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. 2012 Jun 15;287(25):20942-56.
doi: 10.1074/jbc.M112.368654. Epub 2012 Apr 27.

Striatal-enriched Protein-Tyrosine Phosphatase (STEP) Regulates Pyk2 Kinase Activity

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Free PMC article

Striatal-enriched Protein-Tyrosine Phosphatase (STEP) Regulates Pyk2 Kinase Activity

Jian Xu et al. J Biol Chem. .
Free PMC article

Abstract

Proline-rich tyrosine kinase 2 (Pyk2) is a member of the focal adhesion kinase family and is highly expressed in brain and hematopoietic cells. Pyk2 plays diverse functions in cells, including the regulation of cell adhesion, migration, and cytoskeletal reorganization. In the brain, it is involved in the induction of long term potentiation through regulation of N-methyl-d-aspartate receptor trafficking. This occurs through the phosphorylation and activation of Src family tyrosine kinase members, such as Fyn, that phosphorylate GluN2B at Tyr(1472). Phosphorylation at this site leads to exocytosis of GluN1-GluN2B receptors to synaptic membranes. Pyk2 activity is modulated by phosphorylation at several critical tyrosine sites, including Tyr(402). In this study, we report that Pyk2 is a substrate of striatal-enriched protein-tyrosine phosphatase (STEP). STEP binds to and dephosphorylates Pyk2 at Tyr(402). STEP KO mice showed enhanced phosphorylation of Pyk2 at Tyr(402) and of the Pyk2 substrates paxillin and ASAP1. Functional studies indicated that STEP opposes Pyk2 activation after KCl depolarization of cortical slices and blocks Pyk2 translocation to postsynaptic densities, a key step required for Pyk2 activation and function. This is the first study to identify Pyk2 as a substrate for STEP.

