Protein sequence and putative domain structure of SCHIP-1. Line 1, amino acid sequence of human SCHIP-1, deduced from the nucleotide sequence of a SCHIP-1 cDNA, isolated from a λgt10 human fetal brain library. Lines 2 and 3, amino acid sequences of two putative isoforms of human SCHIP-1, named SCHIP-1-Δ(241-253) and SCHIP-1-Δ(22-253). The corresponding cDNAs were identified by reverse transcriptase PCR amplification experiments on human brain poly(A)⁺ RNA. These cDNAs may correspond to in-frame alternative splicing events. Amino acids absent in SCHIP-1-Δ(241-253) and SCHIP-1-Δ(22-253) are replaced by a continuous line. Line 4, amino acid sequence encoded by the partial mouse SCHIP-1 cDNA isolated from the two-hybrid screen. Dashes mark identity between human and mouse sequences; nonconserved amino acids between human and mouse proteins are directly indicated. Yellow and blue boxes indicate the four serine-rich regions and the two aspartic acid/glutamic acid-rich regions of the human SCHIP-1 protein, respectively. Green boxes show the localization of three putative SH3-binding domains conserved between the mouse and human SCHIP-1 proteins, and the orange box shows the localization of a putative tyrosine kinase phosphorylation site in human SCHIP-1. The grey box indicates the region of homology between the SCHIP-1 and FEZ proteins, predicted to adopt a coiled-coil conformation. The red line defines a putative PEST motif conserved in mouse and human SCHIP-1 proteins, and the blue line indicates the leucine zipper pattern in the putative coiled-coil region.

SCHIP-1 can homodimerize through its coiled-coil domain homologous to the FEZ and UNC-76 proteins. (A) Schematic alignment of human SCHIP-1 with human FEZ1 and FEZ2 and C. elegans UNC-76 proteins. Open boxes represent full-length SCHIP-1, FEZ1, and UNC-76 proteins and the 251 C-terminal amino acids (aa) of the FEZ2 protein. The grey color highlights the localization of the coiled-coil domain in each protein. Arrowheads indicate amino acids delimiting coiled-coil domains. Percentages of homology with SCHIP-1 in the coiled-coil domains are indicated. (B) Amino acid alignment of human SCHIP-1 with human FEZ1 and FEZ2 and C. elegans UNC-76 proteins within the predicted coiled-coil domain. Identical amino acids are presented in dark boxes; conserved residues are displayed in grey boxes. (C) The predicted coiled-coil domain is required for SCHIP-1 homodimerization. SCHIP-1 and SCHIP-1 deletion mutants were expressed in vitro in the presence of [³⁵S]methionine and incubated in TKT150 buffer with either GST or GST–SCHIP-1(306-487) bound to glutathione-agarose beads. After washing, retained SCHIP-1 and SCHIP-1 variant proteins were eluted, resolved by SDS-PAGE, and visualized by autoradiography. Aliquots of the labeled proteins corresponding to 1/40 of the input were loaded on the same gel (Translation). The C-terminal region of SCHIP-1 including the coiled-coil domain [GST–SCHIP-1(306-487)] interacts with full-length SCHIP-1 or SCHIP-1-Δ(22-253) but not with a truncated SCHIP-1 missing the predicted coiled-coil domain [SCHIP-1(1-413)].

Expression pattern of SCHIP-1 mRNAs and proteins. (A) Northern blot analysis of SCHIP-1 expression. Human mRNAs from the indicated organs were analyzed by Northern blotting using either SCHIP-1 cDNA bp 1 to 405 (EcoRI/KpnI fragment), SCHIP-1 cDNA bp 402 to 2112 (KpnI/EcoRI fragment), or β-actin cDNA as the probe. Strong expression of SCHIP-1 is detected in skeletal (sk.) muscles, brain, and heart. (B) Immunoprecipitation of endogenous SCHIP-1 in ST88.14 cells. Endogenous SCHIP-1 from the schwannoma cell line ST88.14 was immunoprecipitated (IP) using either a chicken polyclonal antibody detecting a C-terminal region of SCHIP-1 (lane 959) or a polyclonal rabbit antibody detecting a central region of SCHIP-1 (lane 17014). Precipitates were resolved by SDS-PAGE on a 10% gel and detected with either rabbit antibody 17014 (left) or chicken antibody 959 (right). Twenty micrograms of crude protein extract from the ST88.14 cell line (Lysate) was loaded on the same gel. Antibodies used for Western blotting are indicated below the gels. NI, preimmune IgY or rabbit serum. The two independent antibodies 959 and 17014 both immunoprecipitate and detect in Western blots a protein migrating with an apparent molecular mass of 65 kDa. (C) SCHIP-1 expression in five human cell lines. Proteins (50 μg) from each cell line were resolved by SDS-PAGE on a 10% gel and detected with the rabbit polyclonal antibody 17141. Expression of the 65-kDa SCHIP-1 protein is detected in each cell line.

