Pyk2 cytonuclear localization: mechanisms and regulation by serine dephosphorylation
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Cytonuclear signaling is essential for long-term alterations of cellular properties. Several pathways involving regulated nuclear accumulation of Ser/Thr kinases have been described but little is known about cytonuclear trafficking of tyrosine kinases. Proline-rich tyrosine kinase 2 (Pyk2) is a cytoplasmic non-receptor tyrosine kinase enriched in neurons and involved in functions ranging from synaptic plasticity to bone resorption, as well as in cancer. We previously showed the Ca2+-induced, calcineurin-dependent, nuclear localization of Pyk2. Here, we characterize the molecular mechanisms of Pyk2 cytonuclear localization in transfected PC12 cells. The 700–841 linker region of Pyk2 recapitulates its depolarization-induced nuclear accumulation. This region includes a nuclear export motif regulated by phosphorylation at residue S778, a substrate of cAMP-dependent protein kinase and calcineurin. Nuclear import is controlled by a previously identified sequence in the N-terminal domain and by a novel nuclear targeting signal in the linker region. Regulation of cytonuclear trafficking is independent of Pyk2 activity. The region regulating nuclear localization is absent from the non-neuronal shorter splice isoform of Pyk2. Our results elucidate the mechanisms of Ca2+-induced nuclear accumulation of Pyk2. They also suggest that Pyk2 nuclear accumulation is a novel type of signaling response that may contribute to specific long-term adaptations in neurons.
KeywordsNon-receptor tyrosine kinase Cytonuclear localization Nucleus Phosphorylation Protein phosphatase Calcineurin
Proline-rich tyrosine kinase 2 (Pyk2) is a Ca2+-activated non-receptor tyrosine kinase closely related to focal adhesion kinase (FAK) . Pyk2 is enriched in adult neurons and plays an important role in neuronal plasticity [2, 3, 4, 5, 6]. In non-neuronal cells, Pyk2 is involved in osteoclast function , macrophage migration , and focal adhesion disassembly . Neuronal Pyk2 is 5 kDa larger than in cells from the hematopoietic lineage, due to the retention of an additional exon coding for a peptide between the kinase and focal adhesion targeting (FAT) domains [10, 11, 12]. The functional differences between these isoforms are not known. Although the precise mechanism of Pyk2 activation remains unclear, increase in intracellular free Ca2+ can directly or indirectly induce Pyk2 dimerization and trans-autophosphorylation at Y402 creating a Src-homology-2 binding site that recruits Src family kinases and activates various signaling pathways [1, 5, 13, 14, 15].
In most cells, Pyk2 is localized in the cytoplasm [16, 17] and in neurons, in perikarya and dendritic shafts [18, 19]. We have previously demonstrated that Pyk2 activation in neuronal cells is concomitant to its Ca2+-induced, calcineurin-dependent nuclear accumulation following membrane depolarization . Although Pyk2 nuclear accumulation has been serendipitously observed in various cell types or following mutations [21, 22, 23, 24, 25, 26, 27], the physiological relevance and mechanisms of its cytonuclear shuttling are not known. Cytonuclear trafficking of proteins larger than 40 kDa results from an active transport mediated by karyopherins, which facilitate translocation of cargos through the nuclear pore and release them in the nucleus, in the case of importins, or in the cytoplasm in the case of exportins [28, 29]. The best characterized mechanisms for nuclear import and export are based on the association of cargos that contain nuclear localization signal (NLS) or a hydrophobic nuclear export sequence (NES) with importins and the exportin chromosome region maintenance 1 (CRM1), respectively [30, 31, 32]. Here we characterize the role of NLS, NES, and other sequences in Pyk2 cytonuclear trafficking and its control by S778 phosphorylation by PKA and dephosphorylation by calcineurin. Moreover, we show that these regulatory mechanisms are specific for Pyk2 long-splice isoform expressed in neurons.
Materials and methods
Leptomycin B and KN93 were from Calbiochem, cyclosporin A, FK506, forskolin, IBMX, H89, and myristoylated 14–22 fragment of protein kinase A inhibitory peptide (myrPKI14–22) from Sigma. Rabbit anti-Pyk22–18 antibodies were described  or from Sigma, anti-pY402-Pyk2 from Invitrogen, anti-GFP from Roche and anti-DARPP-32 was a gift from P. Greengard (Rockefeller University). Anti-pS778 was raised in rabbit against a pS778-Pyk2773–784 peptide by Eurogentec with a 28-day protocol. Alexa-488- or Cy3-coupled secondary antibodies were from Molecular Probes (Sunnyvale, CA).
