Correspondence to Pietro De Camilli: pietro.decamilli{at}yale.edu
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Introduction |
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Binding of talin to the cytoplasmic domain of integrin ß subunits induces conformational changes in the extracellular portion of the /ß integrin dimer leading to increased affinity for extracellular ligands (the so-called inside-out signaling) (Hynes, 2002; Calderwood and Ginsberg, 2003). Conversely, cell adhesion to substrates induces the nucleation of focal adhesions and the nucleation of the associated actin cytoskeleton. PI(4,5)P2 is known to play an important regulatory role in this bidirectional signaling by its interactions with many proteins of focal adhesions, including talin (Martel et al., 2001; Takenawa and Itoh, 2001; Yin and Janmey, 2003). Talin binds to PIPKI
90 via its band 4.1/ezrin/radixin/moesin (FERM) domain, primarily by its F3 subdomain (Di Paolo et al., 2002; Ling et al., 2002), which also comprises the binding site for ß integrin (Calderwood et al., 1999). In fact, the interactions of PIPKI
90 and ß integrin with the F3 subdomain are mutually exclusive (Barsukov et al., 2003). Accordingly, overexpression of constructs composing the 28-aa tail of PIPKI
90 produces a "trans-dominant inhibition" of integrin function, by competing for its binding to talin (Calderwood et al., 2004). The tyrosine of the sequence WVYSPL undergoes endogenous phosphorylation by Src (Ling et al., 2003). This phosphorylation reaction, which may contribute to the stimulatory effect of focal adhesion kinase on focal adhesions, was reported to enhance the interaction between PIPKI
90 and talin's F3 subdomain, which has a phosphotyrosine binding domain-like fold (Garcia-Alvarez et al., 2003).
PIPKI, in concert with the polyphosphoinositide phosphatase synaptojanin (Cremona et al., 1999), plays an important regulatory function in synaptic physiology. Based on genetic studies and other experimental approaches, it was proposed that PIPKI
controls a plasma membrane pool of PI(4,5)P2 implicated in synaptic vesicle exo-endocytosis (Wenk et al., 2001; Di Paolo et al., 2004) and that synaptojanin dephosphorylates PI(4,5)P2 during the endocytic reaction (Cremona et al., 1999; Wenk and De Camilli, 2004). At synapses, both PIPKI
(Wenk et al., 2001) and synaptojanin (Lee et al., 2004), as well as several other proteins implicated in vesicle recycling and actin function (Bauerfeind et al., 1997; Cousin and Robinson, 2001; Tan et al., 2003; Tomizawa et al., 2003), undergo constitutive phosphorylation and stimulation-dependent, Ca2+-dependent dephosphorylation. At least for some of these proteins, phosphorylation at rest as well as rapid rephosphorylation after a depolarization stimulus is mediated by Cdk5 (Tan et al., 2003; Tomizawa et al., 2003; Lee et al., 2004). Conversely, dephosphorylation is mediated by the Ca2+-dependent phosphatase calcineurin (Bauerfeind et al., 1997; Cousin and Robinson, 2001). In the case of synaptojanin 1, its dephosphorylation by calcineurin triggers its recruitment and activation at endocytic sites from a cytosolic pool (Lee et al., 2004).
The goal of the present study was to examine the regulation by phosphorylation of PIPKI90. Our results demonstrate that S650 within the talin-binding sequence WVYSPL is a main target of regulation by phosphorylation and that S650 phosphorylation blocks talin binding. Furthermore, a mutually antagonistic relationship exists between Src and proline-directed kinases with regard to the phosphorylation of adjacent sites on PIPKI
90, thereby providing a mechanism for the bidirectional modulation of interactions between PIPKI
90 and talin. This regulation is not restricted to synapses and may play an important and general function at focal adhesions.
