Striatal Enriched Phosphatase 61 Dephosphorylates Fyn at Phosphotyrosine 420

Abstract

A family of protein tyrosine phosphatases enriched within the central nervous system called striatal enriched phosphatase (STEP) has been implicated in the regulation of the N-methyl-d-aspartate receptor. STEP61, a membrane-associated isoform located in the postsynaptic densities (PSDs) of striatal neurons, contains two transmembrane domains, two proline-rich domains, and a kinase-interacting motif. This study demonstrates that STEP61 associates with Fyn, a member of the Src family kinases that is also enriched in PSDs. By using human embryonic kidney 293 cells for co-transfection, we determined that a substrate-trapping variant (STEP61 CS) binds to Fyn but not to other members of the Src family present in PSDs. In a complementary experiment, myc-tagged Fyn immunoprecipitates STEP61 CS. STEP61 binds to Fyn through one of its proline-rich domains and the kinase-interacting motif domain, whereas Fyn binds to STEP61 through its Src homology 2 domain and the unique N-terminal domain. STEP61 CS pulls down Fyn when the Tyr420 site is phosphorylated. In vitro, wild-type STEP61 dephosphorylates Fyn at Tyr420but not at Tyr531. These results suggest that STEP regulates the activity of Fyn by specifically dephosphorylating the regulatory Tyr420 and may be one mechanism by which Fyn activity is decreased within PSDs. A family of protein tyrosine phosphatases enriched within the central nervous system called striatal enriched phosphatase (STEP) has been implicated in the regulation of the N-methyl-d-aspartate receptor. STEP61, a membrane-associated isoform located in the postsynaptic densities (PSDs) of striatal neurons, contains two transmembrane domains, two proline-rich domains, and a kinase-interacting motif. This study demonstrates that STEP61 associates with Fyn, a member of the Src family kinases that is also enriched in PSDs. By using human embryonic kidney 293 cells for co-transfection, we determined that a substrate-trapping variant (STEP61 CS) binds to Fyn but not to other members of the Src family present in PSDs. In a complementary experiment, myc-tagged Fyn immunoprecipitates STEP61 CS. STEP61 binds to Fyn through one of its proline-rich domains and the kinase-interacting motif domain, whereas Fyn binds to STEP61 through its Src homology 2 domain and the unique N-terminal domain. STEP61 CS pulls down Fyn when the Tyr420 site is phosphorylated. In vitro, wild-type STEP61 dephosphorylates Fyn at Tyr420but not at Tyr531. These results suggest that STEP regulates the activity of Fyn by specifically dephosphorylating the regulatory Tyr420 and may be one mechanism by which Fyn activity is decreased within PSDs. protein tyrosine phosphatase extracellular signal-regulated kinase glutathione S-transferase human embryonic kidney kinase-interacting motif N-methyl-d-aspartate postsynaptic density striatal enriched phosphatase Src homology phosphate-buffered saline Dynamic regulation of protein tyrosine phosphorylation requires a balance between the activity of protein tyrosine kinases and protein tyrosine phosphatases (PTPs).1 One of the best-characterized families of protein tyrosine kinases is the Src kinase family, which consists of several members including Src, Fyn, Lyn, Lck, and Yes. These proteins are highly conserved in their amino acid sequences and their regulatory domains. Each member also contains a unique domain at the N terminus that distinguishes it from other family members (see review, Ref. 1Thomas S.M. Brugge J.S. Annu. Rev. Cell Dev. Biol. 1997; 13: 513-609Crossref PubMed Scopus (2134) Google Scholar). Src and Fyn are present within postsynaptic densities of central nervous system neurons (2Walikonis R.S. Jensen O.N. Mann M. Provance D.W. Mercer J.A. Kennedy M.B. J. Neurosci. 2000; 20: 4069-4080Crossref PubMed Google Scholar, 3Husi H. Ward M.A. Choudhary J.S. Blackstock W.P. Grant S.G. Nat. Neurosci. 