PC12 cells were grown on type I collagen (BD Biosciences) in RPMI medium containing 10 % horse serum and 5 % fetal calf serum (v/v). COS7 cells were grown in DMEM medium containing 10 % fetal calf serum. Transfections were done with Lipofectamine 2000 (Invitrogen) in cells at about 70 % confluence. For fluorescence analyses, PC12 cells were grown in RPMI on type I collagen-coated glass coverslips after incubation with poly-l-lysine (Sigma). Depolarization was performed by isosmotic replacement of 40 mM NaCl by 40 mM KCl in the extracellular medium as described previously .
Cells were fixed 15 min in a 4 % (w/v) paraformaldehyde solution, permeabilized 12 min on ice with 1/1 methanol/acetone (v/v). Cells were washed with 20 mM sodium phosphate, pH 7.5, 150 mM NaCl (PBS), blocked and incubated 2 h with primary antibodies. After washes, cells were incubated 45 min with Alexa-488- or Cy3-coupled secondary antibodies, washed and mounted in Vectashield (Vector Laboratories) with 4′,6′-diamidino-2-phenylindole (DAPI). PC12 cells transfected with GFP constructs were fixed, washed, and mounted in Vectashield with DAPI. Images were acquired with a Micromax numerical CCD camera (Roper Scientific).
PC12 cells were lysed in a preheated (at 100 °C) solution of 1 % (w/v) SDS, 1 mM Na3VO4, and incubated 5 min at 100 °C. Equal amounts of protein were separated by SDS-PAGE (7–10 % acrylamide, w/v) before electrophoretic transfer onto a nitrocellulose membrane (Hybond Pure; GE Healthcare). Membranes were fixed in 10 % (v/v) acetic acid, 10 % (v/v) isopropanol and washed and blocked 1 h at room temperature in Tris-buffered saline (100 mM NaCl and 10 mM Tris, pH 7.5) with 5 % (w/v) non-fat dry milk for protein detection or with 0.1 % (v/v) Tween 20, 3 % (w/v) BSA for detection of pY402 or pS778. Membranes were then incubated overnight at 4 °C with primary antibodies. Bound antibodies were detected with anti-rabbit IgG IRdye800CW-coupled and anti-mouse IgG IRdye700DX-coupled antibodies (Rockland Immunochemicals). Fluorescence was analyzed at 680 and 800 nm using the Odyssey infrared imager (Li-Cor) and quantified using Odyssey software. Data were normalized to the mean value of untreated controls in the same gels.
GST protein cloning
Pyk2 700–841 WT or S778A and DARPP-32 sequence were inserted into pGEX-6P-2 (Amersham) and expressed in BL-21-competent bacterial cells. The resultant GST-fusion proteins were affinity-purified on glutathione-Sepharose beads (Pharmacia) as described .
In vitro phosphorylation/dephosphorylation assays
Phosphorylation reactions were carried out 10–45 min at 30 °C in 50 mM HEPES (pH 7.4), 10 mM magnesium acetate, 1 mM EGTA, 5 μM ATP, 3 μl [γ-32P]ATP (3 Ci/mmol, 10 μCi/ml), and 10 ng of cAMP-dependent protein kinase catalytic subunit (Millipore). Reactions were stopped by the addition of 25 μl of a stop solution (150 g/l SDS, 0.3 M Tris–Cl, pH 6.8, 25 % (v/v) glycerol, traces of pyronine Y) and heated at 95 °C. For dephosphorylation GST fusion proteins bound to glutathione-Sepharose were washed in PBS containing 1 % Triton-X-100 (v/v), 0.5 mM DTT, and 0.5 mM PMSF, and incubated in a dephosphorylation buffer (Tris 20 mM, NaCl 100 mM, MgCl2 5 mM, DTT 1 mM, BSA 0.5 mg/ml, CaCl2 1 mM) with recombinant calcineurin (Calbiochem) and 1 μM calmodulin. After electrophoresis, polyacrylamide gels were dried and incorporated 32P measured with Fuji Phosphoimager, FLA7000, together with [γ-32P]ATP spotted on bench coat paper for quantification.
