Ca(2+) signaling in nonexcitable cells is typically initiated by receptor-triggered production of inositol-1,4,5-trisphosphate and the release of Ca(2+) from intracellular stores. An elusive signaling process senses the Ca(2+) store depletion and triggers the opening of plasma membrane Ca(2+) channels. The resulting sustained Ca(2+) signals are required for many physiological responses, such as T cell activation and differentiation. Here, we monitored receptor-triggered Ca(2+) signals in cells transfected with siRNAs against 2,304 human signaling proteins, and we identified two proteins required for Ca(2+)-store-depletion-mediated Ca(2+) influx, STIM1 and STIM2. These proteins have a single transmembrane region with a putative Ca(2+) binding domain in the lumen of the endoplasmic reticulum. Ca(2+) store depletion led to a rapid translocation of STIM1 into puncta that accumulated near the plasma membrane. Introducing a point mutation in the STIM1 Ca(2+) binding domain resulted in prelocalization of the protein in puncta, and this mutant failed to respond to store depletion. Our study suggests that STIM proteins function as Ca(2+) store sensors in the signaling pathway connecting Ca(2+) store depletion to Ca(2+) influx.

A typical protein kinase must recognize between one and a few hundred bona fide phosphorylation sites in a background of approximately 700,000 potentially phosphorylatable residues. Multiple mechanisms have evolved that contribute to this exquisite specificity, including the structure of the catalytic site, local and distal interactions between the kinase and substrate, the formation of complexes with scaffolding and adaptor proteins that spatially regulate the kinase, systems-level competition between substrates, and error-correction mechanisms. The responsibility for the recognition of substrates by protein kinases appears to be distributed among a large number of independent, imperfect specificity mechanisms.

The mitogen-activated protein kinase (MAPK) cascade is a highly conserved series of three protein kinases implicated in diverse biological processes. Here we demonstrate that the cascade arrangement has unexpected consequences for the dynamics of MAPK signaling. We solved the rate equations for the cascade numerically and found that MAPK is predicted to behave like a highly cooperative enzyme, even though it was not assumed that any of the enzymes in the cascade were regulated cooperatively. Measurements of MAPK activation in Xenopus oocyte extracts confirmed this prediction. The stimulus/response curve of the MAPK was found to be as steep as that of a cooperative enzyme with a Hill coefficient of 4-5, well in excess of that of the classical allosteric protein hemoglobin. The shape of the MAPK stimulus/response curve may make the cascade particularly appropriate for mediating processes like mitogenesis, cell fate induction, and oocyte maturation, where a cell switches from one discrete state to another.Although the biological responses associated with mitogenactivated protein kinase (MAPK) signaling are highly varied, the basic structure of the MAPK cascade is well conserved (1-3). The cascade always consists of a MAPK kinase kinase (MAPKKK), a MAPK kinase (MAPKK), and a MAPK. MAPKKKs activate MAPKKs by phosphorylation at two conserved serine residues and MAPKKs activate MAPKs by phosphorylation at conserved threonine and tyrosine residues (Fig. 1). The cascade relays signals from the plasma membrane to targets in the cytoplasm and nucleus.A number of other membrane-to-nucleus signaling pathways, such as the Jak/Stat pathways and the cAMP/protein kinase A pathway, employ just a single protein kinase. Why does the MAPK cascade invariably use three kinases instead of one? The possibility that the three kinase arrangement has evolved to allow signal ramification or amplification is attractive but, as yet, not well supported by genetic or biochemical evidence. We have explored the possibility that the cascade arrangement has important consequences for the dynamics of MAPK signaling. Here we shall focus on the steady-state responses of enzymes at each level in the cascade to varying input stimuli. The stimulus/ response curve of a typical Michaelis-Menten enzyme is hyperbolic, and the enzyme responds in a graded fashion to increasing stimuli. An 81-fold increase in stimulus is needed to drive the enzyme from 10% to 90% maximal response (see for example, the MAPKKK curves in Fig. 2). However, some enzymes exhibit stimulus/response curves that are steeper or less steep than the Michaelis-Menten curve. Goldbeter and Koshland have termed these responses "ultrasensitivity" and "subsensitivity," respectively (11-13). An ultrasensitive enzyme requires less than an 81-fold increase in stimulus to drive it from 10% to 90% maximal response (for example, the MAPK and MAPKK curves in Fig. 2); a subsensitive enzyme requires more than an 81-fold increase.The term ultrasensitivity emphasizes the fact that the upstroke of th...