2000; 3: 661-669Crossref PubMed Scopus (1012) Google Scholar). Their presence at the PSD and their direct association with both receptors and key anchoring proteins such as PSD-95 (4Tezuka T. Umemori H. Akiyama T. Nakanishi S. Yamamoto T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 435-440Crossref PubMed Scopus (330) Google Scholar) suggest that they regulate signaling events within these neurons. Indeed, several recent studies show that members of the Src kinase family modulate synaptic transmission by regulating the level of tyrosine phosphorylation of proteins within the PSD including the NR2 subunit of the N-methyl-d-aspartate (NMDA) receptor (5Lau L.F. Huganir R.L. J. Biol. Chem. 1995; 270: 20036-20041Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar, 6Kohr G. Seeburg P.H. J. Physiol. (Lond.). 1996; 492: 445-452Crossref Scopus (279) Google Scholar). The Src family kinases are themselves regulated by tyrosine phosphorylation. They contain two conserved tyrosine residues that are phosphorylated (tyrosine 420 and tyrosine 531; numbering according to the amino acid sequence of human Fyn). Phosphorylation of tyrosine 531 of Fyn by C-terminal Src kinase leads to its inactivation (7Sun G. Sharma A.K. Budde R.J. Oncogene. 1998; 17: 1587-1595Crossref PubMed Scopus (75) Google Scholar, 8Superti-Furga G. Fumagalli S. Koegl M. Courtneidge S.A. Draetta G. EMBO J. 1993; 12: 2625-2634Crossref PubMed Scopus (220) Google Scholar). Dephosphorylation of this site results in a conformational change and activation of the protein through autophosphorylation at the second site, Tyr420. The dynamic balance in the level of phosphorylation at these two sites contributes to the overall level of Fyn kinase activity. Therefore, the identification of tyrosine phosphatases that lead to the dephosphorylation of one regulatory site or the other would add to the understanding of how these kinases are regulated. The striatal enriched phosphatase (STEP) family of PTPs is enriched within specific subsets of neurons in the central nervous system (9Lombroso P.J. Naegele J.R. Sharma E. Lerner M. J. Neurosci. 1993; 13: 3064-3074Crossref PubMed Google Scholar,10Boulanger L.M. Lombroso P.J. Raghunathan A. During M.J. Wahle P. Naegele J.R. J. Neurosci. 1995; 15: 1532-1544Crossref PubMed Google Scholar). STEP61 is one member of this family. It contains two polyproline domains that meet the consensus sequence for motifs involved in protein-protein interactions (11Bult A. Zhao F. Dirkx R., Jr. Sharma E. Lukacsi E. Solimena M. Naegele J.R. Lombroso P.J. J. Neurosci. 1996; 16: 7821-7831Crossref PubMed Google Scholar). In addition, STEP61 contains a kinase-interacting motif (KIM) that has been identified as a domain required for the binding of STEP to extracellular signal-regulated kinase (ERK) 1/2 in vitro(12Blanco-Aparicio C. Torres J. Pulido R. J. Cell Biol. 1999; 147: 1129-1136Crossref PubMed Scopus (141) Google Scholar). STEP61 is regulated, in part, by dopamine signaling through a D1/cAMP/protein kinase A-mediated phosphorylation of the serine residue within the KIM domain. Phosphorylation at that site leads to a decrease in its enzymatic activity against substrates (13Paul S. Snyder G.L. Yokakura H. Picciotto M.R. Nairn A.C. Lombroso P.J. J. Neurosci. 2000; 20: 5630-5638Crossref PubMed Google Scholar). In contrast, this serine is dephosphorylated after the activation of NMDA receptors, leading to an increase in its phosphatase activity 2S. Paul, A. C. Nairn, P. Wang, and P. J. Lombroso, submitted for publication.2S. Paul, A. C. Nairn, P. Wang, and P. J. Lombroso, submitted for publication. and inhibition of ERK1/2-mediated signaling pathways within these neurons. STEP61 is present in the PSDs of neurons that receive both dopaminergic and glutamatergic synaptic input (14Gurd J.W. Bissoon N. Nguyen T.-H. Lombroso P.J. Rider C.C. Beesley P.W. Vannucci S.J. J. Neurochem. 1999; 73: 1990-1994PubMed Google Scholar, 15Nguyen T.