the picture in appearance:
everything seems to modulate everything (Fig.
1). See, for example, a recent title:
"Signaling networks
do all roads lead to the same genes?"
(261). Furthermore, the opposite cross signalings between
cascades, as reported, give a totally incoherent picture
(141). It is striking that excellent reviews on known signal transduction proteins or cascades mention so many demonstrated effects obtained in different cell models that the authors have great
difficulties in drawing a coherent picture or end up by concluding for
each cascade, or even enzyme: "X is regulated by all signal
transduction cascades and regulates almost all cellular processes, from
gene expression to cell death" or "these results suggest that the
pathway linking A to B involves the integration of numerous signal
transduction steps by a highly complex network" (6, 18, 19, 25,
26, 28, 42, 77, 92, 93, 121, 125, 179, 260, 271, 272, 275,
298-300, 359, 360). The very restricted phenotypes of
knockout mice models in which a supposedly essential protein is absent
do not fit with such statements. The impression left was recently
summarized: "elegant complexity coupled with hopeless confusion
better defines our current state of knowledge" (309). In
fact, such reviews give a comprehensive picture of all the interactions
that may exist in mammalian cells, i.e., of the toolkit available for
differentiation. On the other hand, attempts to give a unifying picture
lead to unwarranted generalizations and/or selective consideration of
the literature and scientific myths (e.g., "Ca2+ causes
cell growth"). Thus a first paradox, which has been spelled out for a
few cascades (226, 227, 230), opposes on the one hand the
multiplicity and specificity of signals, their receptors, and their
effects and on the other hand the nonspecificity or promiscuity of the
few signaling cascades (78, 167, 205).
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Another paradox arises from the use of simplified models for the study
of normal physiology, namely, the behavior of the cell within the whole
organism, preferably human. Because of the limitations of clinical
investigation and ever-increasing restrictions of animal
experimentation, one has to rely on models that, from experimental animals to reconstituted systems, may lose in physiological relevance what they gain in simplicity and in definition (Table
1). In the 1960s hundreds of articles,
published in the best journals, on the direct action of thyroid hormone
on mitochondria or on various enzymes were undoubtedly true but
physiologically irrelevant. There is no doubt that work on cell lines
has tremendously enriched our knowledge of signal transduction, of the
actors involved, and of their interactions. However, the literature on
signal transduction shows that similar pathways may have different,
sometimes opposite, effects in different cells. This suggests that, if
we are interested in the human thyroid cell, it is this cell type that
we must study. In fact, the choice of a model dictates our concepts, of
which we become prisoners. If enough researchers use a model, they tend to forget about the caveats and to reject in their research or as
referees the ugly facts that may question their way of life. They
become a constituency of the model. Furthermore, it is easier to
publish clean data and mechanisms on cell lines than more disperse and
less clear-cut in vivo results. But then, as one mentor asked after a
seminar on some peculiar properties of a much-used cell line model,
"So what?" Of course, this should not detract from work on cell
lines because this allows us to identify new partners and interactions
and defines what is possible in signal transduction. On the other hand,
work on physiological models defines what is relevant in the physiology
of a given cell type at a given stage.
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In this review we analyze the physiological relevance of signal transduction data and discuss the mechanisms that, despite the apparently generalized "textbook" schemes, account for the exquisite specificity of in vivo cell signaling. Furthermore, we explore the possible biological consequences of a loosening of this specificity. For graphic representation of signal transduction pathways, we use a recently proposed system (267).
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MANY RELATIONS DESCRIBED IN SIGNAL TRANSDUCTION PATHWAYS MAY NOT BE PHYSIOLOGICALLY RELEVANT |
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TOP
ABSTRACT
INTRODUCTION
MANY RELATIONS DESCRIBED IN...
HOW IS CELL SPECIFICITY...
LOSS OF SPECIFICITY MAY...
UTILITY OF NETWORKS
GENERAL CONCLUSIONS
REFERENCES
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Attempts to synthesize any field of the signal transduction literature these days either simplify it in a personal, selective, and therefore distorted way or end up giving a very confusing picture. Part of the confusion may be clarified by considering the systems used to obtain the data and their possible artifacts (188, 309).
Loss of Biologically Important Information in Simplified Systems
Simplification of the systems used for the study of signal transduction is a double-edged sword. At each level from the in vivo study of humans to the precise molecular definition of proteins by X-ray crystallography, what we gain in precision, rigor, and definition we may lose in relevant biology (Table 1).There are numerous examples of apparent discrepancies between findings
in simplified systems, e.g., in vitro and the situation in
vivo. For instance, expression of the "cell
proliferation signal" epidermal growth factor (EGF) in transgenic
mice causes growth retardation (41). Lack of
phosphoinositol-3-kinase (PI3K)-
, an essential element of
proliferation cascades, leads to colorectal carcinomas
(292). There are great differences, not commented on by
the authors, between the specificities of G proteins and their
-subunits for their controlling receptors in membrane preparations and in whole cells (122). Similarly, the
history of knockout mice is rich in genes whose protein product is
essential for a cell process in vitro but not in vivo.
The crucial fact that teratocarcinoma cells aggregated with normal morulas give rise to normal cells in adult animals (265, 148) or that bone marrow cells injected in heart regenerate myocardium (253) testifies to the importance of the proper tissue environment in the behavior of the cell. Similarly, the interrelations between cancer cells and their neighboring cells are fundamental to their biology (89). Normal ovarian stromal cells promote the growth of normal ovarian epithelial cells but inhibit the corresponding tumor cells in vivo (258). These observations led to the development of new transgenic models in which oncogenes can be activated by a spontaneous recombination event rather than systematically in all cells of a given type (161).
