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NYAP: a phosphoprotein family that links PI3K to WAVE1 signalling in neurons

2011; Springer Nature; Volume: 30; Issue: 23 Linguagem: Inglês

10.1038/emboj.2011.348

1460-2075

Kazumasa Yokoyama, Tohru Tezuka, Masaharu Kotani, Takanobu Nakazawa, Naosuke Hoshina, Yasushi Shimoda, Shigeru Kakuta, Katsuko Sudo, Kazutada Watanabe, Yoichiro Iwakura, Tadashi Yamamoto,

NF-κB Signaling Pathways

Article23 September 2011free access NYAP: a phosphoprotein family that links PI3K to WAVE1 signalling in neurons Kazumasa Yokoyama Kazumasa Yokoyama Division of Oncology, Department of Cancer Biology, Institute of Medical Science, University of Tokyo, Tokyo, Japan Search for more papers by this author Tohru Tezuka Tohru Tezuka Division of Oncology, Department of Cancer Biology, Institute of Medical Science, University of Tokyo, Tokyo, Japan Search for more papers by this author Masaharu Kotani Masaharu Kotani Department of Molecular and Cellular Biology, School of Pharmaceutical Sciences, Ohu University, Fukushima, Japan Search for more papers by this author Takanobu Nakazawa Takanobu Nakazawa Division of Oncology, Department of Cancer Biology, Institute of Medical Science, University of Tokyo, Tokyo, Japan Search for more papers by this author Naosuke Hoshina Naosuke Hoshina Division of Oncology, Department of Cancer Biology, Institute of Medical Science, University of Tokyo, Tokyo, Japan Cell Signal Unit, Okinawa Institute of Science and Technology, Okinawa, Japan Search for more papers by this author Yasushi Shimoda Yasushi Shimoda Department of Bioengineering, Nagaoka University of Technology, Niigata, Japan Search for more papers by this author Shigeru Kakuta Shigeru Kakuta Laboratory of Molecular Pathogenesis, Center for Experimental Medicine and Systems Biology, Institute of Medical Science, University of Tokyo, Tokyo, Japan Search for more papers by this author Katsuko Sudo Katsuko Sudo Laboratory of Molecular Pathogenesis, Center for Experimental Medicine and Systems Biology, Institute of Medical Science, University of Tokyo, Tokyo, JapanPresent address: Animal Research Center, Tokyo Medical University, Tokyo 160-8402, Japan Search for more papers by this author Kazutada Watanabe Kazutada Watanabe Department of Bioengineering, Nagaoka University of Technology, Niigata, Japan Search for more papers by this author Yoichiro Iwakura Yoichiro Iwakura Laboratory of Molecular Pathogenesis, Center for Experimental Medicine and Systems Biology, Institute of Medical Science, University of Tokyo, Tokyo, Japan CREST, Japan Science and Technology Agency, Saitama, Japan Search for more papers by this author Tadashi Yamamoto Corresponding Author Tadashi Yamamoto Division of Oncology, Department of Cancer Biology, Institute of Medical Science, University of Tokyo, Tokyo, Japan Cell Signal Unit, Okinawa Institute of Science and Technology, Okinawa, Japan Search for more papers by this author Kazumasa Yokoyama Kazumasa Yokoyama Division of Oncology, Department of Cancer Biology, Institute of Medical Science, University of Tokyo, Tokyo, Japan Search for more papers by this author Tohru Tezuka Tohru Tezuka Division of Oncology, Department of Cancer Biology, Institute of Medical Science, University of Tokyo, Tokyo, Japan Search for more papers by this author Masaharu Kotani Masaharu Kotani Department of Molecular and Cellular Biology, School of Pharmaceutical Sciences, Ohu University, Fukushima, Japan Search for more papers by this author Takanobu Nakazawa Takanobu Nakazawa Division of Oncology, Department of Cancer Biology, Institute of Medical Science, University of Tokyo, Tokyo, Japan Search for more papers by this author Naosuke Hoshina