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. 2011 Sep 23;30(23):4739-54.
doi: 10.1038/emboj.2011.348.

NYAP: A Phosphoprotein Family That Links PI3K to WAVE1 Signalling in Neurons

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Free PMC article

NYAP: A Phosphoprotein Family That Links PI3K to WAVE1 Signalling in Neurons

Kazumasa Yokoyama et al. EMBO J. .
Free PMC article

Abstract

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 PIP(3) 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.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
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.
Figure 2
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.
Figure 3
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. (FH) 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.
Figure 4
Figure 4
Interaction between the NYAPs and the PI3K p85. (AC) 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.
Figure 5
Figure 5
Activation of the PI3K pathway by the NYAPs. (A) Membrane localization of PI3K p85α and WAVE1 in WT and TKO P1 mouse brains. WT, n=4; TKO, n=4. (B) PI3K activity in WT and TKO P1 mouse brains. PI3K was immunoprecipitated from equal amounts of total lysates obtained from WT and TKO P1 mouse brains with an anti-PI3K p85α antibody, incubated with its substrate phosphatidylinositol and ATP, and subjected to analysis with a PI3K ELISA kit (Echelon). WT, n=6; TKO, n=6. (C) Akt activity in WT and TKO P1 mouse brains. Akt activity was measured in total lysates of WT and TKO brains using an anti-phospho Ser473 Akt antibody. WT, n=3; TKO, n=3. (D) Levels of GTP-bound Rac1 in WT and TKO P1 mouse brains. Due to the low sensitivity of the assay, we pooled four P1 mouse brains per sample. WT, n=3; TKO, n=3. (E) Activation of Akt by phosphorylated NYAPs. Cultured cortical neurons were infected with recombinant adenoviruses expressing EGFP, myc-tagged wild-type NYAP1 and NYAP2, or their YF mutants. Akt activity was measured as in (C). The adenovirus expression system, instead of the sindbisvirus system, was used because of its high expression level. MYO16/NYAP3 was too large to be expressed with the adenovirus system. (F) Contactin5-mediated Akt activation in WT and TKO neurons. Cortical neurons were stimulated with 6 μg/ml C5Fc for the indicated time periods at DIV 2. Akt activation was examined as in (C). A single asterisk (*) and double asterisk (**) indicate P<0.05 and P<0.01 compared with WT, respectively.
Figure 6
Figure 6
Interaction of the NYAPs with the WAVE complex. (A) Proteomic analysis of NYAP2-interacting proteins. P1 mouse brain lysate was incubated with NYAP2-conjugated sepharose in the presence (lane 2, as a negative control) or absence (lane 1) of 1% SDS. Bound proteins were visualized by Coomassie staining. (B) Interactions of the NYAPs with Nap1 in the brain. P1 mouse brains were immunoprecipitated with anti-NYAP1, 2, or 3 antibodies and immunoblotted with an anti-Nap1 antibody. Immunoprecipitated NYAPs were detected with the RC20 anti-phosphotyrosine antibody. (C) Schematic representation of various deletion mutants of NYAP1, NYAP2, and MYO16/NYAP3. (DF) Determination of regions of the NYAPs involved in their interaction with the WAVE complex. HEK293T cells were transfected with GST or various deletion mutants of GST-tagged NYAPs as indicated. The cell lysates were incubated with glutathione sepharose (GE Healthcare) and immunoblotted with anti-Sra1 and anti-Nap1 antibodies, and interactions with endogenous Sra1 and Nap1 were tested. Due to relatively low expression levels of GST–NYAP1(1–832) and NYAP3(1109–1919) (D, F), their interactions with Sra1 and Nap1 seemed faint in these assays. See also Supplementary Figure S7A and B for the interactions.
Figure 7
Figure 7
The PI3K–WAVE1 association bridged by the NYAPs. (A) The neuron-specific and NYAPs-dependent association between PI3K and WAVE1. WT and TKO brains, WT livers, HEK293T cells, and CG4 cells were lysed and immunoprecipitated with an anti-WAVE1 antibody. PI3K p85α was detected only in the immunoprecipitates from WT brains, in which the NYAPs were expressed. WT livers, which do not express WAVE1, were examined as a negative control. (B) Association of Nap1 and Sra1 in the PI3K p85α immunoprecipitates. WT and TKO brains were immunoprecipitated with an anti-PI3K p85α antibody and immunoblotted with anti-Nap1 and anti-Sra1 antibodies. (C) Involvement of all members of the NYAP family in the PI3K–WAVE1 association in the brain. WT, Nyap1, 2, 3 KO, and TKO brains were immunoprecipitated with an anti-WAVE1 antibody and analysed as in (A). The PI3K–WAVE1 association persisted in NYAPs single KO mouse brains. (D) Association between PI3K and WAVE1 in HEK293T cells expressing the NYAPs. HEK293T cells were transfected with FLAG–NYAP1 and PI3K p85α and tested for the PI3K–WAVE1 association as in (A). (E) Models depicting the roles of the NYAPs. The NYAPs are involved in the activation of PI3K and the recruitment of the WAVE1 complex in neurons. The neuronal receptor(s) that mediates Contactin stimulation is currently unknown.
Figure 8
Figure 8
NYAPs-mediated remodelling of the actin cytoskeleton. (AG) Requirement of PI3K- and WAVE1-interacting regions in the NYAPs for remodelling of the actin cytoskeleton in HeLa cells. HeLa cells were transfected with GST (A) or various mutants of GST-tagged NYAPs (BG) as indicated. Cells were fixed and stained with an anti-GST antibody (green) and phalloidin-rhodamine (red). Actin stress fibres are indicated by white arrowheads. Cytosolic accumulation of collapsed actin fibres is indicated by black arrowheads. Scale bar: 50 μm. See also Supplementary Figure S10.
Figure 9
Figure 9
Neuronal hypotrophy in NYAPs triple knockout mice. (A) WT, Nyap1, 2, 3 KO, and TKO adult mouse brains (8 weeks old) were photographed. The genotypes of each brain are indicated in the right panel. (B) The size of the cortex area (excluding the olfactory bulb and cerebellum) of the brains photographed in (A) was measured using Adobe Photoshop. NS: not significant. (C, D) Nissl-stained sagittal (C) and coronal (D) sections of WT and TKO male mouse brains. Scale bar: 1 mm. (E, F) Brain weight (E) and cortical thickness (F) of WT and TKO male mice (WT, n=7; TKO, n=7). (G) Nissl-stained coronal sections of WT and TKO P7 mouse brains. Scale bar: 50 μm. (H) Neurofilament staining. The sections adjacent to those used in (G) were stained with an anti-68 kDa Neurofilament antibody. Scale bar: 50 μm. (IK) Neuronal hypotrophy in TKO brains. Cortical neurons were isolated from WT and TKO E16 mouse brains and cultured for 2 days in the presence (C5Fc) or absence (control) of Contactin5–Fc protein (6 μg/ml) in the culture medium. Neurons were stained with the TuJ1 anti-βIII tubulin antibody and total neurite length was measured. For (J), WT, n=1427 neurons (total); TKO, n=3053 neurons (total). For (K), WT control, n=742; WT+C5Fc, n=1037; TKO control, n=859; and TKO+C5Fc, n=853. NS: not significant. Scale bar: 10 μm. A single asterisk (*) and double asterisk (**) indicate P<0.05 and P<0.01 compared with WT, respectively.

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