Autocrine and paracrine effects may differ in culture and in vivo because of the dilution of factors in vitro, the washout of factors in vivo, or the absence of a necessary factor in the medium. In COS7 cells, for instance, the stimulatory effect of insulin-like growth factor (IGF)-I on the mitogen-activated protein (MAP) kinase (MAPK) cascade is secondary to the autocrine release of EGF (287). The simple manipulation of cells in culture introduces new variables: change of medium or even minor mechanical stress causes ATP release and activation of the ubiquitous purinergic receptors (254, 255). Because many biological effects require the conjunction of several factors, simplified systems may lose properties, including the specificity of interactions. For instance, direct interactions of transcription factors with DNA in acellular systems are much more promiscuous than in vivo (43).
Yet, to define mechanisms, simplified systems and even pure molecular species are necessary. To fully understand the reaction of a pharmacophore with its target one must define the precise structures involved. Still, the biological relevance of the findings needs to be validated up to the level of the human organism. Animal models, transgenics or knockouts, general or local, permanent, permanently or transiently inducible, allow us to test precisely the role of a given gene in vivo. Defined human genetic diseases, when they exist, allow extension of the conclusions to humans.
Arguments In Favor of a Causal Relationship May Not Be Proofs
The best argument in favor of the hypothesis that a biological event is necessary for a signal transduction pathway is to show that its suppression inhibits the downstream events. Suppression can be achieved by pharmacological inhibitors, antibodies, dominant-negative or competing peptides or proteins, deletion of the protein by inhibition of its synthesis (e.g., antisense, RNA interference), or gene knockout. For inhibitors, the postulated suppression must take place in the system studied under the conditions used and it must be specific. Such controls are often missing. Many hormonal effects have been related to protein kinase A (PKA) because they are inhibited by the supposedly specific H89, which, in the same concentration range, inhibits MAP kinase-stimulated kinase (MSK1), a kinase downstream of MAPK (332).In addition, the fact that an event is necessary does not necessarily
imply that it is a required step in the causal relationship sequence
(Fig. 2A). Metabolites or an
O2 supply are necessary for the survival of many cells and
therefore for the operation of their signal transduction pathways, but
they are not part of these pathways!
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Demonstration that experimental induction of what is supposed to be the primary event causes the downstream steps of the pathway is an argument but no more. Constitutively active forms of signal transduction proteins are often used for such purpose, although their specificity may also have been altered, as shown for EGF receptors (EGFRs) (210). Failure to induce the consequences may just indicate that parallel events are also necessary (Fig. 2B). Overexpression of the primary event will be ineffective or inhibitory if the effect is biphasic vs. concentration (182), i.e., in hormesis (36). This is the case for cAMP induction of proliferation in granulosa cells (282) (281) or for p53 and apoptosis (187) (Fig. 2C). Constant expression of the primary event will be ineffective or inhibitory in a sequential process in which each successive step requires the arrest of the previous one (e.g., in phagocytosis with extension of the membrane, engulfment, scission of the vesicle from the membrane, etc.; Refs. 12, 228). Induction per se indicates that the initial event may cause the downstream consequences, but the fact that this event actually takes place in the pathway remains to be proven. Preferably, as laid out by Robison et al. (280) in their rules for cAMP causal relationships, activation/inhibition of each of the actors of a cascade should be shown in the intact cell, direct activation/inhibition of each actor by its upstream modulator should be demonstrated, and the kinetics and concentration effect relationships should be compatible with the proposed scheme.
Validity of Reported Relations May Be Restricted to Artificial Systems
Many interrelations within and between elements of the signal transduction pathways are protein-protein or protein-DNA interactions. The methods used to define such interactions have provided a tremendous yield of new information. However, their results should be properly assessed and their physiological relevance validated. Protein-protein interactions are difficult to study at the low concentrations that prevail in normal cells. For instance, coimmunoprecipitations depend on the affinity and specificity of the antibodies used, on the dissociation rate of the interaction, and on the concentrations of the targets to be demonstrated in Western blots. Of course, the investigator can diversify the antibodies and modulate their concentrations, the washings, etc. To overcome such difficulties the overexpression of one or several proteins in transfected systems has become very general. Factors of overexpressions up to 100-fold are common. At these concentrations, weak, nonphysiological interactions and effects may well take place and the specificity of action of isoforms may disappear (see, for example, pRb and p107; Ref. 158, Fig. 3, A and B). For example, proteins that associate with the GABA receptors and cluster them in transfected cells may not do so in their native neurons (171, 172). Moreover, interactions that are normally impaired by compartmentation may occur in such systems. If an interaction is constrained by stoichiometric binding in a protein scaffold, even a doubling of the concentration of one of the locked proteins may be sufficient to allow spillover outside (Fig. 3C). On the other hand, overexpression of a scaffold protein will segregate its binding proteins in separate complexes and thus have an inhibitory effect (Refs. 197, 252; Fig. 3D). Expression constructs can interfere with nuclear receptors or transcription factors. From such artificial interactions authors infer effects on the corresponding endogenous proteins. In this case, the cell is no more than a glorified test tube.