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Results |
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To further confirm the site of phosphorylation and for use in the studies described later, an antibody that specifically recognizes PIPKI90 phosphorylated at S650 (antiphospho-S650 [pS650] antibody) was raised. The pS650 antibody selectively recognized WT PIPKI
90 phosphorylated in vitro by p35/Cdk5 (Fig. 2 A). Under the same conditions (after 30-min incubation), the S650A mutant was phosphorylated to a much lower extent as revealed by the incorporation of 32P (reflecting one or other phosphorylation sites besides S650), and no signal was detected with the pS650 antibody (Fig. 2 A). WT or S650A HA-PIPKI
90 was transfected into CHO cells with p35 plus Cdk5. Using the pS650 antibody to analyze immunoprecipitated PIPKI
90, the WT, but not the S650 mutant, protein was found to be phosphorylated (Fig. 2 B). Omission of p35 and Cdk5, or transfection with catalytically inactive mutant of Cdk5 (mut-Cdk5; Patrick et al., 1999) resulted in a significantly lower phosphorylation of S650 compared with that observed after transfection with p35/Cdk5 (Fig. 2 C). Under these conditions, S650 may be phosphorylated by low levels of endogenous Cdk5 (Dhavan and Tsai, 2001) and/or by other proline-directed protein kinases (see Figs. 7 and 8).
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We next determined whether a phosphomimetic mutation of S650 to aspartic acid (S650D) affected the interaction with talin. WT and S650D His6-PIPKI90 were overlaid in a "far-Western" assay with a GST fusion protein of the F3 subdomain of the FERM domain of talin (Fig. 4, A and C). The F3 domain clearly bound to the WT protein, as expected (Di Paolo et al., 2002), but no binding was observed for the S650D mutant (Fig. 4 A). Binding of the F3 domain to the S650A mutant was also reduced, although not abolished, and this is in agreement with a potential role of the side chain of serine in the binding (Di Paolo et al., 2002; Liddington et al., 2003). These results were qualitatively confirmed by His6-PIPKI
90 pull-down assays from rat brain extracts (Fig. 4, B and C). In this case, the loss of binding produced by the S650A mutation was nearly as large as that produced by the S650D mutation.
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PIPKI90 and ß integrin share amino acid similarity in the talin-binding region. Within these similar sequences, S650 of PIPKI
90 was proposed to functionally replace the asparagine of the sequence NPXY in ß integrin. When S650 of PIPKI
90 was replaced by an asparagine (S650N), the resulting mutant protein was still able to bind to talin, confirming this prediction. Thus, transfected GFP-PIPKI
90 harboring the S650N mutation colocalized with vinculin at focal adhesions (Fig. 4 F) and coprecipitated with talin from lysates of transfected cells (Fig. 4 G). The presence of a serine at this position in PIPKI
90, but not in ß integrin, allows for a differential regulation of the two interactions by phosphorylation.
If the binding of PIPKI90 to talin is important for focal adhesion dynamics, overexpression of p35/Cdk5 would be expected to affect focal adhesion dynamics by enhancing the phosphorylation state of S650. Consistent with this prediction, overexpression of p35/Cdk5 in NIH3T3 cells disrupted focal adhesions and stress fibers, as detected by antivinculin immunostaining and phalloidin staining, respectively (Fig. 5).
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S650 phosphorylation of PIPKI90 is regulated by Cdk5 and calcineurin at synapses
In the next series of experiments, the in vivo phosphorylation of S650 in endogenous PIPKI90 was investigated. Rat brain synaptosomes, including samples exposed to a 1-h metabolic labeling step with 32Pi, were incubated for 20 min in control buffer, and then were depolarized for 1 min with high K+ (55 mM) in the absence or presence of extracellular Ca2+. As detected by using 32P incorporation or by using the pS650 antibody, we found that PIPKI
90 was constitutively phosphorylated at rest, and dephosphorylated upon high K+ stimulation, but only in the presence of extracellular Ca2+ (Fig. 7 A). The stimulation-dependent dephosphorylation of pS650 was blocked by cyclosporin A, a calcineurin inhibitor (Fig. 7 B). Furthermore, rephosphorylation of S650 upon reexposure to the control buffer was partially inhibited by incubation with butyrolactone I (Fig. 7 C) or roscovitine (unpublished data). The pattern of PIPKI
90 dephosphorylation was qualitatively similar to that of amphiphysin 2, whose upper band (phospho-form) collapses into lower bands (dephospho form) upon calcineurin-dependent dephosphorylation (Fig. 7 A; Bauerfeind et al., 1997; Cousin and Robinson, 2001).
