NR2B-NMDA receptor-mediated increases in intracellular Ca2+ concentration regulate the tyrosine phosphatase, STEP, and ERK MAP kinase signaling

Abstract

NMDA receptors regulate both the activation and inactivation of the extracellular signal-regulated kinase (ERK) signaling cascade, a key pathway involved in neuronal plasticity and survival. This bi-directional regulation of ERK activity by NMDA receptors has been attributed to opposing actions of NR2A- versus NR2B-containing NMDA receptors, but how this is implemented is not understood. Here, we show that glutamate-mediated intracellular Ca(2+) increases occur in two phases, a rapid initial increase followed by a delayed larger increase. Both phases of the Ca(2+) increase were blocked by MK-801, a non-selective NMDA receptor inhibitor. On the other hand, selective inhibition of NR2B-NMDA receptors by Ifenprodil or Ro 25-6981 blocked the delayed larger phase but had only a small effect on the rapid initial increase. The rapid initial increase in Ca(2+), presumably because of NR2A-NMDAR activation, was sufficient to activate ERK, whereas the large delayed increases in Ca(2+) mediated by NR2B-NMDARs were necessary for dephosphorylation and subsequent activation of striatal-enriched phosphatase, a neuron-specific tyrosine phosphatase that in turn mediates the dephosphorylation and inactivation of ERK. We conclude that the magnitude of Ca(2+) increases mediated through NR2B-NMDA receptors plays a critical role in the regulation of the serine/threonine and tyrosine kinases and phosphatases that are involved in the regulation of ERK activity.

Figure 1

Co-expression of STEP and NMDAR subunits in neurons and NR2B-NMDA receptor-mediated dephosphorylation of STEP and ERK2. (A) Immunocytochemical analysis showing the co-expression of STEP and NMDA receptor subunits NR1, NR2A and NR2B in both neuronal somata and processes. High magnification (63×) images of neuronal processes are shown in the horizontal strips below each lower resolution (20×) panel (B) Immunoblot analysis demonstrating that STEP is part of the NMDAR complex. STEP₆₁ was immunoprecipitated from neuronal lysates with anti-STEP antibody, analyzed by SDS-PAGE and probed with anti-NR1 antibody (upper panel). The blot was re-probed with STEP antibody (lower panel). Immunoprecipitation with Protein G-Sepharose beads alone was included as a control. To ensure the presence of both STEP and NR1 in the total lysate (input lysate used for immunoprecipitation), equal amount of protein from the lysates was processed for immunoblot analysis with anti-NR1 and -STEP antibodies. (C) Immunoblot analysis demonstrating the dephosphorylation of STEP following NR2B-NMDAR activation. Neurons were treated with glutamate for 30 min, in the presence or absence of Ro 25–6981 or Ifenprodil. Equal amount of protein from each sample were analyzed by SDS-PAGE and probed with anti-STEP antibody (upper panel). Total ERK2 was also analyzed to indicate total protein loading (lower panel). (D) STEP is endogenously phosphorylated and glutamate exposure leads to its dephosphorylation. Neurons were treated with or without glutamate for 30 min and STEP₆₁ was immunoprecipitated with anti-STEP antibody. The samples were then processed for immunoblotting with the phospho-specific STEP antibody (left panel). The blot was then re-probed with anti-STEP antibody (right panel). (E) Dephosphorylation of ERK2 correlates with activation of STEP. Neuronal cultures were treated with glutamate for the specified time periods in the presence or absence of Ro 25–6981. Phosphorylated ERK2 was identified using a phospho-specific antibody that recognizes ERK1/2 only when phosphorylated at the regulatory tyrosine residue (TEY^PERK, upper panel). Total ERK2 and STEP was analyzed by probing the membrane with either anti-ERK2 antibody (middle panel) or anti-STEP antibody (lower panel). Quantification of phosphorylated ERK2 (active ERK) and dephosphorylated STEP (active STEP) was done by computer-assisted densitometry. Values are mean ± SEM (n = 3). *Indicates significant difference from 5 min glutamate treatment (p < 0.001). ^#Indicates significant difference from 0 min time point (p < 0.001).

Figure 2

Intracellular Ca²⁺ changes measured with Fura-FF following exposure to glutamate. (A) Initial B&W panel: 380 nm excitation fluorescence picture of the field of cells analyzed. Subsequent panels: false color images showing the fluorescence ratio increases over time following exposure to glutamate. (B) Averaged population data showing the time course of glutamate stimulated intracellular Ca²⁺ increase measured at neuronal somata (mean ± SEM, n = 10 cells). (C) Individual responses of the 10 neurons are shown to illustrate the typical range of responses observed in different cells. Data are expressed as both a background-corrected fluorescence ratio (left abscissa) and the estimated Ca²⁺ (right abscissa). Because of the low sensitivity of Fura-FF, Ca²⁺ estimates below 1 μM are not reliable and should not be extrapolated from the right abscissa.

Figure 3

NR2B-NMDA receptor-mediated change in intracellular Ca²⁺ measured with Fura-FF. (A) Population data showing that large, delayed Ca²⁺ increase does not occur in cells pre-incubated with 1μM Ifenprodil or 10 μM Ro 25–6981 before exposure to glutamate (n = 14). (B) Population data showing that 20 μM Ro 256981 even applied after 5 min exposure to glutamate also prevents the delayed Ca²⁺ increase (n = 15). (C) Low Ca²⁺ levels are restored after switch to a Ca²⁺ free medium still containing 100 μM glutamate (n = 15). (D) The NMDAR channel blocker, MK801, eliminates all changes in the Fura-FF signal (n = 9). Values represent mean ± SEM. Similar results to those shown were obtained from at least 3 independent experiments for all treatments.

Figure 4

Ca²⁺ recovery enabled by late application of NR2B-NMDAR blocker Ro 25–6981. (A) Fluorescence image of neuron showing measurement locations. (B) Time courses of soma and dendrite Ca²⁺ changes determined at the locations (see boxes in A). The arrow indicates approximate time of Ro 25–6981 application.

Figure 5

Dephosphorylation of STEP is dependent on the magnitude of intracellular changes in Ca²⁺. Neuron cultures were (A) treated with 10 μM or 100 μM glutamate, (B) treated with 100 μM glutamate or 60 mM KCl, (C) treated with 10 μM glutamate, and (D) treated with 60 mM KCl for the times indicated. (A and B) Equal protein from each sample were analyzed by SDS-PAGE and probed with anti-STEP antibody (upper panel). The blots were re-probed with anti-β tubulin antibody to indicate total protein loading (lower panel). Quantification of phosphorylated (upper band) and non-phosphorylated (lower band) STEP was done using Image J density analysis of immunoblot data obtained from 3 independent experiments. The data is presented as a percentage of the total. Values are mean ± SEM (n = 3). *Indicates significant difference from corresponding control values at 0 min time point (p < 0.001). (C and D) Cells were loaded with Fura-FF prior to treatment with glutamate (n = 13) or KCl (n = 10). Values represent mean ± SEM. Similar results were obtained from at least 3 independent experiments.