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Other confounding factors in transfection studies are the use of constitutively activated mutants whose persistent activity does not reproduce the temporally organized activation of the natural proteins or, conversely, the use of transient transfections that do not reproduce the sustained activity of oncogenes, or the use of dominant-negative mutants whose actions may be much more diverse than foreseen (309). Similarly, the whole field of gene regulation, which has relied on cotransfections of plasmids expressing transcription factors and promoters with reporter genes, is undergoing a reappraisal now that it is realized that genes in normal chromatin may behave differently, or at least in a more sophisticated way, than genes in the naked or poorly "chromatinized" plasmids. In Drosophila, for instance, there is little correlation between binding in vitro of transcription factors to DNA fragments and DNA binding in vivo (21).
In vitro acellular systems with proteins, purified or not, are also
used and thus have little relation to physiology. They may allow us to
estimate thermodynamic properties or to pinpoint possible interactions.
However, the concentrations used may be much higher than in the cell
and may generate many false positives. Moreover, the artificial system
used may lack components that would confer specificity. Interactions of
Hox transcription factors with naked DNA are much less specific than
they are in vivo (150, 151). G complexes lose their
target specificity in acellular systems (73).
Similar reservations hold for yeast double-hybrid systems. For instance, steroid receptors in double-hybrid systems respond to all ligands by activating the downstream promoters, whereas in vivo some ligands act as agonists and others as antagonists. To reproduce the in vivo situation, Yamamoto et al. (371) had to introduce also in yeast the needed coupling factors. Still, as a first step to define the repertoire of possible interactions, this method has allowed gigantic steps (150, 151).
All interactions proposed on the basis of transfection, double-hybrid, or acellular experiments should therefore be validated at the normal concentrations in the cells in which they are supposed to take place, which is easier said than done (247). In each case, of course, the investigator can overexpress to detect unfavorable interactions (for example, interactions that would require the nonexisting phosphorylation of the protein) or underexpress to indicate specificity. Conviction about the validity of interactions demonstrated in vitro arises from the convergence of independent arguments.
Possible Relations May Not Apply Because the Proteins Involved Are Not Expressed in the Same Cells in the Tissue or in the Same Compartment in the Cell
To interact, proteins must be expressed in the same cell. Evidence for this assumption may be invalid for several reasons. The detection by PCR of the corresponding mRNA in the cell may be due to minor "illegitimate transcription" as, with enough cycles of PCR reaction, every mRNA can be detected in every tissue. With such evidence, in the thyroid field, eye muscles would express thyroglobulin, thyroperoxidase, and thyrotropin (TSH) receptor and could be considered as pseudo-thyroid follicular cells! On the other hand, tissue distribution studies (for proteins or mRNA) do not indicate which cells contain the protein studied unless one relies on immunohistochemical or in situ hybridization evidence. Human thyroids contain many muscarinic receptors, but, in contrast to the dog thyroid, the receptors are not in their follicular cells.For an effect to take place (e.g., DNA synthesis after growth factor action), all the elements of the corresponding signal transduction cascade should be present in the cell. In fact, illicit expression of protooncogenes in cells in which they are not normally expressed is a major cause of cancer (274). The finding that EGFR activation by G protein-coupled receptors (GPCRs) and the consequent cell division may require the processing of cell-bound pro-EGF by a metalloprotease, itself activated by the cascade downstream of the GPCR (269), explains why such a mechanism operates only in some cases (58). In addition, quite a few different mechanisms have been proposed in different cell types to explain GPCR activation of the growth factor receptor and MAPK cascades, with no attempt to disprove other hypotheses (120, 125, 170, 214, 215, 231, 294, 354, 382). Most of these articles refer to "the mechanism of EGFR activation by GPCRs."
Similarly, proteins may be restricted to different cell
compartments or even to distinct macromolecular assemblies (scaffolds) that insulate them from others and thus prevent interaction, even though all are present in the same cell. This compartmentation generates functional modules, i.e., discrete entities whose function is
separable from that of other molecules (133). By
generating such a module with the elements of the MAPK cascade, the
yeast scaffold protein Ste 5 confers to nonspecific enzymes the
specificity of action of mating hormones (Fig.
4; Refs. 61,
95).
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Demonstrated Interactions May Only Occur in Some Cell Types, in Some Species, or in Model Cells, Which Explains Why a Cascade May Have Different and Even Opposite Effects in Different Cells
Differentiation in about 200 different cell types implies a specific program of protein expression for each of them. A clear indication of specificity is given by the numerous examples of opposite results of the same cascade in different cells. The same Ras oncogene product blocks proliferation in human fibroblasts and induces it in human thyrocytes and immortalized fibroblasts (57, 112, 113, 250). It induces cyclin D in an intestinal cell line, while causing cyclin D1 phosphorylation and degradation in Rat1 fibroblasts (303). The same cAMP cascade inhibits cell proliferation in many cells (e.g., fibroblasts and other cells of mesodermal origin) but triggers it in some others (thyrocytes, somatotrophs, etc.); depending on the cell type, it activates, inhibits, or does not modulate MAPKs (285). In tadpoles the same hormone, triiodothyronine (T3), acting on the same T3 receptor, induces cell death in the tail and cell proliferation in the rest of the body (308, 328). NF-B inhibits
apoptosis in most normal cells but induces it in some cancer
cells (289). Even supposedly identical cells (e.g.,
endothelial or mesenchymal cells) are different in different tissues
(2, 319). Blood vessels in endocrine tissues have a
specific angiogenic mitogen (190).