Naosuke Hoshina Division of Oncology, Department of Cancer Biology, Institute of Medical Science, University of Tokyo, Tokyo, Japan Cell Signal Unit, Okinawa Institute of Science and Technology, Okinawa, Japan Search for more papers by this author Yasushi Shimoda Yasushi Shimoda Department of Bioengineering, Nagaoka University of Technology, Niigata, Japan Search for more papers by this author Shigeru Kakuta Shigeru Kakuta Laboratory of Molecular Pathogenesis, Center for Experimental Medicine and Systems Biology, Institute of Medical Science, University of Tokyo, Tokyo, Japan Search for more papers by this author Katsuko Sudo Katsuko Sudo Laboratory of Molecular Pathogenesis, Center for Experimental Medicine and Systems Biology, Institute of Medical Science, University of Tokyo, Tokyo, JapanPresent address: Animal Research Center, Tokyo Medical University, Tokyo 160-8402, Japan Search for more papers by this author Kazutada Watanabe Kazutada Watanabe Department of Bioengineering, Nagaoka University of Technology, Niigata, Japan Search for more papers by this author Yoichiro Iwakura Yoichiro Iwakura Laboratory of Molecular Pathogenesis, Center for Experimental Medicine and Systems Biology, Institute of Medical Science, University of Tokyo, Tokyo, Japan CREST, Japan Science and Technology Agency, Saitama, Japan Search for more papers by this author Tadashi Yamamoto Corresponding Author Tadashi Yamamoto Division of Oncology, Department of Cancer Biology, Institute of Medical Science, University of Tokyo, Tokyo, Japan Cell Signal Unit, Okinawa Institute of Science and Technology, Okinawa, Japan Search for more papers by this author Author Information Kazumasa Yokoyama1, Tohru Tezuka1, Masaharu Kotani2, Takanobu Nakazawa1, Naosuke Hoshina1,3, Yasushi Shimoda4, Shigeru Kakuta5, Katsuko Sudo5, Kazutada Watanabe4, Yoichiro Iwakura5,6 and Tadashi Yamamoto 1,3 1Division of Oncology, Department of Cancer Biology, Institute of Medical Science, University of Tokyo, Tokyo, Japan 2Department of Molecular and Cellular Biology, School of Pharmaceutical Sciences, Ohu University, Fukushima, Japan 3Cell Signal Unit, Okinawa Institute of Science and Technology, Okinawa, Japan 4Department of Bioengineering, Nagaoka University of Technology, Niigata, Japan 5Laboratory of Molecular Pathogenesis, Center for Experimental Medicine and Systems Biology, Institute of Medical Science, University of Tokyo, Tokyo, Japan 6CREST, Japan Science and Technology Agency, Saitama, Japan *Corresponding author. Division of Oncology, Department of Cancer Biology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. Tel.: +81 3 5449 5301; Fax: +81 3 5449 5413; E-mail: [email protected] The EMBO Journal (2011)30:4739-4754https://doi.org/10.1038/emboj.2011.348 There is a Have you seen? (November 2011) associated with this Article. PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The phosphoinositide 3-kinase (PI3K) pathway has been extensively studied in neuronal function and morphogenesis. However, the precise molecular mechanisms of PI3K activation and its downstream signalling in neurons remain elusive. Here, we report the identification of the Neuronal tYrosine-phosphorylated Adaptor for the PI 3-kinase (NYAP) family of phosphoproteins, which is composed of NYAP1, NYAP2, and Myosin16/NYAP3. The NYAPs are expressed predominantly in developing neurons. Upon stimulation with Contactin5, the NYAPs are tyrosine phosphorylated by Fyn. Phosphorylated NYAPs interact with PI3K p85 and activate PI3K, Akt, and Rac1. Moreover, the NYAPs interact with the WAVE1 complex which mediates remodelling of the actin cytoskeleton after activation by PI3K-produced PIP3 and Rac1. By simultaneously interacting with PI3K and