-H. Paul S., Xu, Y. Gurd J.W. Lombroso P.J. J. Neurochem. 1999; 73: 1995-2001PubMed Google Scholar, 16Oyama T. Goto S. Nishi T. Sato K. Yamada K. Yoshikawa M. Ushio Y. Neuroscience. 1995; 69: 869-880Crossref PubMed Scopus (51) Google Scholar). STEP61 was recently shown to co-immunoprecipitate as part of the NMDA complex of proteins (17Pelkey K.A. Askalan R. Paul S. Kalia L.V. Nguyen T.-H. Pitcher G.M. Salter M.W. Lombroso P.J. Neuron. 2002; 34: 127-138Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Application of STEP to the cytoplasmic side of membrane patches from embryonic spinal cord neurons led to a depression in NMDA-mediated activity. In contrast, application of functionally inhibiting STEP antibodies led to an increase in NMDA receptor synaptic currents. Finally, application of STEP postsynaptically prevented the induction of long-term potentiation by tetanic stimulation in the CA1 region of hippocampal neurons (17Pelkey K.A. Askalan R. Paul S. Kalia L.V. Nguyen T.-H. Pitcher G.M. Salter M.W. Lombroso P.J. Neuron. 2002; 34: 127-138Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Thus, STEP appears to counter the activity of Src family members known to up-regulate synaptic transmission at excitatory synapses (18Yu X.M. Salter M.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7697-7704Crossref PubMed Scopus (106) Google Scholar). The exact mechanisms by which this occurs remain unclear. Recent studies have shown that the receptor-like PTP, PTPα, plays a role in modulating the activity of Src and Fyn by dephosphorylating phosphotyrosine 531 both in vivo and in vitro(19Bhandari V. Lim K.L. Pallen C.J. J. Biol. Chem. 1998; 273: 8691-8698Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 20Su J. Muranjan M. Sap J. Curr. Biol. 1999; 9: 505-511Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). The identification of additional PTPs that might participate in regulating these kinases was the goal of the present study. Because there are two regulatory tyrosine residues among Src family members, it was hypothesized that a second PTP might independently regulate the phosphorylated tyrosine at amino acid 420. Here, we demonstrate that STEP61 binds to Fyn both in vitro and in vivo. The domains in both proteins that govern this interaction were identified. In STEP61, both the KIM domain and the N-terminal proline-rich domain are involved. In Fyn, the SH2 domain and the unique N-terminal domain are responsible for the observed interaction. Finally, tyrosine 420, but not tyrosine 531, is dephosphorylated by STEP61. These results suggest that STEP61 participates in regulating Fyn activity and that this is one mechanism by which STEP regulates signaling events at excitatory synapses. All experimental procedures used in the present study were approved by the Animal Care and Use Committee at Yale University School of Medicine. Adult, female Long Evans rats were purchased from Charles River Laboratories (Wilmington, MA). Subcellular fractionation was performed with modifications to the methods described in previous publications (11Bult A. Zhao F. Dirkx R., Jr. Sharma E. Lukacsi E. Solimena M. Naegele J.R. Lombroso P.J. J. Neurosci. 1996; 16: 7821-7831Crossref PubMed Google Scholar, 15Nguyen T.-H. Paul S., Xu, Y. Gurd J.W. Lombroso P.J. J. Neurochem. 1999; 73: 1995-2001PubMed Google Scholar). In brief, rats were euthanized, and the striatum was dissected on ice and homogenized in 10-fold (w/v) cold buffer (320 mm sucrose, 4 mm HEPES, pH 7.4, and complete protease inhibitor mixture tablets (Roche Molecular Biochemicals)). Homogenized tissue was centrifuged at 800 × g for 10 min to form S1 and P1 fractions. The S1 fraction was further centrifuged at 9,000 ×g for 15 min to form a crude synaptosomal fraction (P2). This fraction was then washed in homogenization buffer and centrifuged at 10,200 × g for 15 min. The resulting pellet was lysed in a 10× volume of cold water and immediately buffered