Figures

FIGURE 1.
FIGURE 1.
Schematic domain structures used in this study. FERM, band 4.1/ezrin/radixin/moesin homology domain; FAT, focal adhesion-targeting domain; TM1 and TM2, putative transmembrane domains; PTP, protein-tyrosine phosphatase domain; asterisk, cysteine residue in the catalytic core; UN, unique N-terminal domain. Regulatory phosphorylation sites are shown. Numbering refers to amino acid residues within the proteins.
FIGURE 2.
FIGURE 2.
Phosphorylation of Pyk2 at Tyr402 is increased in presence of protein-tyrosine phosphatase inhibitor. A, rat brain extracts were incubated with 1 μg of αN-Pyk2 antibody in the absence of protein A-agarose. Mg-ATP was then added in the presence or absence of pervanadate (Perv) (1 mm) (top, No Resin). In another set of experiments, Pyk2 was first immunoprecipitated (IP) with αN-Pyk2 antibody and protein A-Sepharose and washed before the addition of Mg-ATP in the presence or absence of pervanadate (1 mm) (Resin Bound). After 1 h at 4 °C, EDTA (50 mm) was added to chelate the Mg2+ and halt further phosphorylation. Tyr(P)402 Pyk2 levels were examined by immunoblotting with phosphospecific antibody. B, primary hippocampal cultures were treated with NMDA (50 μm) or ionomycin (1 μm) for 15 min. Pervanadate (1 mm) was added 5 min prior to (T = −5) or at the time of stimulation with the absence of pervanadate serving as a control.
FIGURE 3.
FIGURE 3.
Pyk2 is associated with STEP in vivo. A, total brain homogenates from WT or STEP KO mice were immunoprecipitated (IP) with anti-STEP antibody (upper panel) or anti-Pyk2 antibody (lower panel). Immunoprecipitates were resolved by SDS-PAGE and immunoblotted with anti-Pyk2 or anti-Fyn antibody. Blots were reprobed with anti-STEP antibody. B, primary hippocampal neurons were labeled with anti-STEP (green) and anti-Pyk2 (red) antibodies, and nuclei were stained (DAPI; blue). Some cultures were stained with immunodepleted anti-STEP (ID 4) or anti-Pyk2 (ID 4) or secondary antibodies only. The specificity of immunodepleted antibodies was assessed using hippocampal neuronal lysates with each fraction. GAPDH was probed as the loading control.
FIGURE 4.
FIGURE 4.
STEP dephosphorylates Pyk2 at Tyr402. A, Pyk2 was immunoprecipitated from mouse brain lysates using anti-Pyk2 antibody and protein A/G-agarose in the presence of pervanadate (Perv) (1 mm). Mg-ATP, STEP (10 μm), and pervanadate (1 mm) were added as indicated. The Tyr(P)402 signal was normalized to total Pyk2 levels. Error bars indicate the standard error of the mean (SEM) of at least three independent experiments (in this and subsequent panels). Asterisks indicate statistical significance as compared with control (**, p < 0.01; one-way ANOVA with post hoc Tukey test; n = 4). B, total brain lysates were heated at 65 °C for 20 min to inactive endogenous kinases and phosphatases. Treated lysates (50 μg) were incubated with purified GST-STEP46 WT (active) or GST-STEP46 C/S (inactive) at the indicated concentrations and blotted with Tyr(P)402 Pyk2 or Tyr(P)1472 NR2B. Quantitative analyses for each were normalized to GAPDH (*, p < 0.05; **, p < 0.01; one-way ANOVA with post hoc Tukey test; n = 3). C, full-length pcDNA3-Pyk2 (1 μg) construct was co-transfected with pcDNA3-STEP61 WT (active) or pcDNA3-STEP61 C472S (inactive) construct into HEK293 cells. Thirty-six hours post-transfection, cells were lysed in radioimmune precipitation assay buffer and blotted with antibodies as indicated in the figure.
FIGURE 5.
FIGURE 5.
Interacting regions in STEP and Pyk2. A, HEK293 cells were transfected with full-length Pyk2 along with various STEP mutants (Fig. 1). STEP was immunoprecipitated, and Pyk2 levels were assessed with anti-Pyk2 antibody. B, SYF cells were co-transfected with the same STEP and Pyk2 constructs used in A. Association of Pyk2 with STEP was visualized by immunoprecipitation (IP) of STEP and probing for Pyk2. C, in vitro binding of STEP and Pyk2. GST-tagged Pyk2 fragments (Fig. 1) or GST alone was immobilized on glutathione-Sepharose. After washes, STEP (500 nm) was added for 2 h at 4 °C. Binding of STEP to various fragments was visualized with anti-STEP antibody (upper panel), and Pyk2 proteins were visualized by Ponceau S staining (lower panel). IB, immunoblotting.
FIGURE 6.
FIGURE 6.
PR2 and KIM domains of STEP61 and residues 671–694 of Pyk2 mediate STEP/Pyk2 interaction. A, FITC-conjugated Pyk2-derived peptides 671–694, 689–712, 707–723, and 850–866 were titrated with GST-STEP61 (left) and -STEP46 (middle), and STEP-derived PR2 peptide with GST-Pyk2(671–875) (right). Binding was monitored by FP. For each graph, the signals were normalized, setting as zero the lowest value and as 100% the highest value measured, and fitted to saturation curves. Calculated binding affinities of 671–694 for GST-STEP61 and -STEP46 and of PR2 for GST-Pyk2 671–875 are indicated. AU, absorbance units. Error bars indicate the SEM of at least three independent experiments (in this and subsequent panels). B and C, competition of PR2, KIM, and Myc (control) peptide with GST-STEP61 for Pyk2 and ERK2 (B) and of the Pyk2-derived peptides with GST-Pyk2(671–875) for STEP61 and PSD-95 (C) by increasing concentrations of peptides (as indicated) in pulldown assays with GST-STEP61 (B) and GST-Pyk2(671–875) (C) using brain lysates as the source of native proteins. Pyk2 and ERK2 (B) and STEP61 and PSD-95 (C) were determined by immunoblotting (*, p < 0.05; **, p < 0.01; one-way ANOVA with post hoc Tukey test; n = 4).