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The potential involvement of MAPK as well as of phosphatases other than calcineurin in the regulation of S650 phosphorylation was investigated. Synaptosomes were treated with either okadaic acid, an inhibitor of protein phosphatase 2A (PP2A), or PD98059, an inhibitor of MAPK1/2 (p42/p44). Okadaic acid alone strongly enhanced the phosphorylation state of S650 both at rest and after high K+ depolarization (Fig. 7 D), although a stimulation-dependent dephosphorylation of S650 still occurred upon exposure to high K+ (Fig. 7 D, compare the second and fourth lanes). Okadaic acid also increased the level of phospho-MAPK1/2 (Fig. 7 D). This was expected because PP2A is known to dephosphorylate MAPK, with a resulting inhibition of its activity (Alessi et al., 1995). PD98059 partially prevented the increase in S650 phosphorylation produced by okadaic acid (Fig. 7 E). The increase of pS650 produced by okadaic acid may result both from a direct inhibition of pS650 dephosphorylation by PP2A and/or indirectly from the enhanced MAPK activity caused by inhibition of PP2A. Note that, as previously reported, depolarization in the presence of extracellular Ca2+ increases the phosphorylation state of MAPK1/2 (Fig. 7 D, compare the first and third lanes; Yamagata et al., 2002). This is in contrast to the depolarization-dependent dephosphorylation of S650, thus indicating that calcineurin can override the action of MAPK on S650. Consistent with PIPKI90 being a substrate for MAPK, purified MAPK1 phosphorylated S650 in WT His6-PIPKI
90 in vitro, but did not phosphorylate S650A mutant His6-PIPKI
90 (Fig. 7 F), as determined by anti-p5650 Western blotting.
S650 of PIPKI90 undergoes mitotic phosphorylation
Entry of cells into mitosis is accompanied by massive structural changes. For example, cells grown in culture round up and partially detach from the substrates, a change which correlates with the disruption of focal adhesions (Maddox and Burridge, 2003). The property of S650 of PIPKI90 to function as a substrate for Cdk5, a member of the Cdk family, prompted us to examine its phosphorylation state during mitosis. Disruption of the interaction between talin and PIPKI
90 could be one of the biochemical modifications that correlate with focal adhesion disassembly. CHO cells transfected with WT or mutant HA-PIPKI
90 were either treated with nocodazole to arrest cells in pro-metaphase, or treated with nocodazole and then allowed to progress to G1 interphase by re-addition of serum. pS650 immunoreactivity was detected only in cells expressing WT PIPKI
90 and phosphorylation was dramatically increased in mitotic cells (Fig. 8 A). These results were corroborated by the analysis of endogenous PIPKI
90 in cells (U87MG cells, derived from a human astrocytoma) that express high levels of this enzyme. In this case, cell lysates were subjected to immunoprecipitation with anti-PIPKI
90 antibody prior to Western blotting (Fig. 8 B). Not only S650 phosphorylation was strongly stimulated in mitosis, but coprecipitation of talin with the kinase was nearly abolished in the mitotic state. The mitotic synchronization of these cells was validated by Western blotting of total lysates with antibodies directed against phospho-histone H3, a marker of mitosis (Ajiro et al., 1996). In support of a role for cyclin-activated kinases in the phosphorylation of PIPKI
90 during mitosis, purified cyclin B1Cdk1 complex phosphorylated purified WT His6-PIPKI
90 in vitro but weakly phosphorylated its S650A mutant (Fig. 8 C). Cyclin B1, a physiological substrate of Cdk1 (Borgne et al., 1999), was phosphorylated at roughly similar levels in both samples (Fig. 8 C).
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Discussion |
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At synapses, the generation of PI(4,5)P2 by PIPKI is thought to play an important regulatory role both in exocytosis and in endocytosis (Wenk et al., 2001; Di Paolo et al., 2004; Wenk and De Camilli, 2004). The initial goal of this study was to determine whether PIPKI
90, like several other synaptic proteins that participate in synaptic vesicle traffic, primarily endocytic traffic (Bauerfeind et al., 1997; Cousin and Robinson, 2001; Tan et al., 2003; Tomizawa et al., 2003; Lee et al., 2004), is regulated by the serine/threonine phosphorylation. We have now demonstrated that PIPKI
90 is efficiently phosphorylated at S650 by Cdk5 both in vitro and in vivo, and that this Cdk5 site is dephosphorylated by calcineurin. Importantly, we have shown that phosphorylation of this site negatively regulates the talinPIPKI
90 interaction. Such an interaction is vital for synaptic physiology, because its perturbation by peptides microinjected into giant axons perturbs synaptic vesicle recycling and the dynamics of presynaptic actin (Morgan et al., 2004). Restoration of the interaction by depolarization-triggered dephosphorylation may serve to recruit and activate PIPKI
90 at sites of exo-endocytosis and may underlie, together with interaction of PIPKI
90 with small GTPases (Aikawa and Martin, 2003; Krauss et al., 2003), the increase in PI(4,5)P2 synthesis occurring in stimulated synaptosomes (Di Paolo et al., 2004). Interestingly, in preliminary experiments, we have found that a guanyl nucleotideindependent interaction of PIPKI
90 with Rac1 is also inhibited by Cdk5-dependent phosphorylation at a site distinct from S650 (unpublished data). These findings support a model according to which Cdk5 phosphorylation keeps the synapse in a "resting mode," whereas calcineurin triggers an "active mode" (Lee et al., 2004; Sahin and Bibb, 2004).
