Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 19 (2), 1539-46

SH3P7 Is a Cytoskeleton Adapter Protein and Is Coupled to Signal Transduction From Lymphocyte Antigen Receptors

Affiliations

SH3P7 Is a Cytoskeleton Adapter Protein and Is Coupled to Signal Transduction From Lymphocyte Antigen Receptors

O Larbolette et al. Mol Cell Biol.

Abstract

Lymphocytes respond to antigen receptor engagement with tyrosine phosphorylation of many cellular proteins, some of which have been identified and functionally characterized. Here we describe SH3P7, a novel substrate protein for Src and Syk family kinases. SH3P7 migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a 55-kDa protein that is preferentially expressed in brain, thymus, and spleen. It contains multiple amino acid sequence motifs, including two consensus tyrosine phosphorylation sites of the YXXP type and one SH3 domain. A region of sequence similarity, which we named SCAD, was found in SH3P7 and three actin-binding proteins. The SCAD region may represent a new type of protein-protein interaction domain that mediates binding to actin. Consistent with this possibility, SH3P7 colocalizes with actin filaments of the cytoskeleton. Altogether, our data implicate SH3P7 as an adapter protein which links antigen receptor signaling to components of the cytoskeleton.

Figures

FIG. 1
FIG. 1
SH3P7 is an adapter protein with multiple motifs for protein-protein interactions. (Upper panel) Schematic representation of the protein structures of SH3P7, coactosin, drebrin, and Abp-1. The N-terminal region of sequence similarity was named SCAD. Amino acid (aa) residues 170 to 250 of SH3P7 contain a high content of positively (28.5%) and negatively (27.5%) charged amino acids (indicated by +/−) and may adopt an α-helical structure. This part of the protein contains four repeats of a hexamer which are also present in drebrin and which have the consensus sequence R/KXEEXR. A putative SH3 domain binding site (+PXXP+) and two consensus tyrosine phosphorylation sites (YEVP-YEEP) are at positions 304 to 313, 337 to 340, and 347 to 350, respectively. The C-terminal SH3 domain has been reported previously (35). The positions and sequences of the Lys C-48 and Lys C-15 peptides are shown. Brackets indicate that the N-terminal lysine residue is inferred after Lys-C digestion. Locations of peptide sequences used for immunization are underlined. Protein structures are drawn to scale. (Lower panel) Amino acid sequence alignment of the SCAD regions together with the translated human EST sequence no. AA687496. Related and identical (white dot) amino acid residues are boxed. Gaps are indicated by black dots.
FIG. 2
FIG. 2
Expression of the SH3P7 protein in cell lines and different tissues. Anti-SH3P7 immunoblot analysis of cleared cellular lysates from approximately 106 cells of the indicated cell lines (a) or with 2 mg of total protein from different mouse tissues (b). In the experiments shown, the 87-B2 antibodies (see Materials and Methods) were used. Identical protein patterns were detected by the 85-B2 antibody but not with preimmune serum (data not shown). The apparent molecular masses of marker proteins are indicated in kilodaltons.
FIG. 3
FIG. 3