FIGURE 7.
FIGURE 7.
Fyn and Pyk2 compete for STEP binding. A, constant amounts (1 μg of cDNA) of full-length STEP and Pyk2 constructs were co-transfected with increasing amounts of Fyn construct into HEK293 cells. Thirty-six hours after transfection, cells were lysed before immunoprecipitation (IP) with anti-STEP antibody and immunoblotting (IB) with anti-Pyk2 antibody. Immunoblotting of lysates indicates increasing amounts of Fyn with increasing amounts of Fyn cDNA but unchanged amounts of Pyk2. Immunoblotting of STEP immunoprecipitates indicates unaltered amounts of STEP in all samples. B, constant amounts (1 μg of cDNA) of STEP and Fyn were co-transfected with increasing amounts of Pyk2. Co-immunoprecipitation of Fyn with STEP was measured by immunoblotting with anti-Fyn antibody. C, competitive binding was tested in vitro with all purified proteins. GST-tagged STEP was adsorbed to glutathione beads and incubated with a constant amount of Pyk2 (500 ng) and increasing amounts of Fyn as indicated.
FIGURE 8.
FIGURE 8.
Basal phosphotyrosine levels of Pyk2 and Pyk2 substrates are elevated in STEP KO mouse brains. A, synaptosomal (P2) fractions from WT and STEP KO littermates were used to determine levels of tyrosine phosphorylation of Pyk2 at Tyr(P)402 and total Pyk2 levels. Error bars indicate the SEM of at least three independent experiments (in this and subsequent panels). Quantitative analyses for each were normalized to GAPDH (*, p < 0.05; **, p < 0.01; Student's t test; n = 4). B, phosphorylation of paxillin (Tyr(P)118) and ASAP1 (Tyr(P)782) were determined in P2 fractions with phosphospecific antibodies. Quantitations were normalized to total protein levels, respectively, and then to GAPDH. C, PSD fractions from WT and STEP KO mice were purified. Tyrosine phosphorylation of Pyk2 and ERK1/2 was compared between WT and STEP KO mice (**, p < 0.01; Student's t test; n = 4).
FIGURE 9.
FIGURE 9.
Active TAT-STEP protein blocks phosphorylation and translocation of Pyk2 to PSD upon KCl depolarization. A, WT or STEP KO mouse brain slices were pretreated with TAT-STEP WT (active), TAT-STEP C/S (inactive), or active STEP without TAT for 30 min; stimulated with 40 mm KCl for 2 min; and frozen on dry ice before isolation of PSD fractions. Phosphorylation levels were probed with phosphospecific antibodies (Tyr(P)402 Pyk2 or Tyr(P)204 ERK1/2) and normalized to total Pyk2 and ERK1/2 levels, respectively, and then to β-actin as a loading control. Error bars indicate the SEM of at least three independent experiments (in this and subsequent panels). All values were compared with those in WT aCSF samples (**, p < 0.01; one-way ANOVA with post hoc Tukey test; n = 4). B, primary hippocampal neurons were pretreated with active TAT-STEP WT or inactive TAT-STEP C/S followed by KCl (40 mm; 2 min) stimulations. Colocalization of Pyk2 and PSD-95 was visualized using immunostaining with anti-Pyk2 (red) and anti-PSD-95 (green) antibodies. Arrowheads in the merged images indicate colocalized puncta. The number of Pyk2/PSD-95-colocalized puncta was counted per 10 μm of dendrites; 17 neurons were used for quantification per treatment. All values were compared with control (**, p < 0.01; one-way ANOVA with post hoc Tukey test; n = 17). pERK1/2, phospho-ERK1/2, pPyk2, phospho-Pyk2.
FIGURE 10.
FIGURE 10.
Activation of STEP leads to dephosphorylation of Pyk2 in synaptoneurosomes. A, expression of presynaptic and postsynaptic markers in synaptoneurosome preparation. SYP, synaptophysin; CaMKII, Ca2+/calmodulin-dependent protein kinase II. B, synaptoneurosomes from WT (upper panel) or STEP KO (lower panel) mice were stimulated with 40 mm KCl for the indicated durations. Phosphorylation of Pyk2 was determined with Tyr(P)402 Pyk2 antibody. C, CsA-pretreated synaptoneurosomes (100 nm; 10 min) from WT (upper panel) or STEP KO (lower panel) mouse brains were stimulated with 40 mm KCl as in B. D, quantitation of phospho-Pyk2 from B and C. Phosphorylation levels were first normalized to total protein and then to tubulin as a loading control. Error bars indicate the SEM of at least three independent experiments. All values were expressed as -fold changes compared with WT control levels (*, p < 0.05; **, p < 0.01; one-way ANOVA with post hoc Tukey test; n = 4).

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