the WAVE1 complex, the NYAPs bridge a PI3K–WAVE1 association. Disruption of the NYAP genes in mice affects brain size and neurite elongation. In conclusion, the NYAPs activate PI3K and concomitantly recruit the downstream effector WAVE complex to the close vicinity of PI3K and regulate neuronal morphogenesis. Introduction The phosphoinositide 3-kinase (PI3K) signalling pathway is a fundamental pathway for various cellular events in neurons, including morphogenesis (Rodgers and Theibert, 2002; Shi et al, 2003; Jaworski et al, 2005; Kumar et al, 2005), membrane expansion (Laurino et al, 2005), survival and apoptosis (Brunet et al, 2001), and ion channel regulation (Sanna et al, 2002; Viard et al, 2004). PI3K generates phosphatidylinositol 3,4,5-trisphosphate (PIP3), which then mediates the recruitment and subsequent activation of PH domain-containing effector proteins (Cantrell, 2001). Many different proteins that possess the PH domain, including the Akt and Tec family of kinases, regulators for small G proteins, such as a Rac guanine nucleotide exchange factor, and phospholipase C (PLC) γ isoforms, directly bind to PIP3. Thus, interactions of PIP3 with many different effector proteins enable PI3K to be involved in a number of different cellular responses. Neuronal phenotypes of knockout (KO) mice for PI3K itself include losses of synapses and myelinated axons (Tohda et al, 2006), decreased axonal extension and dampened axonal regeneration (Eickholt et al, 2007), reduction in reproductive hormone secretions in males (Acosta-Martínez et al, 2009), and memory and behavioural phenotypes (Tohda et al, 2006, 2009). Moreover, PTEN, the central negative regulator of the PI3K pathway, is required to obtain proper dendrite morphology, neuronal soma size, and brain size without affecting neuronal cell death during development in mice (Backman et al, 2001; Kwon et al, 2001, 2006). KO mice for Akt3, which is one of the major downstream effectors of PI3K in the brain, demonstrate reduced brain size but do not show aberrant apoptosis (Easton et al, 2005; Tschopp et al, 2005). These reports suggest roles of the PI3K pathway in neuronal morphogenesis rather than regulation of neuronal survival. However, the precise mechanisms leading to the preference of neuronal PI3K for regulation of morphogenesis remain to be identified. Remodelling of the actin cytoskeleton plays a critical role in altering cellular morphology, and it controls a range of cellular events, such as wound healing, immune defense, embryonic development, and neuronal outgrowth (Suetsugu and Takenawa, 2003). The Rho family of GTPases, including Rho, Cdc42, and Rac, mediates these processes by promoting distinct forms of actin remodelling such as stress fibres, filopodia, and lamellipodia (Hall, 1998). Cdc42 and Rac relay signals to cytoskeletal sites through the WASP and WAVE family of proteins, respectively, which then activate the Arp2/3 complex and stimulate actin polymerization (Stradal and Scita, 2006). In fibroblast cells, PDGF modulates WAVE-mediated cell motility through the activation of PI3K and the production of PIP3, which in turn stimulates the formation of lamellipodia (Suetsugu et al, 2003; Oikawa et al, 2004; Sossey-Alaoui et al, 2005). In this process, the PDGF receptor activates PI3K directly, and PI3K-produced PIP3 is shown to be required for the regulation of Rac activity (Shinohara et al, 2002). The activation of Rac is also mediated by the direct interaction between Rac and PI3K p85 (Hawkins et al, 1995; Tolias et al, 1995). Then, WAVE activity towards the Arp2/3 complex is stimulated by binding with PIP3 (Oikawa et al, 2004) and Rac (Steffen et al, 2004) and phosphorylation by the Abl, Src, and