SH3P7 is tyrosine phosphorylated in antigen receptor-stimulated lymphocytes. (a) Antiphosphotyrosine precipitates were prepared from the indicated cell lines, which were either untreated (lanes 1, 3, 5, 7, and 9) or stimulated with 10 μg of anti-mouse IgM antibodies (lanes 2 and 4) per ml, 50 μM pervanadate-H2O2 (lane 6), 7.5 μg of F(ab′)2 fragments of anti-human IgM antibodies (lane 8) per ml, or 10 μg of Okt-3 antibodies (lane 10) per ml. Purified phosphoproteins obtained from 1.5 × 107 cells (lanes 1 to 4 and 7 to 10) or 5 × 106 cells (lanes 5 to 6) were separated by SDS-PAGE and subjected to immunoblot analysis with anti-SH3P7 antibodies (87-B2). (b) By using anti-SH3P7 antibodies (87-B2) coupled to protein G-Sepharose, the SH3P7 protein was precipitated from the indicated cell lines, which were treated as described in the legend for panel a. Purified proteins from 1.5 × 107 cells (lanes 1 to 4) or 5 × 106 cells (lanes 5 to 6) were analyzed by immunoblotting with antiphosphotyrosine antibodies. The position of the endogenous γ2am heavy chain of K46λμm, which is detected by the secondary anti-mouse IgG antibodies, is indicated. (c) Approximately 2 × 107 Ramos B cells (lanes 1 to 4) and 5 × 107 purified splenic B cells (lanes 5 to 8) were either left unstimulated (lanes 1, 3, 5, and 7) or stimulated through their antigen receptors with 7.5 μg of F(ab′)2 fragments from anti-human IgM antibodies (lanes 2 and 4) per ml or with 15 μg of anti-mouse κ antibodies (lanes 6 and 8) per ml, respectively. From these cells, antiphosphotyrosine precipitates (α-YP; lanes 1 to 2 and 5 to 6) and anti-SH3P7 precipitates (α-SH3P7; lanes 3 to 4 and 7 to 8) were prepared and analyzed by antiphosphotyrosine immunoblotting. The apparent molecular masses of marker proteins are indicated in kilodaltons.
FIG. 3
FIG. 3
SH3P7 is tyrosine phosphorylated in antigen receptor-stimulated lymphocytes. (a) Antiphosphotyrosine precipitates were prepared from the indicated cell lines, which were either untreated (lanes 1, 3, 5, 7, and 9) or stimulated with 10 μg of anti-mouse IgM antibodies (lanes 2 and 4) per ml, 50 μM pervanadate-H2O2 (lane 6), 7.5 μg of F(ab′)2 fragments of anti-human IgM antibodies (lane 8) per ml, or 10 μg of Okt-3 antibodies (lane 10) per ml. Purified phosphoproteins obtained from 1.5 × 107 cells (lanes 1 to 4 and 7 to 10) or 5 × 106 cells (lanes 5 to 6) were separated by SDS-PAGE and subjected to immunoblot analysis with anti-SH3P7 antibodies (87-B2). (b) By using anti-SH3P7 antibodies (87-B2) coupled to protein G-Sepharose, the SH3P7 protein was precipitated from the indicated cell lines, which were treated as described in the legend for panel a. Purified proteins from 1.5 × 107 cells (lanes 1 to 4) or 5 × 106 cells (lanes 5 to 6) were analyzed by immunoblotting with antiphosphotyrosine antibodies. The position of the endogenous γ2am heavy chain of K46λμm, which is detected by the secondary anti-mouse IgG antibodies, is indicated. (c) Approximately 2 × 107 Ramos B cells (lanes 1 to 4) and 5 × 107 purified splenic B cells (lanes 5 to 8) were either left unstimulated (lanes 1, 3, 5, and 7) or stimulated through their antigen receptors with 7.5 μg of F(ab′)2 fragments from anti-human IgM antibodies (lanes 2 and 4) per ml or with 15 μg of anti-mouse κ antibodies (lanes 6 and 8) per ml, respectively. From these cells, antiphosphotyrosine precipitates (α-YP; lanes 1 to 2 and 5 to 6) and anti-SH3P7 precipitates (α-SH3P7; lanes 3 to 4 and 7 to 8) were prepared and analyzed by antiphosphotyrosine immunoblotting. The apparent molecular masses of marker proteins are indicated in kilodaltons.
FIG. 3
FIG. 3