Our results suggest that phosphorylation of S650 may also be regulated by other proline-directed kinases and at least one other phosphatase. A potential role of MAPK can be inferred from the inhibitory action of the MAPK inhibitor PD98059 on the levels of pS650 in vivo and from the in vitro phosphorylation of PIPKI90 by purified MAPK1. The powerful stimulatory effect of okadaic acid, an inhibitor of the PP2A (Jovanovic et al., 2001), on the levels of pS650 is also consistent with an action of MAPK, because PP2A could act indirectly by dephosphorylating and, therefore, inhibiting MAPK (Alessi et al., 1995). In the presence of okadaic acid, such activity would be greatly stimulated. Additionally, PP2A may act directly on pS650. If this is the case, the large increase of pS650 observed in the presence of okadaic acid in unstimulated synaptosomes would indicate the occurrence of a very rapid turnover of phosphate on S650 even at rest. Among the questions that remain to be addressed is whether different kinases and phosphatases act on pools of PIPKI
90 with distinct subcellular localization, because PIPKI
90 is concentrated in, but not restricted to, nerve terminals. However, important conclusions from this study are that S650 represents the point of convergence of different regulatory pathways and that its state of phosphorylation controls an important targeting and activation mechanism of this enzyme.
Cdk5 phosphorylation was found to have only a negligible (positive) effect on the catalytic activity of PIPKI90 (unpublished data). Because Cdk5 also phosphorylated PIPKI
87 and slightly activated its catalytic activity (unpublished data), S650 does not seem to participate in this regulation. However, it is possible, that sites phosphorylated by other kinases may directly control its catalytic activity. An inhibitory role on PIPKI
activity produced by protein kinase A phosphorylation at serine 264 (human sequence) was previously suggested, based on studies of the homologous mouse PIPKI
(Park et al., 2001). The precise coordination of different phosphorylation reactions remains to be investigated.
Our study also shows that S650 of PIPKI90 can undergo phosphorylation in nonneuronal cells, where PIPKI
90 is primarily localized at focal adhesions. However, although transfected Cdk5a protein kinase expressed predominantly, but not exclusively, in neurons (Dhavan and Tsai, 2001)can phosphorylate transfected PIPKI
90 in CHO cells, the protein kinase that performs this phosphorylation reaction physiologically in interphase nonneuronal cells remains to be identified. As suggested by the phosphomimetic S650D mutant, phosphorylation of S650 prevents the localization of PIPKI
90 at focal adhesions. In addition, the S650D mutant of PIPKI
90 did not compete with the interaction of talin and ß integrin. Therefore, one can expect that regulation of the turnover of phosphate on S650 may have a key role in the regulation of the balance between adhesion and motility (Calderwood and Ginsberg, 2003; Carragher and Frame, 2004). It is of interest, in this context, that Cdk5 plays a major role in the regulation of cell migration and neurite outgrowth in the nervous system (Dhavan and Tsai, 2001; Xie et al., 2003) and of cell adhesion and migration in nonneuronal cells (Gao et al., 2002). As we have shown here, its overexpression strongly affects focal adhesion.