Cdk5 kinases (Leng et al, 2005; Ardern et al, 2006; Kim et al, 2006; Stuart et al, 2006; Sossey-Alaoui et al, 2007; Lebensohn and Kirschner, 2009; Chen et al, 2010). In neurons, however, precise activation mechanisms of the pathway are not fully understood. Fyn, a member of the Src family of protein tyrosine kinases, is one of the major tyrosine kinases in the brain (Umemori et al, 1992). Substantial genetic evidence suggests that Fyn plays important roles in neuronal morphogenesis (Grant et al, 1992; Sasaki et al, 2002; Morita et al, 2006; Kotani et al, 2007), but the signals that function upstream and downstream of Fyn in neurons are poorly understood. In the course of screening for tyrosine kinase substrates in the brain, we identified a previously uncharacterized family of proteins that we named NYAP—Neuronal tYrosine-phosphorylated Adaptor for the PI 3-kinase. Here, we show that the NYAPs are involved in both the activation of PI3K and the recruitment of the downstream effector WAVE complex to the close vicinity of PI3K, and regulate brain size and neurite outgrowth in mice. Results Identification of the NYAP family of proteins as tyrosine kinase substrates in vitro To identify substrates of the Src family of tyrosine kinases in the brain, we performed solid-phase phosphorylation screening (Yokoyama et al, 2002, 2006). This screening yielded a previously uncharacterized protein, which we termed NYAP1. Extensive screening of the protein database identified two proteins with partial similarity to NYAP1: an uncharacterized protein KIAA1486 (termed NYAP2) and an unconventional myosin MYO16 (termed NYAP3 for descriptive purposes). MYO16/NYAP3 is expressed during brain development and associates with F-actin and the protein phosphatase 1 catalytic subunits 1α and 1γ1 (Patel et al, 2001). Moreover, MYO16/NYAP3 regulates S-phase progression when overexpressed in non-neuronal cells (Cameron et al, 2007); however, physiological roles for MYO16/NYAP3 in the brain remain unclear. The short sequence motif present in NYAP1, 2, and 3 contains conserved tyrosine residues and a proline-rich stretch. We termed this the NYAP Homology Motif (NHM). NYAP1 and NYAP2 do not have any identifiable functional domains, whereas MYO16/NYAP3 contains ankyrin repeats and a myosin motor domain (Figure 1). Based on the partial similarity in the NHM, we considered that NYAP1, 2, and MYO16/NYAP3 form a novel family of proteins. It seems likely that no more member of the NYAP family exists in mouse genome sequences. To examine the physiological roles of the NYAPs, we generated Nyap1, 2, and 3 KO mice (Supplementary Figure S1). NYAPs triple knockout (TKO) mice were apparently healthy and fertile and are currently analysed for behavioural abnormalities. Figure 1.Identification of the NYAP family of proteins. (A) Schematic representation of NYAP1, NYAP2, and MYO16/NYAP3. (B) Mouse NYAPs amino-acid sequence alignment. Identical or similar residues to the column consensus are printed with black or grey backgrounds, respectively. Ankyrin repeats in MYO16/NYAP3 are indicated in red letters, the MYO16/NYAP3 motor domain is in blue letters, NHM motifs are shown in a green box, regions involved in the interaction with the WAVE complex are shown in an orange box, and phosphorylated tyrosine residues are denoted with red arrows. Download figure Download PowerPoint The NYAPs are expressed in developing cortical neurons after migration The Myo16/Nyap3 mRNA is specifically expressed in the developing brain (Patel et al, 2001). Northern blot analysis revealed that Nyap1 and Nyap2 mRNA expression was also specific to the brain among the tissues examined (Figure 2A). Furthermore, among three major brain cell