Cloning and directed mutagenesis
Quantifications and statistical analysis
Pyk2 is usually present in both the cytoplasm and the nucleus, with some heterogeneity in both compartments. This distribution combined with different levels of expression in various cells precluded the use of automatic detection of nuclear localization. To circumvent this difficulty, we have previously shown that it is possible to estimate the cytonuclear distribution of Pyk2 by quantifying the number of cells in which the nuclear fluorescence is superior or equal to cytoplasmic fluorescence (n ≥ c), with results similar to those obtained by confocal microscopy . Thus, cells were classified by an observer blind to the treatment and/or mutation, using micrographs obtained by epifluorescence microscopy. In some experiments, the nuclear/cytoplasmic fluorescence ratio (n/c) was calculated for each cell after measuring the fluorescence intensity at several locations in the nucleus and cytoplasm. Nuclear limits were identified by DAPI staining. The percentage of cells in each category was determined in each coverslip (approximately 50–100 cells per coverslip in 10–20 fields). Data were from three or more independent experiments, each in duplicate. Statistical analysis was done using GraphPad Prism 3.02.
The 700–841 region of Pyk2 recapitulates its depolarization-regulated nuclear localization
Pyk2 700–841 region contains a nuclear targeting sequence (NTS) that plays an accessory role in the nuclear import of the full-length protein
We then investigated the role of this putative NTS motif in the nuclear import of full-length Pyk2 by generating the same double mutation (S747A/T749A) in GFP-Pyk2 (Fig. 2b, d). This mutation did not alter the nuclear accumulation of GFP-Pyk2 induced by membrane depolarization or LMB (Fig. 2d). Since the FERM domain of Pyk2 and FAK contains a classical NLS [23, 35] we inferred that the effects of this NLS might mask those of the NTS. We tested the role of Pyk2 NLS in our experimental conditions by generating a double mutation of two of its basic residues (R184A/R185A). This mutation prevented the nuclear accumulation of GFP-Pyk2 in response to a short depolarization (Fig. 2c, d). This observation revealed that although the FERM domain was not necessary for depolarization-induced nuclear accumulation of GFP-Pyk2421–1009 (Fig. 1c, d), the NLS mutation was sufficient to block the rapid effects of depolarization in the context of the full-length protein. In contrast, the nuclear accumulation of R184A/R185A-GFP-Pyk2 was attenuated but still observed after a 3-h LMB treatment (Fig. 2d), suggesting the existence of additional weaker pronuclear sequence(s). We therefore examined the LMB-induced nuclear accumulation of GFP-Pyk2 containing mutations of both the FERM NLS and the NTS (R184A/R185A/S747A/T749A, Fig. 2b). The LMB-induced nuclear localization of this protein was dramatically reduced as compared to the simple NLS R184A/R185A mutant GFP-Pyk2 (Fig. 2d). This last experiment revealed that Pyk2 NTS in the linker region between the kinase and FAT domains plays an accessory role for the nuclear import of the full-length protein. This role appears critical only following mutation of the NLS motif or in the truncated form of the protein, GFP-Pyk2700–841.
The 700–841 region of Pyk2 contains a nuclear export sequence LL-LFV
We then examined whether the LQFQV motif was sufficient for the nuclear export of GFP-Pyk2700–841 by inserting stop codons in GFP-Pyk2700–841 and generating various truncated forms (GFP-Pyk2700–745, 700–758, 700–767, and 700–793, Fig. 3e, f). Only GFP700–793 was partly excluded from the nucleus but to a lesser extent than the entire GFP-Pyk2700–841 (Fig. 3g). These results suggested that (an) additional residue(s), presumably in the 767–793 sequence, was/were involved in the nuclear export activity of the 700–841 region of Pyk2 and that these residues may be involved in the Ca2+- and calcineurin-dependent regulation of GFP-Pyk2700–841.
