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, 387 (Pt 3), 627-37

Sprouty-4 Negatively Regulates Cell Spreading by Inhibiting the Kinase Activity of Testicular Protein Kinase

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Sprouty-4 Negatively Regulates Cell Spreading by Inhibiting the Kinase Activity of Testicular Protein Kinase

Yoshikazu Tsumura et al. Biochem J.

Abstract

TESK1 (testicular protein kinase 1) is a serine/threonine kinase that phosphorylates cofilin and plays a critical role in integrin-mediated actin cytoskeletal reorganization and cell spreading. We previously showed that TESK1 interacts with Sprouty-4 (referred to as Spry4), an inhibitor of growth factor-induced Ras/MAP (mitogen-activated protein) kinase signalling, but the functional role of this interaction has remained unknown. In the present study, we show that Spry4 inhibits the kinase activity of TESK1 by binding to it through the C-terminal cysteine-rich region. Expression of Spry4 in cultured cells suppressed integrin-mediated cell spreading, and TESK1 reversed the inhibitory effect of Spry4 on cell spreading. Furthermore, Spry4 suppressed integrin- and TESK1-mediated cofilin phosphorylation during the spreading of cells on laminin. These findings suggest that Spry4 suppresses cell spreading by inhibiting the kinase activity of TESK1. Although tyrosine phosphorylation is required for the inhibitory activity of Spry4 on a Ras/MAP kinase pathway, mutation of the corresponding tyrosine residue (Tyr-75 in human Spry4) to an alanine had no apparent effect on its inhibitory actions on TESK1 activity and cell spreading, which suggests a novel cellular function of Spry to regulate the actin cytoskeleton, independent of its inhibitory activity on the Ras/MAP kinase signalling.