SCHIP-1 interacts with two distinct regions of schwannomin in vitro. (A and B) In vitro interaction of schwannomin with SCHIP-1. Either GST or GST–SCHIP-1(120-487) was bound to glutathione-agarose beads and incubated in TKT40 buffer with [³⁵S]methionine-labeled SCH-Iso1 or deletion mutants SCH(1-314), SCH(315-595), SCH(289-595), SCH(1-288), SCH(19-288), and SCH(19-314) as indicated. After washing, retained schwannomin and schwannomin variant proteins were eluted, resolved by SDS-PAGE, and visualized by autoradiography. Aliquots of the labeled proteins corresponding to 1/40 of the input were loaded on the same gel (Translation). SCHIP-1 interacts with SCH-Iso1 in vitro (A). This interaction requires two regions of schwannomin spanning amino acids 1 to 18 and 289 to 314 (B). (C) Map of schwannomin constructs and summary of the properties of the corresponding proteins. (Left) Map of schwannomin constructs. Open boxes indicate the regions of schwannomin that are encoded by the different constructs. Positions of the truncations or amino acids substitutions and deletions are shown by arrowheads. The two regions found to be implicated in the interaction with SCHIP-1 are indicated at the bottom. Relative positions of the NF2 exons and schwannomin amino acid residues are illustrated on the top. (Right) Summary of properties of the various proteins. In the two columns are indicated the ability of the various proteins to interact in GST pull-down experiments with SCHIP-1 (column 1) and the ability of the various proteins to coimmunoprecipitate with SCHIP-1 when the proteins are overexpressed in HeLa cells (column 2). +, interaction in vitro or coimmunoprecipitation detected; −, interaction in vitro or coimmunoprecipitation not detected; ND, ability to coimmunoprecipitate not determined.

Schwannomin region 1 and region 2 are sufficient for interaction with SCHIP-1 in vitro. (A) Map of GST-schwannomin constructs. Open boxes indicate the regions of schwannomin that are encoded by the different constructs. Amino acids delimiting these regions are indicated by arrowheads. (B) In vitro interaction of schwannomin region 1 and region 2 with SCHIP-1. Either GST, GST–SCH(1-314), GST–SCH(1-27), or GST–SCH(280-323) was bound to glutathione-agarose beads and incubated in TKT150 buffer with [³⁵S]methionine-labeled SCHIP-1 as indicated. After washing, retained SCHIP-1 protein was eluted, resolved by SDS-PAGE, and visualized by autoradiography. Region 1 [GST–SCH(1-27)] and region 2 [GST-SCH(280-323)] are able to associate independently with SCHIP-1 in vitro.

The coiled-coil region of SCHIP-1 is required for efficient interaction with schwannomin in vitro. (A) Map of SCHIP-1 constructs. Open boxes indicate the regions of SCHIP-1 encoded by the different constructs. The grey color highlights the localization of the coiled-coil domain. Arrowheads indicate the amino acids delimiting the coiled-coil domain and the positions of truncations and deletions. (B) In vitro interaction of SCHIP-1 with schwannomin. Either GST or GST–SCH(1-314) was bound to glutathione-agarose beads and incubated in TKT150 buffer with [³⁵S]methionine-labeled full-length SCHIP-1, SCHIP-1(1-413), SCHIP-1(1-305), SCHIP-1-Δ(22-253), or SCHIP-1(120-487) as indicated. After washing, retained SCHIP-1 and SCHIP-1 variants were eluted and visualized by autoradiography. Aliquots of the labeled proteins corresponding to 1/40 of the input were loaded on the same gel (Translation). Schwannomin interacts in vitro with full-length SCHIP-1 or SCHIP-1 proteins deleted in the N-terminal domain [SCHIP-1-Δ(22-253) and SCHIP-1(120-487)] but not or poorly with truncated SCHIP-1 proteins missing the coiled-coil region [SCHIP-1(1-413) and SCHIP-1(1-305)].

In vivo interaction of schwannomin and SCHIP-1. HeLa cells were cotransfected with either pCB6-HA-SCHIP-1 expression vector (+) or empty pCB6 vector (−) and vector expressing schwannomin variants SCH(1-314) (A), SCH-Iso1 (B), SCH-Δ(39-121) (C), and SCH-Δ118 (D). Twenty-four hours after transfection, proteins extracts were prepared as described in Materials and Methods, and coimmunoprecipitations were performed with the anti-HA MAb 12CA5. Precipitates were resolved by SDS-PAGE on an 8% gel, electroblotted, and submitted to two rounds of immunoblotting, first with the anti-SCH polyclonal antibody A19 and then with the anti-SCHIP-1 polyclonal antibody 17141. Twenty microliters of crude protein extract (Lysates) was also subjected to immunoblotting with either the anti-SCH polyclonal antibody A19 or the anti-VSV monoclonal antibody MAb P5D4, to verify expression of schwannomin proteins. Antibodies used for Western blotting are indicated below the gels. SCHIP-1 is able to associate in vivo with the schwannomin variant SCH(1-314) (A) and the two naturally occurring mutants SCH-Δ(39-121) and SCH-Δ118 (C and D) but not with SCH-Iso1 (B).

Colocalization of SCHIP-1 and schwannomin in overexpressing cells. HeLa cells were transfected with expression vector pCB6-HA-SCHIP-1 alone (A to C) or together with an SCH-Iso1 expression vector (D). Twenty-four hours later, cells were processed for indirect immunofluorescence using MAb 12CA5 to detect the HA-tagged SCHIP-1 protein (A to D), FITC-phalloidin to reveal F-actin (A to C), and the C14 rabbit polyclonal antibody to detect schwannomin (D). Coverslips were examined with a Leica epifluorescence microscope (A to C, views a and b) and with a Leica confocal microscope (A to C, views c; D). Red indicates anti-mouse secondary antibody staining (SCHIP-1) (A to D), whereas green represents actin staining (A to C) or anti-rabbit secondary antibody staining (schwannomin) (D). Overlays are shown with yellow indicating colocalization (A to D). (A to C) Representative examples of the three main types of localization that have been observed for SCHIP-1 on a single coverslips. Arrowheads indicate regions where SCHIP-1 localizes below the cytoplasmic membrane (B) or colocalizes with cortical actin (C). (D) Cell in which SCHIP-1 localizes beneath the cytoplasmic membrane and colocalizes partially with SCH-Iso1. Scale bars, 5 μm (A to C) and 2 μm (D).