types, Nyap1, 2, and 3 mRNAs were all present specifically in neurons but not in astrocytes or oligodendrocytes (Figure 2B). Figure 2.Neuron-specific expression of the NYAPs. (A) Northern blot analysis of Nyap1 and Nyap2 mRNA in mouse tissues. Vertical dashed lines represent boundaries between different gels. Ethidium bromide-stained images are shown in the lower panels as loading controls (EtBr). (B) Northern blot analysis of NYAPs mRNA in rat cortical neurons and astrocytes, as well as the rat CG4 oligodendrocyte cell line during precursor (OPC) and mature (OL) stages. (C) In situ hybridization analysis of NYAPs mRNA in postnatal day 1 (P1) mouse brains. Scale bar: 2 mm. (D) Magnified images of the boxed areas in (C). Wild-type (WT) P1 mouse brains (upper panels) and each of the Nyap1, 2, or 3 knockout P1 mouse brains (KO, lower panels) were hybridized with Nyap1, 2, or 3 mRNA probes. S, striatum; uc, upper cortical area; lc, lower cortical area; imz, intermediate zone; and vz, ventricular and subventricular zone. Scale bar: 200 μm. Download figure Download PowerPoint In situ hybridization of postnatal day 1 (P1) mouse brains revealed that Nyap1 mRNA was present in the neocortex and the striatum but not in the olfactory bulb. Nyap2 mRNA was present in the neocortex, the striatum, and the olfactory bulb; and Myo16/Nyap3 mRNA was present in the neocortex and the olfactory bulb but not in the striatum. No expression of Nyap1, 2, or 3 was found in the ventricular zone or the corpus callosum (Figure 2C). The highest expression of Nyap1, 2, and 3 was observed in the middle, lower, and upper neocortex, respectively, in P1 mouse brains (Figure 2D). We next examined when neurons began to express the NYAPs (Supplementary Figure S2). Nyap1 expression was detectable in the cortical plate as early as embryonic day 14 (E14); it peaked in the middle neocortex at P1 and then gradually decreased. Nyap2 expression began in the lower neocortex at E18 and decreased thereafter. In contrast to Nyap1, Nyap2 expression persisted in the striatum until at least P7. The expression pattern of Myo16/Nyap3 resembled that of Nyap1 but occurred in slightly upper layers of the neocortex. Because the NYAPs were not expressed in the ventricular zone and intermediate zone during embryonic stages, we concluded that neurons began to express the NYAPs just after their migration into the neocortex. The NYAPs are tyrosine phosphorylated by Fyn upon Contactin stimulation We initially identified NYAP1 as a protein that was directly phosphorylated by Fyn in vitro in the solid-phase phosphorylation screening. To determine whether the NYAPs are genuine tyrosine kinase substrates in vivo, we examined their tyrosine phosphorylation in the brain. When immunoprecipitated from wild-type (WT) P1 mouse brains, the NYAPs were tyrosine phosphorylated (Figure 3A). The phosphorylation levels of the NYAPs were lower in Fyn KO brains (Figure 3A). These data suggest that the Src family of kinases directly phosphorylates the NYAPs in the brain. Figure 3.Tyrosine phosphorylation of the NYAPs. (A) Fyn-dependent tyrosine phosphorylation of the NYAPs. Tyrosine phosphorylation of the NYAPs immunoprecipitated from WT and Fyn KO brains was detected with the 4G10 anti-phosphotyrosine antibody. Amounts of the NYAPs in the immunoprecipitates are shown in the lower panels. (B) Overall tyrosine phosphorylation in total lysates of WT and Nyap1, 2, and 3 KO brains. Amounts of Akt are shown as loading controls. (C) Overall tyrosine phosphorylation in total brain lysates obtained from mice of the indicated ages. Amounts of βIII-tubulin are shown as loading controls. E12.5: embryonic day 12.5. (D) Quantification of the recombinant Contactin5–Fc