Activation of the same pathway in the same cell type in different species may also lead to opposite results (209). The TSH receptor in human thyroid cells activates both the cAMP and phospholipase C cascades and accordingly activates thyroid secretion by the former and thyroid hormone synthesis by the latter, whereas in dog thyrocytes it only activates the cAMP pathway, which activates both functions (348). The same end result of TSH is obtained by different pathways in dog and human. In vertebrates as in viruses, evolution often conserves the function but may achieve it by different mechanisms.
Effects May Not Occur at All Times in a Given Cell
Receptors and signal transduction proteins are differentially expressed during embryogenesis, growth, and even under different physiological conditions. Effects may therefore differ at different stages. In fact, because signals during embryogenesis act through a few cascades, embryogenic development could not occur if these cascades were not interpreted differently by the target cells at different stages (62, 102). The whole early tissue organization in Xenopus development depends on the well-defined, orderly time sequence of the action of four inducing factors on cells, which becomes different at each stage (314). The same Ras activation applied to Drosophila imaginal tissue at different stages leads to proliferation, apoptosis, or suppression of apoptosis (162). The evolution of cells in embryogenesis is now mimicked in vitro in embryonic stem (ES) cells (212). A maturating dendritic cell or T lymphocyte changes its panel of secreted cytokines and cytokine receptors, i.e., its signaling systems, within a few hours (27, 249).The same stimulus may achieve the same result by different mechanisms
at different times of the life of a cell, e.g., protein kinase C
(PKC)-
is required for T cell receptor-induced NF-B activation in
mature but not immature T lymphocytes (323).
Many effects depend on the cellular environment. Vasopressin, acting
through its V1a receptor, activates Gq in proliferating Swiss 3T3 cells but Gq and G13 in cells in the
G0/G1 phase of the cell cycle (1).
In hepatocytes in culture, norepinephrine stimulates DNA synthesis
through -receptors at low density and through 
-receptors at high
density (166). Muscarinic receptors stimulate growth of
quiescent NIH 3T3 cells but inhibit it when the cells are growing
(245). The same increase in cytosolic Ca2+ may
push or retract the growth cone of a nerve, depending on preexisting
Ca2+ or cAMP level (378). Malignant mammary
epithelial cells may revert to a normal phenotype in a specific
intracellular matrix environment (23).
Cell Lines May Not Be Good Models of Their In Vivo Counterparts
Most articles on cell lines extrapolate their findings to the in vivo cell counterpart, e.g., extrapolating to the human thyroid cell what has been found in FRTL5 rat thyroid cell line. An ever greater part of the literature on cell signaling bears on cell lines as models in part because of the ease of working with them. In fact, cell lines are poor models of their in vivo counterparts. First, by definition, contrary to normal somatic cells, they are immortal, i.e., they reproduce indefinitely. The process by which they are obtained implies a selection over several generations of genetically altered cells having the desired properties, very different from those of the cells of origin. For instance, whereas tyrosine kinases of the Src family are necessary for the proliferating effect of platelet-derived growth factor (PDGF) in normal fibroblasts, they are not in NIH 3T3 cells or other cell lines (32). It is striking that similar rat thyroid cell lines selected by different criteria, the PCCl3 and the FRTL5 cell lines, exhibit quite different properties. For example, they require one (FRTL5) or two (PCCl3) oncogenes to become transformed. Finally, cell lines may evolve: the FRTL5 cells described at their origin required insulin and TSH to grow. Presently available samples require only one of the two or, in some laboratories, only insulin, TSH having just a complementary role. Some FRTL5 cells used in different laboratories are stimulated by EGF, some not; in some, serum enhances TSH mitogenic action, in some not. What is the interest for physiologists of the differences between two thyroid cell lines or between the same cell line having evolved in different laboratories? Another example of a molecular evolution of cell lines is the progressive accumulation and selection of cells with larger alleles due to unstable triplet repeats (117).Similarly, cancer cell lines are often studied as representative of in vivo cancer cells even though they have developed new characteristics: p53 is mutated in thyroid and esophageal carcinoma cell lines but not in the corresponding primary tumors (327, 368). p16INK4 is inactivated in thyroid tumor cell lines but not in thyroid tumors (37). PTEN mutations are detected in melanoma cell lines but little in melanomas (379-381). Even in cancer cells the characteristics of disseminated cells can be very different from those of the tumor that releases them (177). How relevant to metastasis are human cancer cell lines injected in animals (318)?
Intuitively, one would guess that the variations between similar cells (e.g., thyrocytes of different species) would be much greater at the initial steps of the cascades than at their core mechanisms: there are hundreds of receptors modulating cAMP levels but only six isoenzymes of cAMP-dependent kinase, and these have the same substrates. However, this is not always true, as variations may also occur at the core of signal transduction pathways: in dog thyrocytes cAMP activates cyclin D/cyclin-dependent kinase (CDK)4 complexes without inducing cyclin Ds, whereas in FRTL5 cells it does so by inducing cyclin D1 (65).