As we also show here, S650 of PIPKI90 undergoes phosphorylation in mitosis and is a very good in vitro, and a likely in vivo, substrate for the cyclin B1Cdk1 complex. It will be of interest to determine whether phosphorylation of PIPKI
90 at S650 helps drive focal adhesion disassembly during mitosis, or simply correlates with this process. We note that other proteins implicated in membrane traffic at synapses are also physiological substrates for both cyclin B1/Cdk1 and p35/Cdk5. These include epsin, Eps15, and amphiphysin (Chen et al., 1999; Floyd et al., 2001). Mechanisms that regulate entry into the mitotic state, where much of the exo-endocytic traffic is blocked (Pypaert et al., 1991), may be closely related to mechanisms that limit exo-endocytic traffic at neuronal synapses at rest.
At variance with what was reported previously (Ling et al., 2003), studies with purified peptides, including the same peptides used by Ling et al. (2003), did not reveal an effect of Y649 phosphorylation on the interaction of PIPKI90 with talin. Furthermore, structural data indicate that phosphorylation of Y649 would be unlikely to directly perturb the interaction between these two proteins (Liddington et al., 2003; de Pereda et al., 2004). In contrast, the negative effect of S650 phosphorylation on the interaction of PIPKI
90 with talin is consistent with structural predictions pointing to an intimate interaction of S650 with talin (Liddington et al., 2003) and with the mutagenesis experiments reported here.
However, we did find that the presence of phosphate on Y649 strongly inhibits the phosphorylation of S650, and vice versa. Thus, Src-dependent phosphorylation of Y649 may act indirectly to enhance talin binding by inhibiting phosphorylation of S650. Conversely, in addition to directly blocking the interaction with talin, phosphorylation of S650, in a feed-forward mechanism, blocks the phosphorylation reaction at Y649 that inhibits S650 phosphorylation. Src family kinases have been implicated both in synaptic function and in focal adhesion dynamics (Purcell and Carew, 2003; Carragher and Frame, 2004), thus supporting a physiological significance of this bidirectional control of PIPKI90.
While our manuscript was in review, a study by de Pereda et al. (2004) reported a crystallographic analysis of the interaction between the F3 subdomain of talin and the COOH-terminal tail of mouse PIPKI. The structure is consistent with the results of our phosphorylation studies. It predicts that phosphorylation of serine 650 of human PIPKI
90 would block the interaction, and explains the lack of effect that we report here for the phosphorylation of tyrosine 649. Dissociation constant values determined by ITC for the talinPIPKI
90 peptide interaction in this new study (see Fig. S1 in de Pereda et al., 2004) are in the same range of those reported by us and show only a minor (1.6-fold) positive effect of phosphorylation of tyrosine 644, which corresponds to human tyrosine 649, on the interaction (de Pereda et al., 2004). Overall, these new findings are in very good agreement with our results.
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Materials and methods |
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DNA constructs and protein expression
Constructs of human PIPKI87 (640 aa), PIPKI
90 (668 aa) and 28--aa tail (641668) of PIPKI
90, and the F3 subdomain and head domain of talin were subcloned into pcDNA3-HA, pGEX-6P-1, or pEGFP-C2 vectors, as described previously (Di Paolo et al., 2002; Krauss et al., 2003). We also subcloned PIPKI
87 and PIPKI
90 into a modified version of pET15 vector (Novagen) with an NH2-terminal His6-tag. S650 in the PIPKI
90 constructs was mutated to alanine, aspartic acid, or asparagine by a QuikChange site-directed mutagenesis kit (Promega), and the mutations were confirmed by DNA sequencing. Full-length GST- and His6-PIPKI
90 fusion proteins expressed in Sf9 cells and Escherichia coli strain BL21, respectively, were purified using glutathione-Sepharose beads (Amersham Biosciences) and Talon metal affinity resin (BD Biosciences) following the manufacturer's protocols and dialyzed against TBS. Expression constructs encoding p35, Cdk5, and mut-Cdk5 in the pCMV vector were gifts from L.-H. Tsai (Harvard Medical School, Boston, MA). A construct of GST-integrin ß1 tail was a gift from J. Ylanne (University of Oulu, Oulu, Finland).
