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. 2007 Dec 12:1184:17-27.
doi: 10.1016/j.brainres.2007.09.065. Epub 2007 Oct 4.

Brevetoxin-induced phosphorylation of Pyk2 and Src in murine neocortical neurons involves distinct signaling pathways

Zhengyu Cao  1 , Joju George, Daniel G Baden, Thomas F Murray
Affiliations

Brevetoxin-induced phosphorylation of Pyk2 and Src in murine neocortical neurons involves distinct signaling pathways

Zhengyu Cao et al. Brain Res. .

Abstract

Brevetoxins (PbTx-1 to PbTx-10) are potent lipid soluble polyether neurotoxins produced by the marine dinoflagellate Karenia brevis. Brevetoxins bind to site 5 of the alpha-subunit of voltage-gated sodium channels (VGSCs) and augment Na(+) influx. In neocortical neurons brevetoxins elevate intracellular Ca(2+) and augment NMDA receptor signaling. In this study, we explored the effects of PbTx-2 on Pyk2 and Src activation in neocortical neurons. We found that both Pyk2 and Src were activated following PbTx-2 exposure. PbTx-2-induced Pyk2 Tyr402 phosphorylation was dependent on elevation of Ca(2+) influx through NMDA receptors. Moreover, Pyk2 Tyr402 phosphorylation was also found to require PKC activation inasmuch as RO-31-8425 and GF 109203x both attenuated the response. In contrast, PbTx-2-induced Src Tyr416 phosphorylation involved a Gq-coupled receptor inasmuch as U73122, a specific PLC inhibitor, abolished the response. This Gq-coupled receptor appears to be mGluR 5. The PKCdelta inhibitor rottlerin abolished PbTx-2-induced Src activation demonstrating that this isoform of PKC is involved in the activation of Src by PbTx-2. Considered together these data suggest that although activation of neuronal Pyk2 and Src result from PbTx-2 stimulation of VGSC, engagement of these two non-receptor tyrosine kinases involves distinct signaling pathways.