Conclusions on Physiological Relevance of Reported Effects
It is therefore dangerous to extrapolate to other systems the data obtained in one species. One sees articles in which data on human, dog, or pig thyroid cells in primary cultures or rat thyroid cell lines are combined in reasoning about the nonexistent paradigmatic "the thyroid cell." Although the existence of a given mechanism is often demonstrated only in one system, articles imply, implicitly or explicitly, that the mechanism described is general (e.g., "activation of the EGFR by seven transmembrane receptor," "cAMP activates MAPK"). The specificity of cell signaling in different cells, even for the same or similar extracellular signals, and even through the same initial receptor, is demonstrated a contrario in reviews that attempt to synthesize our present knowledge. In a recent article on seven transmembrane receptors and cell proliferation, no two of the systems described work in the same way, and even when one receptor is considered, its mitogenic cascade differs from one model to another (125). The buzzwords of such reviews are "complex, pivotal, subtle..." (260, 261). This explains the wrong impression of confusion emerging from such reviews.The map of all possible interactions and causal relations in signal transduction should therefore be considered as a map of possibilities, only few of which really take place at a given time in a given cell type. The exquisite cell- and stage specificity in signal transduction is fortunate for the pharmacologist (364) who aims at such a specificity for his drugs.
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HOW IS CELL SPECIFICITY OF ACTION OF SIGNAL TRANSDUCTION CASCADES ACHIEVED? |
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ABSTRACT
INTRODUCTION
MANY RELATIONS DESCRIBED IN...
HOW IS CELL SPECIFICITY...
LOSS OF SPECIFICITY MAY...
UTILITY OF NETWORKS
GENERAL CONCLUSIONS
REFERENCES
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Cell Responses Depend on the Pattern of Their Protein and Isoform Expression
The specificity of response to one cascade in different cell types depends on its differentiation, i.e., on its protein composition and, therefore, on the genes whose promoters are accessible. For example, in kinase cascades the response to the same cAMP and cAMP-dependent kinase depends on the population of phosphorylable proteins in a specific cell type, i.e., on its differentiation. Similarly, the response to a similar transcription factor in different cells depends on the nature of the gene promoters that are accessible.At each step of most cascades, several isoforms of proteins perform overlapping functions. They may be encoded by different genes or result from different mRNA splicing of the same gene or from postranslational processing. In mammalian cells there are at the present time 10 adenylate cyclases, more than 40 cyclic nucleotide phosphodiesterases (315), 70 A-kinase-anchoring proteins (AKAPs) (74), and 11 families of nonreceptor tyrosine kinases (279). Evolution multiplies the varieties of possible isoforms at each stage, from one kinase at each step of the MAPK STE pathway in yeast, Caenorhabditis elegans, and Drosophila to several kinases in mammalian cells (61). This explains why work on such simple model organisms is fundamental to demonstrate basic mechanisms and schemes, their actors, and their interactions and thus give the foundation of signal transduction. It also explains why direct extrapolation to mammalian cells is risky.
Because the isoforms have relatively similar properties, they are
often, when discovered, lumped together as though interchangeable. In
fact, more detailed investigations reveal different (sometimes opposite) and overlapping regulatory properties, e.g., the positive or
negative effect of phosphorylation by MAPK on phosphodiesterase 4D
isoforms (217) and the opposite and qualitatively
different effects of p53 isoforms (239, 240). They also
present cell type-specific expressions (30), intracellular
localizations (206) as determined by specific docking
domains (305), effectors (e.g., for receptors or kinases;
Ref. 126), and controls in expression at the
transcriptional, translational, and posttranslational level (70,
123). These differences may give the isoforms entirely different
physiological roles. They may also respond differently to direct and
cross signalings. Isoforms of glucose transporters are usually tissue
specific, with a conserved transmembrane catalyzing transport domain
and different cytoplasmic tails allowing specific regulations
(236-238, 333). The same Ca2+ signal will
enhance or decrease cAMP accumulation depending on the type of
adenylate cyclase present (147). Whereas the - and -subunits of the G proteins transducing the action of seven
transmembrane receptors were long thought to be interchangeable, it
appears more and more that the response to a given receptor requires
the presence of a defined set of -, -, and -subunits
(140, 160, 278). E2F2 and E2F4 have opposite roles
on cell differentiation (257). With only some of the
possible isoforms present in a given cell, of all the possible controls
only the few permitted by this selection will operate in this cell.
This has been well demonstrated, e.g., for G protein -subunits in
the different cells of human fetal adrenal gland (30) or
for AU-rich element (ARE) binding proteins that regulate the stability
and translation of mRNA in embryos (142).
The nature of the expressed isoforms may itself depend on the physiological state of the cell: depolarization induces, through Ca2+ and Ca2+/calmodulin-dependent protein kinase, a different splicing of the pre-mRNA of the Slo channels and therefore the expression of different proteins with different allosteric properties (369).
The relatively low number of genes in the human genome seems to put a
lid over the number of possible isoforms of any protein. In fact, any
gene coding for an isoform of a signal transduction protein may also
code for several isoforms by mRNA alternative splicing (24,
119), e.g., each cyclic nucleotide phosphodiesterase gene for
3-10 alternatives (24). In these, the presence or
absence of one motif of protein-protein interaction in an alternative form of a protein may channel a pathway in a given direction or not
(24, 119) according to the protein recognition code
(321). The presence of one or another intraprotein
signaling module such as a protein phosphorylation motif may confer
positive or negative regulation by PKA (Ref. 66; Fig.
5) or by MAPK (340). A
splice variant of phospholipase C behaves as a negative regulator of phospholipase C
(242). Truncation by alternative
splicing of fosB into 
fosB leads to a different transcriptional
repertoire (290). Splice forms of an Eph receptor inverse
the adhesion/repulsion response caused by this receptor
(137). Variations in the upstream open reading frames of
mRNAs also greatly change the life of the mRNA and its translocation
efficiency (236-238).