In vitro phosphorylation
p35Cdk5 and cyclin B1Cdk1 complexes, expressed and purified from Sf9 cells coinfected with baculovirus encoding each component, were gifts from Y. Kim and P. Greengard (The Rockefeller University, New York, NY) and Y. Wang and G. Warren (Yale University), respectively. Purified GST-PIPKI90 or His6-PIPK
87/90 fusion proteins (200300 ng) were incubated with p35Cdk5 complex (Lee et al., 2004) or cyclin B1Cdk1 complex (Wang et al., 2003) at an
1:1 stoichiometric ratio in the presence of 10 µCi
-[32P]ATP (1 Ci = 37 GBq) for up to 30 min at 30oC. PIPKI
90 phosphorylation by MAPK1 (Upstate Biotechnology) was performed according to the manufacturer's protocol. Kinase reactions (3040 µl final volume) were stopped by the addition of SDS-PAGE sample buffer, and samples were analyzed by SDS-PAGE and autoradiography. In case of peptide phosphorylation by c-Src (Upstate Biotechnology) and p35/Cdk5, each kinase was mixed with the 12-mer peptides (each 25 µM) in the presence of 400 µM ATP and 40 µCi
-[32P]ATP. c-Src phosphorylation was performed in 25 mM Tris-HCl, pH 7.2, 30 mM MgCl2, 0.5 mM EGTA, and 62.5 µM Na3VO4, and p35/Cdk5 phosphorylation was assayed as described above. After a 20-min incubation at 30oC, reaction mixtures (40 µl) were stopped by adding 20 µl of 40% TCA solution, and a portion of reaction mixtures (25 µl) was spotted onto P81 phosphocellulose paper (Whatman). All reactions were in the linear range and consumed <1% of peptide substrates. After washing five times with 0.75% phosphoric acid and once with acetone, radiolabeling of the peptides was quantified by Cerenkov counting.
Identification of phosphorylation sites and 2-D peptide mapping
12 µg His6-PIPK90 labeled with
-[32P]ATP by p35Cdk5, as described in the previous paragraph, was excised from SDS-PAGE and digested with trypsin (Lee et al., 2004). The resulting peptides were separated by HPLC and detected by Cerenkov counting. Peptide masses containing a phosphate group were determined by MALDI/MS, and the phosphorylation sites were confirmed by further radio sequencing of the peptides. 2-D phosphopeptide mapping was performed as previously described (Hsieh-Wilson et al., 2003). In brief, after centrifugation of the trypsin-digested radiolabeled material, supernatants were collected and lyophilized. Pellets were then washed with distilled water and running buffer (10% acetic acid and 1% pyridine in water) several times. Lyophilized pellets were dissolved in running buffer and spotted onto cellulose TLC plates. The plates were sequentially subjected to electrophoresis in the running buffer, pH 3.5, at 400 V, and then to chromatography in a buffer (25% 1-butanol, 7.5% acetic acid, and 37.5% pyridine in water). After drying the plates, autoradiography was performed.
In vitro talin-binding assay and ITC
The talin overlay assay was performed as described previously (McPherson et al., 1994). 100200 ng of purified His6-PIPKI90 fusion proteins transferred onto nitrocellulose membranes were incubated without or with GST-F3 fusion protein (2 µg/ml). Membranes were blotted with anti-GST antibody. For the pull-down assay from rat brain extracts, His6-PIPKI
90 fusion proteins (45 µg) coupled to Talon metal affinity beads in TBS containing 1% (vol/vol) Triton X-100 were mixed with rat brain extracts (6.4 mg) prepared in buffer A (20 mM Hepes, pH 7.4, 120 mM KCl, 1 mM DTT, 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 1 mM PMSF, and 1% [vol/vol] Triton X-100) for 3 h at 4oC. ITC measurements were performed using a Microcal VP-ITC isothermal titration calorimeter equipped with a PC running VPViewer software. Dissociation constant values were obtained from the data collected automatically and analyzed with Origin software. In each experiment, 3-µl aliquots of peptide solution (1 mM) were injected into a calorimetric cell preloaded with 1.4267 ml GST-talin head domain (16.4 µM) using a rotating stirrer syringe (250-µl vol) every 250 s at 37°C. All solutions included 20 mM sodium phosphate, pH 7.4, and 150 mM NaCl and were degassed. To estimate a blank heat effect associated with dilution and mechanical phenomena, peptide injections were performed after saturation of binding.
