Tyrosine 402 phosphorylation of Pyk2 is involved in ionomycin-induced neurotransmitter release

Abstract

Protein tyrosine kinases, which are highly expressed in the central nervous system, are implicated in many neural processes. However, the relationship between protein tyrosine kinases and neurotransmitter release remains unknown. In this study, we found that ionomycin, a Ca²⁺ ionophore, concurrently induced asynchronous neurotransmitter release and phosphorylation of a non-receptor protein tyrosine kinase, proline-rich tyrosine kinase 2 (Pyk2), in clonal rat pheochromocytoma PC12 cells and cerebellar granule cells, whereas introduction of Pyk2 siRNA dramatically suppressed ionomycin-induced neurotransmitter release. Further study indicated that Tyr-402 (Y402) in Pyk2, instead of other tyrosine sites, underwent rapid phosphorylation after ionomycin induction in 1 min to 2 min. We demonstrated that the mutant of Pyk2 Y402 could abolish ionomycin-induced dopamine (DA) release by transfecting cells with recombinant Pyk2 and its mutants (Y402F, Y579F, Y580F, and Y881F). In addition, Src inhibition could prolong phosphorylation of Pyk2 Y402 and increase DA release. These findings suggested that Pyk2 was involved in ionomycin-induced neurotransmitter release through phosphorylation of Y402.

Figure 1. Pyk2 phosphorylation is consistent with ionomycin-induced neurotransmitter release.

(A) PC12 cells were incubated for 2 min in low-K⁺ solution. DA release during this period represented the basal release. The buffer was immediately changed every 2 min with the low-K⁺ solution with or without 1 μM ionomycin to stimulate Ca²⁺ influx. Samples were collected and tested according to the release assay in Materials and Methods. The amount of DA release in the medium was expressed as the percentage of total cellular content (n = 8/sample). (B) Cerebellar granule cells were performed similar to PC12 cells, and the amount of Glu released in the medium was expressed as the percentage of total cellular content (n = 8/sample). (C) PC12 cells were incubated with or without 1 μM ionomycin, lysed, and subjected to western blot using an anti-phosphotyrosine (P-Tyr, 4G10) antibody. Arrows indicate the major immunoreactive bands co-migrating with Pyk2 (upper) and paxillin (lower). (D) PC12 cell lysates were subjected to IP with anti-Pyk2 antibody, followed by IB with anti-phosphotyrosine and anti-Pyk2 antibodies. (E) PC12 cells were incubated for 2 min at 37°C in low-K⁺ solution with or without 1 μM ionomycin. Equal amounts of cell lysates were immunoprecipitated with anti-Pyk2 antibody, and immunoblotted with phosphotyrosine antibody. Phosphorylation levels were studied in different experiments from 0 min to 5 min (right, inset) or from 1.5 min to 60 min (left, main graph). Pyk2 phosphorylation at each indicated time is expressed as a percentage of the maximal level of induced phosphorylation. Note: Duration of ionomycin treatment is indicated by the horizontal bar, which represents 2 min. The values in the results are expressed as mean ± S.E.M. from four representative experiments (^##p<0.01, **p<0.01, ***p<0.001).

Figure 2. Pyk2 siRNA abolishes ionomycin-induced neurotransmitter release.

(A) PC12 cells were transfected with 50 nM Pyk2 siRNA or control siRNA for 48 h, and tested for the expression of Pyk2 and FAK. PC12 cells were transfected with 50 nM Pyk2 siRNA for 24, 48, and 72 h or 50 nM control siRNA for 72 h, and harvested for western blot. PC12 cells were transfected with 10, 50, and 100 nM Pyk2 siRNA or 100 nM control siRNA for 48 h, and harvested for western blot. (B) PC12 cells were transfected with 100 nM Pyk2 siRNA or control siRNA for 48 h, and used for release assay with or without 1 μM ionomycin for 2 min. Samples were collected and tested for DA release. The amount of DA released in the medium was expressed as the percentage of total cellular content (n = 8/sample). The values are expressed as mean ± S.E.M. from four representative experiments (**p<0.01, ***p<0.001).

Figure 3. Ionomycin-induced Pyk2 tyrosine phosphorylation is site-specific only for Tyr-402.