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Figures

Fig. 1
Fig. 1
PbTx-2 increases Pyk2 Tyr-402 and Src Tyr-416 phosphorylation. (a) Concentration–response profile for PbTx-2 effects on Pyk2 Tyr-402 and Src Tyr-416 phosphorylation. Neocortical neurons were exposed to 1–1000 nM PbTx-2 for 10 min. The phosphorylation levels of Pyk2 and Src were detected by western blot using phosphospecific antibodies against the active forms of the kinases (pPyk2(pY402), and pSrc(pY416)). The relative densities of phosphotyrosine were normalized to the total protein densities of both Pyk2 and Src for each treatment. Individual bars represent the mean±S.E.M. from four independent experiments (*, p<0.05; **, p<0.01, PbTx-2 vs. control by ANOVA). (b) Time course for PbTx-2 (100 nM)-induced stimulation of Pyk2 Tyr-402 and Src Tyr-416 phosphorylation. Cells were exposed to PbTx-2 for 1–30 min. Bars indicate mean values of five independent experiments (*, p<0.05; **, p<0.01, PbTx-2 vs. control by ANOVA).
Fig. 2
Fig. 2
Effect of tetrodotoxin (TTX) on PbTx-2-induced Pyk2 and Src activation. Representative western blotting and quantification of Pyk2 Tyr-402 phosphorylation (a) and Src Tyr-416 phosphorylation (b) after exposure of neocortical cells to PbTx-2 for 5 min in the presence or absence of TTX (1 μM). Bars indicate mean values from six independent experiments (**, p<0.01, PbTx-2 vs. control; aa, p<0.01, PbTx-2 vs. PbTx-2+TTX by ANOVA).
Fig. 3
Fig. 3
Effects of PP-2 and PP-3 on PbTx-2-induced Pyk2 and Src phosphorylation. Representative western blotting and quantification of Pyk2 Tyr-402 phosphorylation (a) and Src Tyr-416 phosphorylation (b) after exposure of neocortical cells to PbTx-2 for 5 min in the presence or absence of the Src-family kinase inhibitor PP-2 (1 μM) or the inactive analog PP-3 (1 μM). Bars depict mean values derived from six independent experiments (**, p<0.01, PbTx-2 vs. control; aa, p<0.01, PbTx-2 vs. PbTx-2+ PP-2 by ANOVA).
Fig. 4
Fig. 4
Influence of Ca2+ dynamics on PbTx-2-induced Pyk2 Tyr-402 and Src Tyr-416 phosphorylation. (a) Representative western blotting and quantification of Pyk2 Tyr-402 phosphorylation after exposure of neocortical cells to PbTx-2 for 5 min in the presence or absence of the Ca2+ chelator BAPTA/AM (40 μM) or low Ca2+ (0.2 mM). Bars depict mean values derived from four independent experiments (**, p<0.01, PbTx-2 vs. control; aa, p<0.01, PbTx-2 vs. PbTx-2/0.2 mM Ca2+; bb, p<0.01, PbTx-2 vs. PbTx-2+BAPTA by ANOVA). (b) Representative western blotting and quantification of Pyk2 Tyr-402 phosphorylation after exposure of neocortical cells to PbTx-2 for 5 min in the presence or absence of MK-801 (1 μM) or nifedipine (1 μM). Bars depict mean values derived from five independent experiments (**, p<0.01, PbTx-2 vs. control; *, p<0.05, PbTx-2 vs. PbTx-2+MK-801 by ANOVA). (c) Representative western blotting and quantification of Src Tyr-416 phosphorylation after exposure of neocortical cells to PbTx-2 for 5 min in the presence or absence of the Ca2+ chelator BAPTA/AM (40 μM) or low Ca2+ (0.2 mM). Bars depict mean values derived from four independent experiments (**, p<0.01, PbTx-2 vs. control; aa, p<0.01, PbTx-2 vs. PbTx-2+BAPTA by ANOVA).
Fig. 5
Fig. 5
Effect of a PLC inhibitor U73122, a mGluR 1 antagonist S-4-CPG, and the mGluR 5 antagonist MTEP on PbTx-2-induced Src Tyr-416 phosphorylation. (a) Representative western blotting and quantification of Src Tyr-416 phosphorylation after exposure of neocortical cells to PbTx-2 for 5 min in the presence or absence of U73122 (3 μM). Bars depict mean values derived from six independent experiments (**, p<0.01, PbTx-2 vs. control; aa, p<0.01, PbTx-2 vs. PbTx-2+U73122 by ANOVA). (b) Representative western blotting and quantification of Src Tyr-416 phosphorylation after exposure of neocortical cells to PbTx-2 for 5 min in the presence or absence of S-4-CPG (500 μM) or MTEP (1 μM). Bars depict mean values derived from five independent experiments (**, p<0.01, PbTx-2 vs. control; aa, p<0.01, PbTx-2 vs. PbTx-2+MTEP by ANOVA).
Fig. 6
Fig. 6
Effects of the PKC inhibitors on PbTx-2-induced Pyk2 Tyr-402 and Src Tyr-416 phosphorylation. (a) Representative western blotting and quantification of Pyk2 Tyr-402 phosphorylation after exposure of neocortical cells to PbTx-2 for 5 min in the presence or absence of RO-31-8425 (1 μM) or GF 109203x (1 μM). Bars depict mean values derived from five independent experiments (**, p<0.01, PbTx-2 vs. control; aa, p<0.01, PbTx-2 vs. PbTx-2+RO-318425; bb, p<0.01, PbTx-2 vs. PbTx-2+GF 109203x by ANOVA). (b) Representative western blotting and quantification of Src-416 phosphorylation after exposure of neocortical cells to PbTx-2 for 5 min in the presence or absence of RO-31-8425 (1 μM) or GF 109203x (1 μM). Bars depict mean values derived from five independent experiments (**, p<0.01, PbTx-2 vs. control; aa, p<0.01, PbTx-2 vs. PbTx-2+GF 109203x by ANOVA). (c) Effects of the PKCδ inhibitor rottlerin on PbTx-2-induced Src Tyr-416 phosphorylation. Representative western blotting and quantification of Src-416 phosphorylation after exposure of neocortical cells to PbTx-2 for 5 min in the presence or absence of rottlerin (5 μM). Bars depict mean valuesderived from five independent experiments (**, p<0.01, PbTx-2vs. control; aa, p<0.01, PbTx-2 vs. PbTx-2+rottlerin by ANOVA).
Fig. 7
Fig. 7
Schematic diagram showing proposed pathways underlying PbTx-2-induced Src Tyr416 and Pyk2 Tyr402 phosphorylation in mouse neocortical neurons.
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