fusion protein. Contactin5–Fc (C5Fc) was captured from the conditioned medium with protein G-sepharose and quantified by Coomassie staining. Protein concentration of C5Fc was estimated by comparing with SDS–PAGE Standards (high range, Bio-Rad). (E) C5Fc-induced tyrosine phosphorylation of the NYAPs. Cultured cortical neurons obtained from WT and NYAPs triple knockout (TKO) brains were stimulated with 6 μg/ml C5Fc for the indicated time periods. Tyrosine phosphorylation of the NYAPs was examined in total cell lysates. TKO neurons were used as negative controls, ensuring that phosphorylation signals in WT neurons were predominantly phosphorylated NYAPs. Upregulation of tyrosine phosphorylation of the NYAPs after 30 min stimulation with C5Fc was quantified (WT, n=8; TKO, n=8). A single asterisk (*) and double asterisk (**) indicate P<0.05 and P<0.01 compared with control neurons, respectively. (F–H) Determination of phosphorylated tyrosine residues in the NYAPs in HEK293T cells. HEK293T cells were transfected with Fyn (YF, the constitutively active mutant; KM, the kinase inactive mutant) and FLAG–NYAP1 (F), FLAG–NYAP2 (G), FLAG–MYO16/NYAP3 (H), or their YF mutants, as indicated. NYAP1 Y1F indicates that Tyr212 was mutated to phenylalanine in mouse NYAP1; NYAP1 Y2F, Tyr257; NYAP2 Y1F, Tyr277; NYAP2 Y2F, Tyr300; NYAP3 Y1F, Tyr1416; and NYAP3 Y2F, Tyr1441. Y1F–Y2F indicates that both tyrosine residues (Y1 and Y2) were mutated. The cell lysates were immunoprecipitated with the M2 anti-FLAG antibody and immunoblotted with the RC20 anti-phosphotyrosine antibody. Although HEK293T cells express Src-family kinases endogenously, the constitutively active mutant of Fyn (FynYF) was introduced to facilitate comparison of phosphorylation levels. (I) Determination of phosphorylated tyrosine residues in the NYAPs in neurons. Cultured cortical neurons were infected with recombinant sindbisviruses expressing EGFP, EGFP-tagged wild-type NYAPs (WT), or their YF mutants. The neurons were lysed, immunoprecipitated with an anti-EGFP antibody, and immunoblotted with the 4G10 anti-phosphotyrosine antibody. Download figure Download PowerPoint In WT P1 mouse brains, five conspicuous tyrosine-phosphorylated proteins with sizes of 240, 180, 120, 100, and 90 kDa were present (Figure 3B). Interestingly, the sizes of NYAP1, 2, and 3 are 100, 90, and 240 kDa, respectively, which match three of the five phosphorylated proteins. In addition, the bands at 100, 90, and 240 kDa were absent in the lysates of Nyap1, 2, and 3 KO brains, respectively, indicating that these bands represented tyrosine phosphorylation of NYAP1, 2, and 3. The 180- and 120-kDa phosphoproteins have not yet been identified. Next, tyrosine phosphorylation levels of the NYAPs were quantitatively examined during brain development (Figure 3C; see also Supplementary Figure S11 for protein expression levels of the NYAPs). Phosphorylation of NYAP1 and MYO16/NYAP3 began at E14, peaked during perinatal days in the whole brain, and then gradually decreased. Phosphorylation of NYAP2 began at E16; in the adult brain, phosphorylated NYAP2 expression persisted. These results indicated that the NYAPs were heavily phosphorylated on tyrosines, accounting for a large percentage of tyrosine phosphorylation in the brain throughout the entire life of a mouse. The Src family of kinases is activated in response to various stimuli, so we tested whether these stimuli induced tyrosine phosphorylation of the NYAPs. Recently, members of the Contactin family of GPI-anchored membrane proteins have been demonstrated to activate the Src family of tyrosine kinases directly (Kasahara et al, 2002) or indirectly via PTPα (Ponniah et al, 1999; Su et al, 1999; Ye et