Figures

Figure 1
Figure 1. TESK1 interacts with Spry4 through the C-terminal cysteine-rich region
(A) Schematic representation of HA–Spry4, Myc–TESK1 and their mutants. Numbers above the boxes indicate amino acid residue numbers. CR1–CR3 indicate the highly conserved regions between TESK1 and TESK2 [34]. (B) Co-precipitation assay. Myc–TESK1 and HA–Spry4 mutants were co-expressed in COS-7 cells. Cell lysates were immunoprecipitated with anti-Myc antibody and analysed by immunoblotting with anti-HA antibody (top panel) or anti-Myc antibody (middle panel). Expression of HA–Spry4 and its mutants was analysed by immunoblotting cell lysates with anti-HA antibody (bottom panel). IP, immunoprecipitation; blot, immunoblotting. (C) In vitro pull-down assay. COS-7 cell lysates expressing Myc–TESK1 or its mutants were incubated with GST–Spry4 bound to glutathione–Sepharose, and precipitates were analysed by immunoblotting with anti-Myc antibody (top panel) and Coomassie Brilliant Blue (CBB) staining (middle panel). Expression of Myc–TESK1 or its mutants was analysed by immunoblotting cell lysates with an anti-Myc antibody (bottom panel).
Figure 2
Figure 2. Phosphorylation of Spry4 at Tyr-75 is required for the inhibitory activity on FGF-induced ERK activation, but not for TESK1 binding
(A) Effects of expression of Spry4 or Spry4(Y75A) on FGF-induced ERK activation. 293T cells expressing YFP, YFP–Spry4 or YFP–Spry4(Y75A) were serum-starved and then stimulated with basic FGF (bFGF) for 30 min. Cell lysates were subjected to SDS/PAGE and analysed by immunoblotting with anti-P-ERK1/2 antibody to detect ERK1/2 activation (top panel), anti-ERK1/2 antibody (middle panel) and anti-GFP antibody (bottom panel). (B) Co-precipitation of Spry4(Y75A) with TESK1. COS-7 cells were co-expressed with Myc–TESK1 and HA–Spry4 or HA–Spry4(Y75A). Cell lysates were immunoprecipitated with anti-Myc antibody and analysed by immunoblotting with anti-HA antibody (top panel) or anti-Myc antibody (middle panel). Expressions of HA–Spry4 and HA–Spry4(Y75A) were analysed by immunoblotting cell lysates with anti-HA antibody (bottom panel).
Figure 3
Figure 3. Co-localization of TESK1 and Spry4 in HeLa cells
HeLa cells were co-transfected with plasmids for HA–Spry4, Myc–TESK1 or their mutants, as indicated. Cells were fixed and co-immunostained with anti-HA (green) and anti-Myc (red) antibodies. In merged images, co-localization appears in yellow. Scale bar, 20 μm.
Figure 4
Figure 4. Spry4 inhibits the kinase activity of TESK1
Effects of GST, GST–Spry4 (A) or its mutants (B) on the kinase activity of TESK1. Myc–TESK1 expressed in COS-7 cells was immunoprecipitated with anti-Myc antibody, incubated in the absence (−) or presence of the indicated amounts of GST or GST–Spry4 (A) or 100 μg/ml GST–Spry4 or its mutants (B) and then subjected to in vitro kinase assay, using His6–cofilin as a substrate. (A, B) Reaction mixtures were separated by SDS/PAGE and cofilin was analysed by autoradiography (top panels) and Amido Black staining (middle panels). Myc–TESK1 was analysed by immunoblotting with anti-Myc antibody (bottom panels). The right panels show the relative kinase activities of TESK1 expressed as means±S.E.M. for triplicate experiments, with the activity of TESK1 in the absence of GST protein taken as 1.0.
Figure 5
Figure 5. Spry4 suppresses cell spreading
(A) Time-lapse analyses of the spreading of C2C12 cells after replating on laminin. Cells transfected with CFP or CFP–Spry4 together with YFP–actin were cultured for 18 h, suspended and replated on laminin-coated coverglasses. Cells were analysed by time-lapse fluorescence microscopy, making use of YFP fluorescence. Scale bar, 40 μm. (B) Three categories of spreading cells. C2C12 cells transfected with YFP or YFP–Spry4 were cultured and replated on laminin-coated coverglasses. After incubation for 30 min, cells were fixed and stained with rhodamine–phalloidin for F-actin. On the basis of the area of spreading cells, cells were categorized into three classes; cell area<800 μm2 (class 1), 800 μm2<cell area<1600 μm2 (class 2) and cell area>1600 μm2 (class 3). Scale bar, 40 μm. (C) Quantitative analysis of the effects of Spry4 expression on the spreading of C2C12 cells. Cells transfected with YFP or YFP–Spry4 were cultured for 18 h, suspended and replated on laminin-coated coverglasses. After incubation for 30 min, cells were fixed and stained with rhodamine–phalloidin. Cells were classified into three categories and percentages of these cells in YFP-positive cells (at least 200 cells) were calculated. The results are the means±S.E.M. for triplicate experiments. (D) Cell spreading morphologies of YFP- or YFP–Spry4-expressing cells. C2C12 cells transfected with YFP or YFP–Spry4 were replated on laminin and cultured for 30 min. Cells were fixed and analysed by YFP fluorescence (upper panels) and staining with rhodamine–phalloidin for F-actin (lower panels). Arrows indicate the YFP-positive cells. Scale bar, 40 μm.
Figure 6
Figure 6. TESK1 rescues the inhibitory effect of Spry4 on cell spreading
(A) C2C12 cells were transfected with YFP–TESK1 or YFP–TESK1(D170A). Cells were cultured for 18 h, suspended and replated on laminin-coated coverglasses. After 30 min, cells were fixed and analysed by YFP fluorescence (upper panels) and rhodamine–phalloidin staining for F-actin (lower panels). Arrows indicate YFP-positive cells. Scale bar, 40 μm. (B) Quantitative analysis of the effects of expression of TESK1 and TESK1(D170A) on the spreading of C2C12 cells. Percentages of the cells classified into the three categories (shown in Figure 5B) in YFP-positive cells (at least 100 cells) were calculated. The results are the means±S.E.M. for triplicate experiments. (C) C2C12 cells were co-transfected with YFP–Spry4 plus CFP or CFP–TESK1, as indicated. Cells were cultured and replated on laminin-coated coverglasses, as in (A). After 30 min, cells were fixed and analysed by YFP (top panels) and CFP fluorescence (middle panels) and rhodamine–phalloidin staining (bottom panels). Arrows indicate YFP- and CFP-positive cells. Scale bar, 40 μm. (D) Quantitative analysis of the effects of co-transfection with YFP–Spry4 plasmid plus CFP or CFP–TESK1 plasmid at the indicated ratio on the spreading of C2C12 cells. Percentages of the cells classified into the three categories are expressed as the means±S.E.M. for triplicate experiments. (E) Immunoblot analysis of the expression of CFP, CFP–TESK1 and YFP–Spry4. Lysates of C2C12 cells transfected with CFP, CFP–TESK1 and YFP–Spry4 plasmids, as in (D), were analysed by immunoblotting with anti-GFP antibody.
Figure 7
Figure 7. Effects of expression of Spry4 mutants and treatment with PD98058 on cell spreading
(A) C2C12 cells were transfected with YFP–Spry4 mutants or pretreated with 20 μM PD98059 for 20 min and then replated on laminin-coated dishes. After 30 min, cells were fixed and stained with rhodamine–phalloidin for F-actin. Arrows indicate cells expressing YFP-fusion proteins. Scale bar, 40 μm. (B) Quantitative analysis of the effects of expression of Spry4 mutants or treatment with PD98059 on the spreading of C2C12 cells. Percentages of the cells classified into the three categories (shown in Figure 5B) in YFP-positive cells (at least 100 cells) were calculated. The results are the means±S.E.M. for triplicate experiments.
Figure 8
Figure 8. Spry4 inhibits integrin- and TESK1-mediated cofilin phosphorylation
(A) C2C12 cells transfected with HA–Spry4 or control vector were suspended and replated on laminin-coated dishes. At the indicated times, cells were lysed and the lysates were analysed by immunoblotting with anti-P-cofilin and anti-cofilin antibodies. Expression of HA–Spry4 was analysed by immunoblotting with anti-HA antibody. In the right panel, the relative amounts of P-cofilin in C2C12 cells are plotted against the time after plating cells on laminin. The results are the means±S.E.M. for triplicate experiments, with the amount of P-cofilin in mock-transfected cells at zero time of plating taken as 1.0. (B) C2C12 cells transfected with Myc–TESK1 with or without HA–Spry4 were suspended and replated on laminin-coated dishes. At the indicated times, cells were lysed and the lysates were analysed as in (A). Expression of Myc–TESK1 was analysed by immunoblotting with anti-Myc antibody. In the right panel, the relative amounts of P-cofilin in C2C12 cells are plotted against the time after plating cells on laminin, as in (A).

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