to 1 mm HEPES, pH 7.4. Lysed synaptosomes were centrifuged at 25,000 × g for 20 min to form LS1 and LP1 fractions. The LP1 fraction was resuspended in 250 mm sucrose and HEPES, pH 7.4, and additional protease inhibitors were added before overlaying the sample on a gradient consisting of 4 ml of 0.8m sucrose, 1 ml of 1.0 m sucrose, and 4 ml of 1.2 m sucrose. The gradient was centrifuged at 65,000 × g for 2 h. Samples from the 1.0–1.2 msucrose interface were collected and washed in a 10× volume of cold PBS at 48,000 × g for 10 min. The resulting PSDs were resuspended in PBS and frozen at −80 °C until needed. pGEX-STEP46 was transformed into BL21 cells and grown at 37 °C until log phase before induction with 0.1 mmisopropyl-β-d-thiogalactopyranoside. Cells were harvested, resuspended in 0.01 culture volume with PBS, and lysed in B-Per according to manufacturer's instructions (Pierce). Cell lysate was centrifuged, and the resulting supernatant was passed through a glutathione-Sepharose column (Amersham Biosciences). The column was washed extensively with PBS, and recombinant protein was eluted by using 10 mm glutathione in 50 mmTris-HCl, pH 8.0. STEP-GST recombinant protein was concentrated with a Centriplus-30 column (Millipore) before passing through Sephadex G-50 equilibrated with PBS to remove the glutathione peptide. GST-STEP fusion protein was coupled toN-hydroxysuccinimide-activated Sepharose-4 Fast Flow (Amersham Biosciences) overnight at 4 °C. The next day, unconjugated protein was removed in cold PBS, and nonreactive were with buffer m pH for 1 at protein was washed with buffer and buffer m m pH before a with cold The was then used to an The fraction was in by to remove The supernatant was collected and with cold PBS before passing through the which was then extensively washed with by PBS with or protein was eluted with pH buffer m pH and in the presence of 1.0 m pH was concentrated with and by using and STEP was in as described P.J. G. Lerner M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The GST-STEP was by using with and STEP the the sites and and the the in which and sites were and into In addition, a sequence was added within the for in The was also using the and a Fyn was a of M. The of Fyn was by and into was performed to or Fyn by using STEP and Fyn were performed according to instructions (see 1 for were with the phosphorylated with and by using cells were from and in with were performed as described T.-H. Paul S., Xu, Y. Gurd J.W. Lombroso P.J. J. Neurochem. 1999; 73: 1995-2001PubMed Google Scholar). In brief, a before cells were at 1 × The next day, was with and All were on ice or at 4 cells were in buffer mm pH 7.4, mm 1 mm 2 mm and 10 of both and Samples were centrifuged at × g for 10 min. The supernatant was with 50 of protein (Amersham Biosciences) for 1 and then with of of 50 of protein was added the next to the sample and for 1 h. The were washed in were eluted with 50 of buffer and on cells were with or after cells were lysed with 50 mm pH 7.4, mm 2 mm mm and 10 of both and The lysate was and centrifuged to remove of glutathione-Sepharose 4 were added to the and for 1 by with were eluted from the with 50 of sample buffer and cells were with wild-type after cells were and lysed in 50 mmTris-HCl, pH 7.4, mm and complete protease inhibitor mixture (Roche Molecular was performed as described the complex was washed with 50 mm pH 7.4, and was added to and at °C for min. were washed in buffer 1 mm and in Samples were by was performed as described by PubMed Scopus Google Scholar). were membrane were with in 20 for min and with in overnight at 4 °C or for 1 at and then with antibodies for 1 at The membrane was with from was used at with an overnight antibodies were from Cell whereas was from The against STEP was used at in at as described L.M. Lombroso P.J. Raghunathan A. During M.J. Wahle P. Naegele J.R. J. Neurosci. 1995; 15: 1532-1544Crossref PubMed Google Scholar, 15Nguyen T.