al, 2008). Contactins (Contactin1–6) are a subgroup of molecules belonging to the immunoglobulin superfamily that are expressed exclusively in the nervous system. Contactin1 and 2 have been studied extensively in neurite extension and myelination (Shimoda and Watanabe, 2009). We found that the Contactin5–Fc protein induced the phosphorylation of the NYAPs in cortical neurons (NYAP1, 147.7±8.7%; NYAP2, 198.1±23.9%; NYAP3, 223.9±45.9%; Figure 3D and E). Surprisingly, other stimuli we examined (NGF, BDNF, GDNF, EGF, PDGF, IGF, Insulin, Neuregulin, Reelin, Semaphorin-3A, Ephrin-A3, Ephrin-B2, NMDA, KCl, and glutamate) had no effect on the phosphorylation of the NYAPs in our assays. Next, we determined the phosphorylation sites on the NYAPs. Based on the sequence similarity in the family, we chose to mutate conserved tyrosine residues in the NHM (indicated by red arrows in Figure 1B: NYAP1 Tyr212 and Tyr 257, NYAP2 Tyr277 and Tyr300, and NYAP3 Tyr1416 and Tyr1441) to phenylalanine (YF mutants). Mutation of each tyrosine residues in the NYAPs slightly reduced their phosphorylation in HEK293T cells (Figure 3F–H). When both tyrosine residues were mutated, their phosphorylation was almost abrogated in HEK293T cells (Figure 3F–H) and in neurons (Figure 3I), indicating that most of phosphorylation occurred at these tyrosine residues in the NHM motifs. The NYAPs are the major binding partners of PI3K p85 in the brain The phosphorylated tyrosine residues in the NYAPs lie in the consensus sequence (YxxM motif) for the binding by SH2 domains of the PI3K p85 subunit (Songyang et al, 1993). Immunoprecipitation analyses revealed that PI3K p85α interacted with the NYAPs in HEK293T cells (Figure 4A–C) and in the brain (Figure 4D). Further, these interactions depended on the phosphorylation state of the NYAPs because the YF mutations abrogated the interactions (Figure 4A–C). Moreover, PI3K p85β interacted with the NYAPs (Supplementary Figure S3). NYAP1 Y1F and NYAP2 Y1F did not bind at all with PI3K p85α, and NYAP1 Y2F and NYAP2 Y2F displayed reduced ability to interact with PI3K p85α (Figure 4A and B). These data suggest that both tyrosine residues participate in the binding with PI3K p85α, but the Y2-mediated binding requires phosphorylation of Y1 and/or the binding of Y1 with PI3K p85α, which might cause conformational changes of the NYAPs or PI3K p85α. Figure 4.Interaction between the NYAPs and the PI3K p85. (A–C) Tyrosine phosphorylation-dependent interaction of the NYAPs with the PI3K p85α subunit. HEK293T cells were transfected with Fyn, PI3K p85α, and FLAG-NYAP1 (A), FLAG–NYAP2 (B), FLAG–MYO16/NYAP3 (C), or their YF mutants, as indicated. The cell lysates were immunoprecipitated with the anti-FLAG antibody, immunoblotted with an anti-PI3K p85α antibody, and examined for co-precipitation of PI3K p85α with the NYAPs. The asterisk (*) in (C) represents crossreactive bands, which are faintly apparent in (A) and (B). (D) Interaction between the NYAPs and the PI3K p85 in the brain. WT and Nyap1, 2, and 3 KO brains were immunoprecipitated with anti-NYAP1, 2, or 3 antibodies and immunoblotted with an anti-PI3K p85α antibody. Nyap1, 2, and 3 KO brains were used as negative controls. (E) Phosphoproteins associated with PI3K p85α in the brain. The PI3K p85α immunoprecipitates from WT, Nyap1, 2, 3 KO, and TKO brains were immunoblotted with an anti-phospho(Tyr) p85 PI3K binding YxxM motif antibody (pYxxM). The similar results were obtained when another anti-PI3K p85α antibody (from Millipore) and the 4G10 anti-phosphotyrosine antibody were used. (F) Developmental changes in tyrosine phosphorylated binding partners of PI3K p85α. The brain lysates used in Figure 3C were immunoprecipitated with