Cell culture, transfection, and immunoprecipitation
CHO cells were grown in DMEM supplemented with 10% FBS and antibiotics and transfected for 24 h with expression constructs of WT or mutants of HA-PIPKI90 and of GFP-PIPKI
90, p35, Cdk5 or mut-Cdk5 using Lipofectamine 2000 (Life Technologies) according to the instruction manual. For immunoprecipitation of PIPKI
90 and talin, the cell lysates prepared in the buffer A were incubated with anti-PIPKI
90 and antitalin antibodies for 3 h at 4oC, and then were incubated with protein A and protein GSepharose beads (Amersham Biosciences) for 2 h, respectively. Cell lysates and bound materials washed with the buffer A five times were analyzed by SDS-PAGE and Western blotting. A similar protocol was used for the affinity chromatography of talin from transfected CHO cells on the GST-integrin ß1 tail, except that glutathione Sepharose beads were used.
Synaptosomes
Rat brain synaptosomes were prepared as described previously (Bauerfeind et al., 1997; Lee et al., 2004). In brief, 200-µl aliquots of synaptosomes were preincubated in the "control buffer" with or without Ca2+ for 20 min at 37oC, and then diluted with an equal volume of either control buffer or stimulation buffer containing high K+. When inhibitors of protein kinases and phosphatases were used, the inhibitors were present during both the preincubation and the stimulation steps. For analysis of total phosphorylation of PIPKI90, synaptosomes were labeled with 2 mCi/ml
-[32P]orthophosphate for 1 h prior to the preincubation. After a 1-min stimulation, 100 µl of buffer B (20 mM Hepes, pH 7.4, 50 mM NaCl, 50 mM Na3PO4, 50 mM NaF, 5 mM EDTA, 5 mM EGTA, 5 mM ß-glycerophosphate, 1 mM PMSF, and 1% Triton X-100 [vol/vol]) supplemented with 10% SDS (final 2% SDS) was directly added to synaptosomes for lysis and incubation mixtures were boiled for 10 min. For repolarization, synaptosomes stimulated by high K+ were rapidly harvested, resuspended in control buffer, and were further incubated for 15 min before lysis. For PIPKI
90 immunoprecipitation, synaptosomal lysates were diluted with 9 vol of the buffer B and added to protein ASepharose beads precoated with anti-PIPKI
90 antibody for 2 h at 4oC. Western blotting or autoradiography of synaptosomal lysates and PIPKI
90 immunoprecipitates were performed after SDS-PAGE.
Mitotic synchronization
U87MG cells were grown in minimum essential medium with Earle's salts supplemented with 10% FBS, 0.1 mM nonessential amino acids, 2 mM L-glutamine, 1 mM sodium pyruvate, 0.15% sodium bicarbonate, and antibiotics. To prepare both mitotic and G1 interphase cells, subconfluent U87MG cells were treated with 250 ng/ml nocodazole (Sigma-Aldrich) for 18 h (Chen et al., 1999). Mitotic cells released into culture medium were collected by mechanical shake-off and washed with PBS. To harvest G1 interphase cells, the mitotic cells were replated into fresh culture medium and further incubated for 10 h. CHO cells transfected with HA-PIPKI90 constructs were processed as described above. Preparation of cell lysates in the buffer A, PIPKI
90 immunoprecipitation, SDS-PAGE, and Western blotting were performed as described above.
Immunofluorescence
Immunofluorescence of transfected NIH3T3 cells was performed as described previously (Di Paolo et al., 2002). Fluorescence was visualized with a microscope (Axioplan 2; Carl Zeiss MicroImaging, Inc.) equipped with a cooled CCD camera (Orca ER2; Hamamatsu) using a Plan-Apochromat (63x, 1.4 NA) oil immersion objective at room temperature. Images were acquired with Metamorph software (Universal Imaging) and processed using Adobe Photoshop.
Miscellaneous
Amounts of proteins were calculated by a BCA assay kit (Pierce). SDS-PAGE and Western blotting were conducted using standard procedures.
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Acknowledgments |
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This work was supported in part by grants from the National Institutes of Health (NS36251 and CA46128 to P.D. Camilli; DA10044 to A.C. Nairn; Yale/National Institute on Drug Abuse Neuroproteomics Center, P30 DA018343 to P.D. Camilli and A.C. Nairn) and from the Yale Center for Genomics and Proteomics to P.D. Camilli., a Brown-Coxe fellowship to S. Voronov, a Howard Hughes Medical Institute fellowship to K. Letinic, and grants from the Yale Diabetes and Endocrinology Center to P. Di Camilli and G. Di Paolo.
Submitted: 7 September 2004
Accepted: 18 January 2005
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