-H. Paul S., Xu, Y. Gurd J.W. Lombroso P.J. J. Neurochem. 1999; 73: 1995-2001PubMed Google Scholar). were for min at with buffer mm pH and and then with to for the of A was used in an to substrates of have been used to proteins that with a of PTPs after amino are in to the PTP A. C. J. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, Biol. 1996; 16: PubMed Scopus Google Scholar). were passed a column and washed extensively mm and proteins were eluted with pH buffer and for the presence of protein kinase has been to with STEP and as a for these R. A. A. EMBO J. 1998; 17: PubMed Scopus Google Scholar) next for the association of STEP with members of the Src kinase family, including Fyn, Src, and determined of these Fyn was present in the a tyrosine kinase present within was also that Src or to the column for results to for Src and not These results that the used in these Fyn binds to a substrate-trapping of The next determined the association of Fyn with STEP be in and one or of the STEP to GST-STEP were with Fyn into and were performed The results that Fyn was down to by STEP the CS STEP61 and its The to down is an variant within the STEP family of and STEP61 contain the KIM domain. They by the presence in STEP61 of a acid region at the N terminus that contains two transmembrane and two polyproline domains. down Fyn in the is with the from the column the STEP61 variant down Fyn its In addition, the of Fyn were with the STEP61 CS These results suggest that Fyn associates with STEP61. were STEP61 was with a myc-tagged was used to and the were for the presence of STEP STEP61 CS was with the sample but was not in the of STEP61 and Src was Src was not to with the wild-type or the substrate-trapping these results suggest that STEP61 with the tyrosine kinase These were to the Fyn binding domain in STEP61. that of of the two polyproline domains and or the KIM domain (see were with Fyn The of the resulting to with Fyn was 4 STEP61 CS with of the or the KIM domain in the variant decreased the of Fyn of in the wild-type variant the binding of STEP to These results suggest that the KIM and polyproline domains are involved in the binding of STEP61 with In a complementary of Fyn domains were and with STEP61 CS. the STEP61 isoform with It also with the Fyn that an The Fyn with an SH2 or the unique domain to to STEP61 4 These results suggest that the domain of Fyn is not for the binding to STEP61, whereas the SH2 and the unique domains of Fyn to be for the observed interactions with regulatory are phosphorylated in Fyn and regulate its activity. These two tyrosine residues as for In an of we the association of the substrate-trapping STEP61 CS isoform to wild-type Fyn, with its association to the Fyn that contain at tyrosine 420 or tyrosine 531 co-transfection, STEP61 fusion protein was down and for the presence of Fyn these antibodies were used that specifically of the STEP61 CS down wild-type Fyn and the The is not phosphorylated at the 531 site but is phosphorylated at the Tyr420 residue In contrast, when tyrosine 420 was STEP61 was to with Fyn These results suggest that phosphorylation at tyrosine 420 of Fyn is for the observed association with the substrate-trapping STEP61 CS next determined Fyn was dephosphorylated at of these sites by These were performed in the presence of to autophosphorylation of fusion protein was added to the wild-type The phosphotyrosine level at site was then by using and antibodies was used to demonstrate in and was used to show the of These results a decrease in with of STEP In contrast, there was in that specifically and dephosphorylating in is the excitatory in the central nervous system Annu. Rev. Physiol. 1995; PubMed Scopus Google Scholar). The NMDA receptor is one of several receptors that have been implicated in key signaling pathways including synaptic and D.W. Neurosci. Full Text PDF PubMed Scopus Google Scholar, J.W. Rev. 15: PubMed Scopus Google Scholar, G.L. Neurosci. 