an anti-PI3K p85α antibody and immunoblotted with the 4G10 anti-phosphotyrosine, anti-PI3K p85α, and anti-p110α antibodies. The bottom panel showing the amounts of βIII-tubulin is the same as that shown in Figure 3C. Download figure Download PowerPoint Because PI3K is activated when the regulatory p85 subunit binds to tyrosine-phosphorylated proteins, we evaluated all tyrosine-phosphorylated proteins co-immunoprecipitated with PI3K p85. To carry out this experiment, an anti-phospho(Tyr) p85 PI3K binding YxxM motif antibody (Figure 4E) and the anti-phosphotyrosine antibody 4G10 (Figure 4F) were used for detection. Surprisingly, only four phosphoproteins of 240, 150, 100, and 90 kDa were associated with PI3K p85α in the brain. Three of these (the 100, 90, and 240 kDa proteins), which were missing in the immunoprecipitates from Nyap1, 2, and 3 KO brains, corresponded to phosphorylated NYAP1, 2, and 3, respectively (Figure 4E). These proteins accounted for the majority of the PI3K p85-associated tyrosine phosphorylation in the brain throughout the entire life of a mouse (Figure 4F). The 150-kDa phosphoprotein has not been identified. Interestingly, PI3K expression levels in the brain gently peaked around perinatal days, when the expression and phosphorylation of the NYAPs were high (Figure 4F), supporting the cooperative roles of PI3K and the NYAPs. An essential process for PI3K activation is its recruitment to the plasma membrane, whereby PI3K accesses its substrates such as PIP2 (Klippel et al, 1996). To examine this process, we isolated the membrane fraction from WT and TKO P1 mouse brains. In WT brains, a small amount of PI3K p85α was localized in the membrane fraction (27.4±4.5% of total PI3K p85α). However, this recruitment was markedly attenuated in TKO brains (52.5±6.8% of WT; Figure 5A). A certain amount of the NYAPs was also localized in the membrane fraction (Supplementary Figure S4). These results implicated that the NYAPs participate in the recruitment of PI3K to the neuronal plasma membrane. Next, we measured the activity of PI3K and its downstream effector proteins. Activity of PI3K isolated from TKO P1 mouse brains was attenuated (68.1±3.8% of WT; Figure 5B). Activated PI3K produces PIP3, which then activates PH domain-containing effector proteins including the Akt and Tec family of kinases, regulators for small G proteins, and PLCγ (Cantrell, 2001). Akt activation, evaluated as phosphorylation of Akt Ser473, was attenuated in TKO P1 mouse brains (pAkt/Akt, 60.7±1.4% of WT; Figure 5C), whereas expression levels of Akt were unaffected (Supplementary Figure S5). Rac1 activity, which is regulated by PI3K (Hawkins et al, 1995; Tolias et al, 1995; Shinohara et al, 2002), was also attenuated in TKO brains (71.5±10.5% of WT; Figure 5D). Moreover, adenovirus-mediated expression of WT NYAP1 or NYAP2, but not their YF mutants, enhanced Akt activity in neurons (Figure 5E), suggesting that tyrosine phosphorylation of the NYAPs was required for PI3K activation. To further ensure that the NYAPs directly acted in this pathway, we examined activation of the PI3K pathway after induction of NYAPs phosphorylation by Contactin stimulation. Cortical neurons isolated from WT and TKO mice were cultured for 2 days in vitro and stimulated with Contactin5–Fc fusion protein. In WT neurons, Akt was activated after the addition of Contactin5–Fc, but Akt activation did not occur in TKO neurons (Figure 5F). On the other hand, BDNF, which did not induce NYAPs phosphorylation, activated Akt even in the absence of the NYAPs (Supplementary Figure S6). Collectively, these data indicated that the phosphorylated NYAPs activate PI3K and its downstream effectors. Figure 5.Activation of the