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.