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Finally, the repertoire of possible effectors of each signal transduction protein, as presently known, will probably expand in the future. When an action of such a protein is discovered, it is often assumed to be the only one until we are shown otherwise. Thus we restrict the role of GPCRs to their effects on G proteins (129, 134) and the role of PTEN to its protein phosphatase activity (68) to discover later that other effects exist. The specificity of isoform expression thus goes a long way in explaining cell specificity of responses.
Operation of a Pathway May Depend on Spatial Structure of Its Constitutive Elements: Subcellular, Membrane Localizations, Multiprotein Complexes
Spatial structure in the cell refers to cell compartments and to supramolecular complexes. Nuclear or cytoplasm compartmentation prevents many possible interactions, sequestrating active molecules from each other. For example, sequestration of MDM2 in the nucleolus by p19ARF blocks its inhibition of p53. The regulation of the cell cycle and of transcription represents a ballet of nuclear to cytosol import-export dynamics (110, 232, 266). Similar dynamic controls of protein localization operate even in bacteria (154, 155, 304). Thus the existence or not of a translocation mechanism or of its regulation may greatly differentiate the effects of a signal transduction pathway in different cell types. Loss of spatial structure of signal transduction pathways is a cause of several diseases (243).The targeting or nontargeting of a protein at the membrane may change
the whole pattern of its interactions. Such targeting may involve
protein-lipid or protein-protein interactions (193). It
may require specified mRNA localization and protein production (313). Nonprenylated Ras or, in some cells,
nonmyristoylated cGMP-activated kinase does not activate its cascade
(136, 219). Insulin and WNT both inhibit glycogen synthase
kinase 3, but this leads exclusively to increased glycogen synthesis
for insulin and exclusively to increased availability of -catenin
for WNT (71). Integrin necessarily stimulates at
defined contacts (298-300). Compartmentalization goes
further with the segregation of some membrane proteins within or
without lipid rafts and caveolae (107). GPCRs may have
only access to their compatible G proteins in "raft" subdomains of
the membrane (254). This could explain the discrepancy between TSH promiscuous effects on G proteins in isolated membranes and
its more restricted effects in intact cells (5).
The numerous proteins whose main function is to anchor signal
transduction proteins (e.g., the AKAPs for cAMP-dependent protein
kinase) to definite structures show the importance of such
localizations (86).
Similarly, scaffold proteins also have the role of assembling supramolecular complexes, bringing together signal transduction proteins in one permanent or transitory functional unit, module, or "signalosome" (35, 133, 260, 261). Such complex functional multiprotein assemblies were first described for metabolic enzymes, accounting for metabolic channeling (256). Their properties are more than the sum of the properties of their individual constituents (106). For instance, by associating tightly phospholipase C and its G protein, the INAD scaffold protein allows the former to activate the GTPase activity of the latter and thus to shorten the signaling of the photoreceptor (51, 95). For cAMP, inositol 1,4,5-trisphosphate (IP3), and even phosphatidylinositol-3,4,5-trisphosphate (PIP3), colocalization of the signal generation effector and remover allows highly localized effects in dendritic spines (169). The synapse or the neuromuscular junction is a permanent multimeric complex constituted sequentially (84, 329-331) and dependent on specific targeting (44-46, 132, 171, 172). In yeast the Ste5 scaffold protein channels the activation of the MAPK pathway by mating factor to mating-specific genes (Refs. 61, 108; Fig. 4). 14-3-3 Proteins have a similar role in vertebrates (241, 370). Activation of the T cell or B cell receptor requires the constitution of a large integrated multimeric complex in membrane rafts (181, 326, 336, 358). The stability of such complexes varies from very transient to quasi-permanent (97).
Specificity of Response of Different Cell Types May Result from a Combinatorial Logic
From specific combination of elements involved in parallel.
In this type of regulation, a few factors may combine to specify many
different instructions. A signal can be compared to a letter in a word:
it has no meaning per se, only the combination of several letters has a
meaning and the same letter used in a different word or combination may
have a different or opposite meaning. Such regulations have been
demonstrated, for example, for gene expression and for odor
discrimination by the olfactory system (34, 109, 220). In
the former, it is generally the combination of several complementary
DNA regulatory elements and their specific transcription factors that
confers specificity and strength to a promoter (204). Thus
even broadly overlapping sets of regulated transcription factors may
have very different end effects (Refs. 94,
371; Figs. 6 and
7). Assuming that in humans, as in other
species, transcription factors may represent 5% of the 100,000 expressed mRNAs, the number of possible combinations of three (with
repetitions) is 2 × 1010! The distribution of the 30 Ets transcription factors in different cell types can be considered as
a fingerprint of each type (223). The expression of
thyroid-specific genes depends on three transcription factors [thyroid
transcription factor (TTF)1, TTF2, Pax8], each of which is expressed
in the thyrocyte and in at least one other cell type, but all three are
only coexpressed in the thyrocyte (60). Similarly
different cascades with partially overlapping sets of induced genes may
also exhibit the same combinatorial logic (Ref. 94; Fig.
8).
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), 5 signals could allow 25 = 32 combinations.
Examples abound in physiology and pathology. Different receptors acting
on the Janus kinase (JAK) system act on different combinations of Jaks
that will activate different signal transducer and activator of
transcription (STAT) transcription factors (275). In
macrophages, inflammation results from the activation of different
Toll-like receptors, each triggering secretion of specific sets of
chemokines with partially overlapping target receptors differentially
expressed in target cells (Ref. 33; Fig.