(A) PC12 cells were exposed to 1 μM ionomycin at 37°C for 90 s. The cells were fixed, and phosphotyrosine proteins were labeled by phosphorylation site-specific antibodies against Tyr-402 (pY402), Tyr-579 (pY579), Tyr-580 (pY580), or Tyr-881 (pY881) and TRITC-conjugated second antibody (red), respectively. Pyk2 expression was labeled by FITC (green). Scale bar, 5 μm. (B) PC12 cells were treated with or without 1 μM ionomycin at 37°C for 90 s, then lysed, and subjected to western blot using phosphorylation site-specific antibodies. Pyk2 transfected 293T cell lysate was used as a positive control. The total amount of Pyk2 was used as an internal control. (C) The time course of Pyk2 phosphorylation at Tyr-402 by ionomycin in PC12 cells. PC12 cells were exposed to 1 μM ionomycin at 37°C for the indicated times. The cells were labeled with pY402 site-specific polyclonal antibody, followed by a secondary layer of TRITC-conjugated second antibody to rabbit IgG (red). Scale bar, 10 μm.

Figure 4. Pyk2 Tyr-402 autophosphorylation is essential in ionomycin-induced neurotransmitter release.

(A) Phosphorylation of Pyk2 Tyr-402 in PC12 cells transfected with Pyk2-related plasmids. PC12 cells were transfected with 4 μg of empty vectors (Mock), Pyk2-WT, Pyk2-Y402F, Pyk2-Y579F, Pyk2-Y580F, and Pyk2-Y881F for 48 h. The cells were treated with 1 μM ionomycin for 2 min, and immediately harvested for analysis of Pyk2 Tyr-402 phosphorylation by western blot. Actin was used as an internal control. The average Pyk2 phosphorylation in the Mock group was set as 100% for all experiments to standardize the results with the same plasmids from repeated experiments (n = 4/sample). (B) PC12 cells were transfected with 4 μg of empty vector (Mock), Pyk2-WT, Pyk2-Y402F, Pyk2-Y579F, Pyk2-Y580F, and Pyk2-Y881F for 48 h, and used for DA release assay with 1 μM ionomycin. The amount of DA released in the medium was expressed as the percentage of the total cellular content (n = 8/sample). The values are expressed as means ± S.E.M. from four representative experiments (*p<0.05, **p<0.01, ^###p<0.001).

Figure 5. PP2-enhanced neurotransmitter release is Pyk2 Tyr-402-dependent.

(A) PC12 cells were transfected with 4 μg of empty vectors (Mock), Pyk2-Y402F, Pyk2-Y579F, Pyk2-Y580F, and Pyk2-Y881F for 48 h, respectively. The cells were washed thrice with low-K⁺ solution, and incubated for 20 min in low-K⁺ solution with or without (Mock) 20 μM PP2 for 20 min. The cells were washed thrice, and sequentially incubated for 2 min in low-K⁺ solution with 1 μM ionomycin. The amount of DA release in the medium was expressed as the percentage of the total cellular content (n = 8/sample). (B) Another parallel performed groups after stimulated by ionomycin for 2 min were immediately harvested, and lysed for western blot using phosphorylation site-specific antibodies against Tyr-402 (pY402), Tyr-579 (pY579), Tyr-580 (pY580), or Tyr-881 (pY881). Actin was used as an internal control (n = 4/sample). (C) Equal amount of cell lysates harvested at different incubation period were immunoprecipitated with anti-Src antibody, then immunoblotted with anti-phosphotyrosine antibody. The total amount of Src was used as an internal control. The values are expressed as means ± S.E.M. from four representative experiments (*p<0.05, ^#p<0.05, ^##p<0.01, ^###p<0.001).

Figure 6. Src siRNA prolongs Pyk2 autophosphorylation and increases DA release.

(A) PC12 cells were transfected with 100 nM Src siRNA or control siRNA for 48 h, then harvested, and lysed for western blot using anti-Src antibody and anti-Pyk2 antibody. (B) PC12 cells were transfected with 100 nM Src siRNA or control siRNA for 48 h, and incubated with 1 μM ionomycin for 2 min. Cells were harvested at 0, 2, 4, and 6 min after ionomycin treatment, then immunoprecipitated with anti-Src antibody, then immunoblotted with anti-phosphotyrosine antibody. The total amount of Src was used as an internal control. (C) PC12 cells were transfected with 100 nM Src siRNA or control siRNA for 48 h, and incubated with 1 μM ionomycin for 2 min. Cells were harvested at 0, 2, 4, and 6 min after ionomycin treatment for western blot to test the expression of pY402 and total Pyk2. Pyk2 Y402 phosphorylation at each indicated time is expressed as a percentage of the maximal level of induced phosphorylation. (D) PC12 cells were transfected with 100 nM Src siRNA or control siRNA for 48 h, and incubated with 1 μM ionomycin for 2 min. The amount of DA released in the medium was expressed as the percentage of the total cellular content. The values are expressed as mean ± S.E.M. from four representative experiments (*p<0.05).