SH3P7 is tyrosine phosphorylated in antigen receptor-stimulated lymphocytes. (a) Antiphosphotyrosine precipitates were prepared from the indicated cell lines, which were either untreated (lanes 1, 3, 5, 7, and 9) or stimulated with 10 μg of anti-mouse IgM antibodies (lanes 2 and 4) per ml, 50 μM pervanadate-H2O2 (lane 6), 7.5 μg of F(ab′)2 fragments of anti-human IgM antibodies (lane 8) per ml, or 10 μg of Okt-3 antibodies (lane 10) per ml. Purified phosphoproteins obtained from 1.5 × 107 cells (lanes 1 to 4 and 7 to 10) or 5 × 106 cells (lanes 5 to 6) were separated by SDS-PAGE and subjected to immunoblot analysis with anti-SH3P7 antibodies (87-B2). (b) By using anti-SH3P7 antibodies (87-B2) coupled to protein G-Sepharose, the SH3P7 protein was precipitated from the indicated cell lines, which were treated as described in the legend for panel a. Purified proteins from 1.5 × 107 cells (lanes 1 to 4) or 5 × 106 cells (lanes 5 to 6) were analyzed by immunoblotting with antiphosphotyrosine antibodies. The position of the endogenous γ2am heavy chain of K46λμm, which is detected by the secondary anti-mouse IgG antibodies, is indicated. (c) Approximately 2 × 107 Ramos B cells (lanes 1 to 4) and 5 × 107 purified splenic B cells (lanes 5 to 8) were either left unstimulated (lanes 1, 3, 5, and 7) or stimulated through their antigen receptors with 7.5 μg of F(ab′)2 fragments from anti-human IgM antibodies (lanes 2 and 4) per ml or with 15 μg of anti-mouse κ antibodies (lanes 6 and 8) per ml, respectively. From these cells, antiphosphotyrosine precipitates (α-YP; lanes 1 to 2 and 5 to 6) and anti-SH3P7 precipitates (α-SH3P7; lanes 3 to 4 and 7 to 8) were prepared and analyzed by antiphosphotyrosine immunoblotting. The apparent molecular masses of marker proteins are indicated in kilodaltons.
FIG. 4
FIG. 4
Src and Syk family kinases can phosphorylate SH3P7 at the YXXP motif. (a) Antiphosphotyrosine immunoblot analysis of proteins precipitated from approximately 2.5 × 106 unstimulated J558Lδm7.1 cells with either rabbit control serum (control; lanes 1 to 4) or anti-SH3P7 antibodies (α-SH3P7; lanes 5 to 8) and subsequently subjected to an in vitro kinase assay in buffer alone (lane 1 and 5) or together with baculovirus-expressed Syk (lanes 2 and 6), Lyn (lanes 3 and 7), or Blk (lanes 4 and 8). (b) Baculovirus-expressed Lyn (lanes 1 to 5), Blk (lanes 6 to 10), and Syk (lanes 11 to 15) were used in an in vitro kinase assay without additional proteins (lanes 5, 10, and 15) or together with either GST (lanes 1, 6, and 11) or GST-SH3P7 fusion proteins that contain amino acids 1 to 337 (lanes 2, 7, and 12), 1 to 433 (lanes 3, 8, and 13), or 303 to 433 (lanes 4, 9, and 14) of SH3P7. Phosphorylated proteins were visualized by antiphosphotyrosine immunoblotting. (c) K46 cells were transiently transfected with expression vectors bearing the gene encoding wild-type flagged SH3P7 (wt-Flag; lane 2) or a flagged SH3P7 mutant with Y→F substitutions at position 337 (FY-Flag; lane 3) or 347 (YF-Flag; lane 4) or positions 337 and 347 (FF-Flag; lane 5). After stimulation of untransfected (lane 1) and transfected (lanes 2 to 5) cells with 50 μM pervanadate-H2O2 for 5 min, proteins were precipitated with anti-Flag antibodies and analyzed by antiphosphotyrosine and anti-Flag immunoblotting (upper and lower panel, respectively). The apparent molecular masses of marker proteins are indicated in kilodaltons.
FIG. 4
FIG. 4