9). Heterodimerization of EGFR-like
receptors generates many different receptors that discriminate between
ligands and between effectors (251, 324). Control of
cyclin-CDKs in different tissues results from various combinations of
the CDK inhibitors, which explains why the different combinations of
knockouts generate different types of tumors (101).
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to induce maturation of human
dendritic cells (295). Another example is the necessary
complementarity of PI 3-kinase and Ras activation in the induction of
metastasis by MET-hepatocyte growth factor (HGF) receptor
(13). Such a requirement explains why general overexpression of only one protooncogene leads only to a few types of
tumors. An example at the level of protein direct activation is the
necessary coincidence of RacGTP and p67phox to activate
O
From combination of unregulated parallel factors and of one
regulated factor in each cell type: the triggering reaction or
"switch."
Many different signals operate in their target cell by inducing one or
several rather ubiquitous transcription factors, i.e., early-immediate
genes. C-Fos and Egr1, which are induced in many cells in response to
all sorts of signals, are an obvious example (372).
Cell-specific response is given by the other cell-specific transcription factors that are also necessary for induction of a
specific gene in the cell. The nonspecific Egr1, in conjunction with
SF1, leads in gonadotroph cells to the subsequent induction of LH
gene (165, 338) and, in conjunction with WT1, induces Mullerian inhibiting substance in Sertoli cells (Ref. 310;
Fig. 10). The interleukin (IL)-6
promoter in monocytes requires cAMP response element-binding protein
(CREB), AP1, and cellular enhancer-binding protein (CEBP) but is
activated by the newly released NF-B (344). Just as in an electric circuit the response to an electric switch corresponds to the system downstream (light, air conditioning, heating,
etc.), the transcription response to a signaling pathway and its
general triggering transcription factor depends on the existing
tissue-specific transcription factors and their regulatory elements.
This concept would account for synexpression, i.e., the expression with
a similar pattern of a set of genes in a cell type in response to a
stimulus (124, 246), and in some cases for cell
differentiation (124). It goes a long way in explaining the puzzling question of how a promiscuous signaling cascade can achieve unique effects in a given cell type. The phosphorylation of
histones H1 by cAMP-dependent kinases in different cell types presumably causes a general loosening of the chromatin structure, which
might facilitate later, more promoter-specific transcription effects,
fits with the same concept. This concept of a single switch whose
meaning is only determined by the specific existing elements of the
cell also applies to early steps of signal transduction cascades. The
effect of a general signal such as Ca2+ or cAMP in a cell
depends on its complement of existing protein substrates of calmodulin
or PKA (Fig. 11). Similarly, the
pattern of gene expression induced by EGF in a single cell line depends on the composition of the cell matrix (373).
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From specific combination of sequential factors.
In the specificity of nuclear receptors action at least six different
factors are involved, each of which can switch the sign (+ or ) of
the response: the regulatory element in the promoter DNA, the nuclear
receptor or the transcription factor binding to the regulatory element,
the hormone binding to the receptor, the phosphorylation of the
receptor, the other transcription factors present on the promoters, the
coactivator or corepressor binding to the receptor, and their
modulators and adaptors (157, 192, 221, 283, 371).
Other independent or dependent factors regulating the outcome are the
various feedbacks between transcription factors (276), the
methylation of the promoter and enhancer, and the state of the
chromatin (histone acetylation and phosphorylation, high-mobility
group (HMG) protein binding, polycomb group protein binding, etc.), DNA
methylation and chromatin structure being generally linked (40,
47, 152, 277, 297, 339, 353). "Coordination of large
sets of genes could be accomplished by affecting the function of
specific components of the transcriptional machinery" itself
(139). Moreover, some genes like that of the human
fibroblast growth factor (FGF)-1 have four different promoters,
differently regulated, directing the expression of four alternatively
spliced transcript variants (48).
Specificity of Response of a Cell to Different Cascades Having Apparently Similar Consequences May Result From Specific Combination of Biochemical Effects of Each Cascade: Common "Awakening" Reaction and Specific Effects
Some cell responses are common to many if not all signaling pathways in a given cell type. At the posttranslational level CREB and cAMP-responsive element modulator (CREM) phosphorylation and activation are caused by the cAMP cascade, intracellular Ca2+, and growth factors acting through MAPK, stress, and p38 and MAPK-activated protein (MAPKAP) kinase, etc. (63). Glycogen synthase kinase 3 is phosphorylated on the same serine and
inactivated by protein kinase B (PKB) and PKA
(202-204). At the transcription level the
early-immediate gene c-Fos is induced by almost any cell stimulant from
the growth factors inducing mitogenesis in fibroblasts to repetitive
activation of nondividing neurons (145, 235). Such common
responses to cascades having very different effects imply by definition
that these responses are general and nonspecific. In fact, they suggest
a sort of undifferentiated "awakening" reaction. Other more
specific effects of the cascades or the specific complement of
transcription factors (see From combination of unregulated
parallel factors and of one regulated factor in each cell type: the
triggering reaction or "switch") will determine the direction
or response the "awakened cell" will choose. For instance in the
thyroid, as in other cells (25, 26), each cascade, besides
its common activation of Rap1, also activates specific kinases with
more or less specific substrates (PKA for cAMP, PKC for diacylglycerol,
Ca2+/calmodulin-dependent protein kinases for
Ca2+ and Ras MAPK and PKB for insulin and growth factors),
which confers specificity to its action (Fig.
12; Refs. 55,
79, 81). Each cascade also induces a more or
less specific panel of early-immediate genes besides c-Fos. At the
level of chromatin, similar general rapid modifications suggested that
"diverse pathways can then contribute to the modification of these
proteins without the necessity of targeting these pathways to a
particular chromosomal site" (365).