The cytonuclear localization of Pyk2-700–841 region is regulated by calcineurin
S778 is a substrate of PKA and is dephosphorylated by calcineurin in vitro
S778 is phosphorylated by PKA and dephosphorylated by calcineurin in cells
S778 phosphorylation controls Pyk2 nuclear export but not its autophosphorylation on Y402
To further investigate the contribution of PKA to the cellular localization of Pyk2, we examined the effects of two unrelated pharmacological inhibitors of PKA, H89 (100 μM, 30 min) and a cell-permeable form of the protein kinase A inhibitory peptide (myrPKI14–22, 10 μM, 30 min). These two molecules increased the number of cells with Pyk2 in the nucleus from 15 to approximately 55 % and pS778 labeling was virtually absent in these conditions (Fig. 7d, e). The effects of PKA inhibitors were almost as pronounced as those of depolarization (Fig. 7d, e). These results showed that the cyto/nuclear localization of Pyk2 is strongly dependent on the activity of PKA in these experimental conditions.
Although our results provided strong evidence that S778 dephosphorylation was necessary to allow Pyk2 nuclear accumulation, this effect could result from a regulation of Pyk2 nuclear export or import. To address this point, we reasoned that the inhibition of the nuclear export by LMB should have less effect on S778A-GFP-Pyk2 localization if S778 phosphorylation is necessary for Pyk2 nuclear export. Conversely, if S778 phosphorylation increases Pyk2 nuclear import, LMB effects and S778A mutation should be additive and lead to a further nuclear accumulation of S778A which has actually been observed for other mutants of Pyk2 . PC12 cells transfected with WT or S778A-GFP-Pyk2 were treated or not with LMB. LMB induced a much stronger increase in the nuclear localization of GFP-Pyk2, than of S778A-GFP-Pyk2 localization (Fig. 7f), suggesting a regulation of Pyk2 nuclear export through S778 phosphorylation.
Since calcineurin is required for both Pyk2 nuclear translocation and autophosphorylation , it was important to determine whether the regulation of S778 phosphorylation was also involved in the regulation of Pyk2 Y402 autophosphorylation, the essential step in its functional activity. We measured Pyk2 autophosphorylation by pY402-specific immunoblotting in PC12 cells transfected with WT or S778A-GFP-Pyk2, treated or not with forskolin or high K+. Membrane depolarization induced phosphorylation of S778A-GFP-Pyk2 on Y402 to the same extent as WT GFP-Pyk2 (Fig. 7g, h). Forskolin did not alter this phosphorylation. These results indicated that the role of calcineurin in Pyk2 autophosphorylation is not mediated by S778 dephosphorylation.
Pyk2 short-splice isoform does not undergo Ca2+-induced cytonuclear shuttling
In the present study, we describe a novel mechanism by which Pyk2 intracellular localization is regulated. Using various deletions we show that depolarization-induced nuclear accumulation of Pyk2 is recapitulated by a region located in the kinase-FAT linker region, encompassing residues 700–841. Fused to GFP this polypeptide was excluded from the nucleus, whereas it was predominantly nuclear following depolarization or LMB treatment. Conversely, deletion of this polypeptide from Pyk2 resulted in a predominantly nuclear localization of the enzyme. The LMB-sensitivity suggested a CRM1-mediated nuclear export, and we identified a sequence possibly corresponding to a NES, L730LAPKLQFQVP. Mutation of L735, F737, and V739 induced a complete relocalization of GFP-Pyk2700–841 to the nucleus. This result demonstrated that L735QFQV is necessary to maintain Pyk2 in the cytoplasm in basal conditions. However, a region encompassing residues 768–793 was also necessary for the nuclear export to be active. Since we suspected that nuclear trafficking was regulated by phosphorylation/dephosphorylation, we systematically mutated Ser/Thr residues in this region and found that S778 played a critical role. S778 is located in a canonical PKA phosphorylation site and mutation of either S778 or the two basic residues involved in kinase interaction resulted in GFP-Pyk2700–841 nuclear relocalization. Large-scale analysis of protein phosphorylation using mass spectrometry has found that S778 is one of the phosphorylated residues of Pyk2 in vivo [47, 48]. However, until now, nothing was known about the specific function of S778 and this is the first time a role of Pyk2 S/T phosphorylation has been identified. We found that S778 was an excellent substrate for PKA and was dephosphorylated by calcineurin. The S778A mutation resulted in a depolarization-insensitive nuclear localization of full-length