The frequencies of calcium oscillations are optimized for efficient calcium-mediated activation of Ras and the ERK/MAPK cascade

Abstract

Ras proteins are binary switches that, by cycling through inactive GDP- and active GTP-bound conformations, regulate multiple cellular signaling pathways, including those that control growth and differentiation. For some time, it has been known that receptor-mediated increases in the concentration of intracellular free calcium ([Ca(2+)](i)) can modulate Ras activation. Increases in [Ca(2+)](i) often occur as repetitive Ca(2+) spikes or oscillations. Induced by electrical or receptor stimuli, these repetitive Ca(2+) oscillations increase in frequency with the amplitude of receptor stimuli, a phenomenon critical for the induction of selective cellular functions. Here, we show that Ca(2+) oscillations are optimized for Ca(2+)-mediated activation of Ras and signaling through the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) cascade. We present additional evidence that Ca(2+) oscillations reduce the effective Ca(2+) threshold for the activation of Ras and that the oscillatory frequency is optimized for activation of Ras and the ERK/MAPK pathway. Our results describe a hitherto unrecognized link between complex Ca(2+) signals and the modulation of the Ras/ERK/MAPK signaling cascade.

Fig. 1.

The use of the Ca²⁺ clamp to generate synchronous Ca²⁺ oscillations within a cellular population. (A) Single-cell imaging of four HeLa cells randomly chosen from a cellular population during stimulation with 10 μM histamine. Although each individual cell responds to the addition of histamine by increasing intracellular free Ca²⁺, each does so with distinct temporal kinetics inducing Ca²⁺ oscillations that vary in their frequency and amplitude. (B) The use of the Ca²⁺ clamp for generating synchronous Ca²⁺ oscillations of varying frequency. Here, HeLa cells were treated with thapsigargin in Ca²⁺-free extracellular media before eliciting Ca²⁺ oscillations by exposure for 15 s to Ca²⁺-containing media at a frequencies of 120 s (Bi) and 200 s (Bii). Traces are the average of 15 cells randomly imaged within the population. (C) By altering the duration of exposure to Ca²⁺-containing media from 5 through 10, 15, and 20 s, synchronous oscillations of varying amplitude can be generated in a population of cells. For each condition, traces are the average of 15 cells randomly imaged within the population. (D) The temporal dynamics of individual Ca²⁺ oscillations generated by agonist stimulation and through the Ca²⁺ clamp are similar. Here, single-cell Ca²⁺ imaging was performed on HeLa cells stimulated with either ATP or histamine, comparing the kinetics of the response with that elicited by a 15-s exposure to Ca²⁺-containing media in the Ca²⁺ clamp after treatment with thapsigargin. Each trace is the average of 15 cells randomly imaged within the population.

Fig. 2.

Complex Ca²⁺ signals regulate Ras activation. (A) Generation of Ca²⁺ oscillations within a population of HeLa cells. Before recording, serum-starved HeLa cells were incubated for 15 min with thapsigargin in media lacking extracellular Ca²⁺ to deplete internal Ca²⁺ stores. Ca²⁺ oscillations of a fixed interspike interval (60 s) were induced for a period of 10 min by exposure for 10 s to Ca²⁺-containing media. Under these conditions, the average increase in [Ca²⁺]_i was 178 nM. To generate the nonoscillatory increase in [Ca²⁺]_i, the cells were exposed to extracellular media-containing 0.1 mM Ca²⁺ for the entire duration of the experiment. The average constant [Ca²⁺]_i under these conditions was 157 nM. Each trace is the average of 15 cells randomly imaged within the population. (B) [Ca²⁺]_i oscillations enhance the Ca²⁺ sensitivity of endogenous H-Ras activation at low-level stimulation. The comparative ability of [Ca²⁺]_i oscillations (•) or constant [Ca²⁺]_i elevation (○) to couple to Ras activation was determined under two conditions, oscillating Ca²⁺ (165 and 364 nM average) and constant Ca²⁺ (185 and 383 nM, respectively). Activation of Ras was determined as described in Methods after 10 min of Ca²⁺ manipulation.

Fig. 3.

The frequency of Ca²⁺ oscillations is optimized for activation of Ras and the MAPK pathway. (A) Activation of Ras after generation of [Ca²⁺]_i spikes with varying interspike intervals. HeLa cells were transiently transfected with 2 μg of H-Ras cDNA for 24 h. Serum-starved cells were treated with thapsigargin for 15 min, after which Ca²⁺ spikes of a fixed amplitude (365 ± 44 nM) were generated for 10 min over a range of interspike intervals (60–600 s). Activation of Ras was determined as described in Methods. Similar data were obtained in an examination of endogenous Ras activation (data not shown). (B) Activation of MAPK after the generation of [Ca²⁺]_i spikes with varying interspike intervals. Ca²⁺ spikes of fixed amplitude were generated for 10 min over a range of interspike intervals (60–600 s). Activation of MAPK was determined as described in Methods. (C) Ca²⁺-mediated activation of the ERK/MAPK pathway depends on MEK. Serum-starved HeLa cells were treated with U0126, STO-609, KN-93, and KN-92 as described in Methods. The cells were treated with thapsigargin for 15 min, after which Ca²⁺ spikes of fixed amplitude and interspike interval (120 s) were induced over a period of 10 min. Activation of ERK/MAPK was determined as described in Methods.

Fig. 4.

Ca²⁺ signals have a modulatory rather than a decisive role in regulating Ras signaling. (A) Activation of ERK/MAPK after generation of rapid [Ca²⁺]_i oscillations compared with ERK/MAPK activation after stimulation with 100 ng/ml EGF. Serum-starved HeLa cells were treated with thapsigargin. After 15 min, Ca²⁺ oscillations of fixed amplitude and interspike interval (60 s) were generated over a period of 10 min. Alternatively, cells were treated with 100 ng/ml EGF for 1 min. Activation of ERK/MAPK was determined as described in Methods. (B) Ca²⁺ oscillations can potentiate ERK/MAPK activation induced by suboptimal EGF concentration. Serum-starved HeLa cells were treated with thapsigargin for 15 min, stimulated with 300 pg/ml EGF, and either left untreated (TG) or exposed to fixed-amplitude Ca²⁺ oscillations over a range of interspike intervals (60–600 s). Activation of ERK/MAPK was determined as described in Methods and is plotted as fold activation over the control treatment, where cells were left untreated.