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Specificity of Response to a Cascade May Depend on Timing of Stimulus
Timing may also explain specificity. A signal may be short or long, immediate or delayed, continuous or oscillatory. The multiple qualitative differences in effect that such modalities confer have been well studied in the case of the intracellular signal Ca2+ (18, 19, 75, 82, 83, 99, 202-204, 233). For example, the frequency of calcium transients dictates NF-B transcriptional activity and neuronal differentiation (143, 144, 317).
Calcium influx and consequent pituitary hormone secretion depend on the step duration of spontaneous depolarization (346). Insulin
stimulation of mitogenesis requires a longer occupancy of the receptor
than its metabolic action (311). In dog thyroid cells
IGF-I, which activates MAPK kinase for a short time and PI3K for
longer, has a permissive effect on TSH mitogenic action, whereas EGF,
which activates PI3K for a short time and MAPK for longer, has a
mitogenic effect in the presence of IGF-I (55). Short-term
stimulation of the MAPK pathway by EGF causes proliferation, whereas
longer stimulation by nerve growth factor (NGF) causes differentiation in PC12 cells (337). Similarly, short-term activation of
MAPK in Drosophila ommatidia causes growth and survival,
whereas longer activation causes differentiation (128) and
sometimes growth arrest (362). In regenerating liver, two
waves of MAPK activation are necessary for the division of hepatocytes:
an early wave for the awakening competence acquisition and a late wave
for progression and cyclin D induction (325). In T cells
long-term stimulation of Rap1 enhances the activation of Elk by the Ras
cascade, whereas short-term stimulation inhibits it (59).
The main mechanisms involved in the timing of effects in a signaling
cascade are the positive and negative feedbacks and feedforward
regulations (103). The presence of one such regulation may
completely modify the timing of a cascade. For example, negative
feedbacks and feedforward controls translate a constant stimulus in a
sequence of wavelike causally related events (Fig.
13). In a
biphasic effect the relative strength of a negative feedback on an
initial step may transform a positive to a globally negative effect and
conversely. Constant infusion of the hypothalamic stimulatory hormone
gonadotropin-releasing hormone (GnRH) leads, through desensitization of
its cascade, to an overall inhibition of gonadotropic pituitary cells.
Many cell processes imply an orderly causal sequence of events, some of
which require the extinction of previous steps and the absence of
subsequent events. This is evident for cell motion or phagocytosis (176). In a sequential process by which -adrenergic
receptor first activates Gs, then through arrestin
Gi and Src (213-215), because Gs and Gi have opposite effects, differences in
the kinetics of the sequence will lead to opposite results
(231). The presence of a single positive feedback
can confer bistability to a system (i.e., it is either on or off) and
thus permanence to an effect (334). Similarly, the
hysteresis of calmodulin-dependent protein kinase converts a short
Ca2+ signal in a much longer protein phosphorylation
(18, 19, 104). Thus differences in timing of a response to
a signal result from the characteristics of the proteins involved,
i.e., from the pattern of the proteins present.
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Cell Specificity of Response May Depend on Qualitative Difference of Protein Expression of Modulators
Modulators are proteins not involved in the signal transduction cascade themselves but which positively or negatively modulate the proteins of this cascade (Table 2). When a given protein has opposite actions, differential modulations of them will lead to one result or the opposite, e.g., cMyc, which induces cell proliferation or apoptosis (50). Examples of such inhibitory modulating proteins abound: starting from natural antagonists of extracellular signals, soluble receptors that compete with extracellular signals for membrane-bound receptors, kinase inhibitors, G protein inhibitors [regulator of G protein signaling (RGS) proteins; Ref. 312], and Bin1 or Groucho inhibitors of MYC or LLT1/TCF
transactivating action
(85), among others. An excess of scaffold proteins can
also cause inhibition (35). Other modulators enhance the
activation of a cascade, e.g., DARPP32, which after phosphorylation by
PKA inhibits PP1 and thus enhances and prolongs the phosphorylation and
thus the activity of CREB (306). Tyrosine phosphorylation of calmodulin increases many of its activations (53).
Targeting of a signal transduction protein, for example to the
membrane, may also greatly modify its activity.
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Some modulators even change the receptivity of a protein from one
signal to another. Receptor activity-modifying protein (RAMP) proteins
switch the specificity of the cGRP receptor to one or the other
hormone, RAMP1 to CGRP, RAMP2 to adrenomodulin (196, 236-238, 302). For hormone nuclear receptors, besides the
controls mentioned above, there are modulators of coactivators [e.g.,
pCIP for CREB binding protein (CBP)] and competitors.
There are even modulators of modulators such as p34SEI-I,
which antagonizes the inhibition by p16INK4a of
cyclin-CDK complexes (322), and prothymosin-, which
sequestrates the repressor of estrogen activity (224).
Finally, signal transduction cascades may induce, depending on the cell
type, the synthesis and secretion of autocrine or paracrine factors or
of proteases
) of genes by the cAMP cascade and the
diacylglycerol (DAG) protein kinase C cascade. Both cascades activate
(arrowheads) the cAMP response element-binding protein (CREB)
transcription factors. Each cascade also activates distinct
transcription factors X for cAMP and Y for DAG.
Both cascades induce B through CREB, but cAMP also induces A, which
requires both X and CREB, and DAG specifically induces C,
which requires both Y and CREB.