No abstract

Xenopus oocytes convert a continuously variable stimulus, the concentration of the maturation-inducing hormone progesterone, into an all-or-none biological response-oocyte maturation. Here evidence is presented that the all-or-none character of the response is generated by the mitogen-activated protein kinase (MAPK) cascade. Analysis of individual oocytes showed that the response of MAPK to progesterone or Mos was equivalent to that of a cooperative enzyme with a Hill coefficient of at least 35, more than 10 times the Hill coefficient for the binding of oxygen to hemoglobin. The response can be accounted for by the intrinsic ultrasensitivity of the oocyte's MAPK cascade and a positive feedback loop in which the cascade is embedded. These findings provide a biochemical rationale for the all-or-none character of this cell fate switch.

It is becoming increasingly clear that bistability (or, more generally, multistability) is an important recurring theme in cell signaling. Bistability may be of particular relevance to biological systems that switch between discrete states, generate oscillatory responses, or ''remember'' transitory stimuli. Standard mathematical methods allow the detection of bistability in some very simple feedback systems (systems with one or two proteins or genes that either activate each other or inhibit each other), but realistic depictions of signal transduction networks are invariably much more complex. Here, we show that for a class of feedback systems of arbitrary order the stability properties of the system can be deduced mathematically from how the system behaves when feedback is blocked. Provided that this open-loop, feedback-blocked system is monotone and possesses a sigmoidal characteristic, the system is guaranteed to be bistable for some range of feedback strengths. We present a simple graphical method for deducing the stability behavior and bifurcation diagrams for such systems and illustrate the method with two examples taken from recent experimental studies of bistable systems: a two-variable Cdc2͞Wee1 system and a more complicated five-variable mitogenactivated protein kinase cascade.O ne of the most important and formidable challenges facing biologists and mathematicians in the postgenomic era is to understand how the behaviors of living cells arise out of the properties of complex networks of signaling proteins. One interesting systems-level property that even relatively simple signaling networks have the potential to produce is bistability. A bistable system is one that toggles between two discrete, alternative stable steady states, in contrast to a monostable system, which slides along a continuum of steady states (1-9). Early biological examples of bistable systems included the lambda phage lysis-lysogeny switch and the hysteretic lac repressor system (10-12). More recent examples have included several mitogen-activated protein kinase (MAPK) cascades in animal cells (13-15), and cell cycle regulatory circuits in Xenopus and Saccharomyces cerevisiae (16)(17)(18). Bistable systems are thought to be involved in the generation of switch-like biochemical responses (13,14,19), the establishment of cell cycle oscillations and mutually exclusive cell cycle phases (17, 18), the production of self-sustaining biochemical ''memories'' of transient stimuli (20,21), and the rapid lateral propagation of receptor tyrosine kinase activation (22).Bistability arises in signaling systems that contain a positivefeedback loop (Fig. 1a) or a mutually inhibitory, doublenegative-feedback loop (which, in some regards, is equivalent to a positive-feedback loop) (Fig. 1b). Indeed, Thomas (23) conjectured that the existence of at least one positive-feedback loop is a necessary condition for the existence of multiple steady states; alternative proofs of this conjecture are given in refs. 24-27. However, the existence of positive loop...

A simple negative feedback loop of interacting genes or proteins has the potential to generate sustained oscillations. However, many biological oscillators also have a positive feedback loop, raising the question of what advantages the extra loop imparts. Through computational studies, we show that it is generally difficult to adjust a negative feedback oscillator’s frequency without compromising its amplitude, whereas with positive-plus-negative feedback, one can achieve a widely tunable frequency and near-constant amplitude. This tunability makes the latter design suitable for biological rhythms like heartbeats and cell cycles that need to provide a constant output over a range of frequencies. Positive-plus-negative oscillators also appear to be more robust and easier to evolve, rationalizing why they are found in contexts where an adjustable frequency is unimportant.

In the early embryonic cell cycle, Cdc2-cyclin B functions like an autonomous oscillator, whose robust biochemical rhythm continues even when DNA replication or mitosis is blocked. At the core of the oscillator is a negative feedback loop; cyclins accumulate and produce active mitotic Cdc2-cyclin B; Cdc2 activates the anaphase-promoting complex (APC); the APC then promotes cyclin degradation and resets Cdc2 to its inactive, interphase state. Cdc2 regulation also involves positive feedback, with active Cdc2-cyclin B stimulating its activator Cdc25 (refs 5-7) and inactivating its inhibitors Wee1 and Myt1 (refs 8-11). Under the correct circumstances, these positive feedback loops could function as a bistable trigger for mitosis, and oscillators with bistable triggers may be particularly relevant to biological applications such as cell cycle regulation. Therefore, we examined whether Cdc2 activation is bistable. We confirm that the response of Cdc2 to non-degradable cyclin B is temporally abrupt and switch-like, as would be expected if Cdc2 activation were bistable. We also show that Cdc2 activation exhibits hysteresis, a property of bistable systems with particular relevance to biochemical oscillators. These findings help establish the basic systems-level logic of the mitotic oscillator.