Pyk2. The “occlusion” of the LMB effects in this mutant further indicates that S778 regulates nuclear export of Pyk2. Our results suggest that S778 phosphorylation is necessary for the NES activity of the L730LAPKLQFQV motif, and that its dephosphorylation by calcineurin is a major trigger of the nuclear localization of Pyk2. A strong and delayed activation of nuclear PKA occurs following stimulation of membrane receptors in neurons . Thus, PKA may phosphorylate S778 in the nucleus allowing the export of Pyk2 to the cytoplasm. Experiments with a phospho-specific antibody targeting this residue supported this hypothesis since phosphorylated S778 was found only in the cytoplasm of unstimulated cells and depolarization-induced nuclear relocalization was concomitant with the disappearance of pS778 immunocytofluorescence and with a decrease in the pS778 signal in immunoblots. Although we demonstrated that S778 is phosphorylated by PKA in vitro and in response to forskolin in intact cells, we cannot exclude that other kinase(s) is(are) also active on this residue. In fact the predominantly cytoplasmic localization of Pyk2 and the cytoplasmic pS778 immunofluorescence suggest a basal S778 phosphorylation, which may result from the activity of other kinase(s) and/or to a remarkable resistance of pS778 to dephosphorylation by phosphatases other than calcineurin. S778 also belongs to a consensus sequence for casein kinase 2 (CK2) (3 acidic residues at positions +3–5). However, CK2 inhibition did not interfere with Pyk2 localization (Faure and Girault, unpublished observation). In contrast, mutation of the basic residues defining PKA consensus recognition site (K775R776) or pharmacological inhibition of PKA activity both induced a nuclear localization of Pyk2, supporting the important role of PKA in PC12 cells. It is worth mentioning that induction of nuclear localization of other proteins such as NFAT by calcineurin involves dephosphorylation of several residues . The partial increase of nuclear localization induced by the T765A mutation suggests that T765 might also contribute to nuclear exclusion of Pyk2.
It is interesting to note that the NLS is conserved between FAK and Pyk2, suggesting that the ability to be enriched in the nucleus is common to these kinases. In contrast, the region containing the NES, NTS, and S778 is specific to Pyk2 and these motifs are conserved in Pyk2 from various species from fish to mammals, suggesting that the Ca2+/calcineurin regulated nuclear localization is an important and specific feature of Pyk2. It is remarkable that these sequences are absent from the short isoform of Pyk2, which is predominantly found in hematopoietic cells, whereas the long isoform is highly expressed in the brain [10, 12]. In PC12 cells the short isoform was found in the nucleus in basal conditions. Thus the regulated nuclear export of Pyk2 appears to be specific to the neuronal isoform.
What is the function of Pyk2 in the nucleus? Interesting clues are available from various models. Nuclear Pyk2 activates keratinocytes differentiation by increasing the expression of Fra-1 and JunD transcription factors . The autophosphorylated, active form of Pyk2 accumulates in the nucleus of depolarized neurons , although Pyk2 nuclear accumulation is independent of its autophosphorylation and kinase activity ( and present study). Pyk2 could directly regulate tyrosine phosphorylated resident proteins of the nucleus such as histone variant H2AX  or H3/H4 . Pyk2 associates with Src-family kinases, which localize to the nucleus in specific conditions, regulating euchromatin decondensation  and protection against oxidative stress through phosphorylation of Nrf2 by Fyn [54, 55]. Nuclear Pyk2 may also play a role of a scaffolding protein for p53 . Our findings will allow designing the tools necessary for addressing these possibilities.
In conclusion, the present study shows that the Pyk2 long isoform expressed in neurons can undergo a Ca2+- and calcineurin-regulated nuclear accumulation. Evidence from other cell types indicates possible functional targets of nuclear Pyk2 and suggests that its regulated cyto-nuclear traffic may be involved in various brain functions or dysfunctions.
This work was supported by Inserm, UPMC, and grants from Agence nationale de la recherche (ANR-08-BLAN-0287-02), Fondation pour la recherche médicale (FRM), Association pour la recherche contre le cancer (ARC), Fondation pour la recherche sur le cerveau (FRC), Framework Program 7 (SynSys), and European research council (ERC). The group of JAG is affiliated with the Paris School of Neuroscience (ENP).
Conflict of interest