1995; Full Text PDF PubMed Scopus Google Scholar). phosphorylation of NMDA receptors is one mechanism by which the activity of these receptors be regulated. phosphorylation NMDA receptors and through these members of the Src family of tyrosine kinases have been implicated in these including Src, Fyn, and G. Seeburg P.H. J. Physiol. (Lond.). 1996; 492: 445-452Crossref Scopus (279) Google Scholar, K. Y. G. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar, J.A. K. Gurd J.W. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar, X.M. Askalan R. Salter M.W. 1997; PubMed Scopus Google Scholar, N. H. K. 1998; Google Scholar, T. S. T. C. Umemori H. K. M. T. Yamamoto T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). These kinases are themselves phosphorylated on two regulatory tyrosine residues that have kinase and the other kinase Thus, the identification of tyrosine phosphatases that specifically on one or the other phosphorylation site would add an additional of of the kinases involved in key regulatory receptors, and other substrates within the The in the present study was that STEP with Fyn and dephosphorylated one of its two regulatory tyrosine residues of this The using as the that Fyn to a substrate-trapping variant of A for the was that also to the column these proteins been shown to with STEP C. Torres J. Pulido R. J. Cell Biol. 1999; 147: 1129-1136Crossref PubMed Scopus (141) Google Scholar, R. A. A. EMBO J. 1998; 17: PubMed Scopus Google Scholar). In the present the was further for the of the results by for other kinase members also present in such as Src, Lyn, or these proteins are not By using we that STEP and Fyn in These show that STEP In STEP61 down Fyn The complementary of down myc-tagged Fyn and demonstrate a interaction with STEP61. The substrate-trapping variant of STEP61 to the wild-type and to the form of Fyn, but not the that the used in these there is of in that STEP requires one but not the other tyrosine residue to be present and phosphorylated in to to also specific binding domains within STEP61 and the N-terminal proline-rich domain and the KIM domain in STEP61 to be for binding with a of to with The KIM domain been identified as a binding domain in STEP required for association with protein kinase C. Torres J. Pulido R. J. Cell Biol. 1999; 147: 1129-1136Crossref PubMed Scopus (141) Google Scholar). The present results suggest that this domain may also be for interaction with additional including the tyrosine kinase The domains in Fyn that are for binding to to the SH2 and the unique N-terminal domains. the unique domain of Fyn is Fyn to with STEP61. This is in with that both Src and with unique domains to with STEP These results suggest that Fyn is in an or when with In an or the SH2 domain binds to its phosphorylated through an the phosphorylated is dephosphorylated by a Fyn the Tyr420 site and the SH2 domain from STEP61 dephosphorylates Tyr420. This the SH2 domain to to other and to the PTP domain of STEP P. S. S. M.J. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, M.B. Nat. Rev. Biol. 2002; 3: PubMed Scopus Google Scholar). The domain and the KIM motif of STEP61 the association between STEP and In results suggest that STEP binds to Fyn and specifically dephosphorylates the regulatory tyrosine at amino acid 420. A recent that also binds to Fyn but in this specifically dephosphorylates J. Muranjan M. Sap J. Curr. Biol. 1999; 9: 505-511Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). The binding of to Fyn is of its phosphatase domain and requires an interaction between and the SH2 domain of Fyn V. Lim K.L. Pallen C.J. J. Biol. Chem. 1998; 273: 8691-8698Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, J. Sap J. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). It to be determined the further regulation of Fyn by STEP described in this study is responsible for the regulation of NMDA receptors at the for C. and for on the