Src and Syk family kinases can phosphorylate SH3P7 at the YXXP motif. (a) Antiphosphotyrosine immunoblot analysis of proteins precipitated from approximately 2.5 × 106 unstimulated J558Lδm7.1 cells with either rabbit control serum (control; lanes 1 to 4) or anti-SH3P7 antibodies (α-SH3P7; lanes 5 to 8) and subsequently subjected to an in vitro kinase assay in buffer alone (lane 1 and 5) or together with baculovirus-expressed Syk (lanes 2 and 6), Lyn (lanes 3 and 7), or Blk (lanes 4 and 8). (b) Baculovirus-expressed Lyn (lanes 1 to 5), Blk (lanes 6 to 10), and Syk (lanes 11 to 15) were used in an in vitro kinase assay without additional proteins (lanes 5, 10, and 15) or together with either GST (lanes 1, 6, and 11) or GST-SH3P7 fusion proteins that contain amino acids 1 to 337 (lanes 2, 7, and 12), 1 to 433 (lanes 3, 8, and 13), or 303 to 433 (lanes 4, 9, and 14) of SH3P7. Phosphorylated proteins were visualized by antiphosphotyrosine immunoblotting. (c) K46 cells were transiently transfected with expression vectors bearing the gene encoding wild-type flagged SH3P7 (wt-Flag; lane 2) or a flagged SH3P7 mutant with Y→F substitutions at position 337 (FY-Flag; lane 3) or 347 (YF-Flag; lane 4) or positions 337 and 347 (FF-Flag; lane 5). After stimulation of untransfected (lane 1) and transfected (lanes 2 to 5) cells with 50 μM pervanadate-H2O2 for 5 min, proteins were precipitated with anti-Flag antibodies and analyzed by antiphosphotyrosine and anti-Flag immunoblotting (upper and lower panel, respectively). The apparent molecular masses of marker proteins are indicated in kilodaltons.
FIG. 4
FIG. 4
Src and Syk family kinases can phosphorylate SH3P7 at the YXXP motif. (a) Antiphosphotyrosine immunoblot analysis of proteins precipitated from approximately 2.5 × 106 unstimulated J558Lδm7.1 cells with either rabbit control serum (control; lanes 1 to 4) or anti-SH3P7 antibodies (α-SH3P7; lanes 5 to 8) and subsequently subjected to an in vitro kinase assay in buffer alone (lane 1 and 5) or together with baculovirus-expressed Syk (lanes 2 and 6), Lyn (lanes 3 and 7), or Blk (lanes 4 and 8). (b) Baculovirus-expressed Lyn (lanes 1 to 5), Blk (lanes 6 to 10), and Syk (lanes 11 to 15) were used in an in vitro kinase assay without additional proteins (lanes 5, 10, and 15) or together with either GST (lanes 1, 6, and 11) or GST-SH3P7 fusion proteins that contain amino acids 1 to 337 (lanes 2, 7, and 12), 1 to 433 (lanes 3, 8, and 13), or 303 to 433 (lanes 4, 9, and 14) of SH3P7. Phosphorylated proteins were visualized by antiphosphotyrosine immunoblotting. (c) K46 cells were transiently transfected with expression vectors bearing the gene encoding wild-type flagged SH3P7 (wt-Flag; lane 2) or a flagged SH3P7 mutant with Y→F substitutions at position 337 (FY-Flag; lane 3) or 347 (YF-Flag; lane 4) or positions 337 and 347 (FF-Flag; lane 5). After stimulation of untransfected (lane 1) and transfected (lanes 2 to 5) cells with 50 μM pervanadate-H2O2 for 5 min, proteins were precipitated with anti-Flag antibodies and analyzed by antiphosphotyrosine and anti-Flag immunoblotting (upper and lower panel, respectively). The apparent molecular masses of marker proteins are indicated in kilodaltons.
FIG. 5
FIG. 5
Subcellular localization of SH3P7. NIH 3T3 fibroblasts were transiently transfected with an expression vector bearing the gene encoding a GFP-SH3P7 fusion protein (a to c). Forty-eight hours after transfection, cells were fixed in 3% paraformaldehyde and stained with TRITC-labelled phalloidin to visualize the actin-containing cytoskeleton. Subsequently, confocal microscopy was performed by using the FITC and TRITC filter settings to detect either GFP-SH3P7 (a) or phalloidin–F-actin (b) conjugate, respectively. (c) Overlay of panels a and b.

Similar articles

See all similar articles

Cited by 29 articles

See all "Cited by" articles

Publication types

MeSH terms

Feedback