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First published online 5 May 2004
doi: 10.1242/jcs.01148

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Recruitment of Pyk2 and Cbl to lipid rafts mediates signals important for actin reorganization in growing neurites

¹ Institute for Biochemistry II, Building 75, Goethe University Medical School, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany
² Ludwig Institute for Cancer Research, Box 595, Husargatan 3, Uppsala, 75124, Sweden
³ Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, PA 19111, USA

View larger version (37K): [in a new window]   Fig. 1. Constitutive association between Pyk2 and Cbl in mammalian cells. (A) PC12-PDGFR cells were starved for 16 hours and were left untreated (–) or stimulated with 100 ng/ml EGF (E) or PDGF (P) for 5 minutes. Following cell lysis, equal amounts of cell lysates were subjected to immunoprecipitation (IP) with antibodies to Pyk2 (600) or Cbl (RF) or pre-immune sera (pre-im) and immunoprecipitates were analyzed by western blotting (WB) with anti-Cbl (TL) and anti-Pyk2 (N-19) antibodies. TCL, total cell lysate. (B) Pyk2 or a kinase inactive mutant of Pyk2 (PKM) were overexpressed in HEK 293T cells alone or together with Cbl, or with Cbl and Src kinase (K+) or Cbl and a kinase inactive mutant of Src (K–). Cbl was immunoprecipitated (IP) with anti-Cbl (RF) antibodies and co-precipitation was monitored by western blotting (WB) with anti-Pyk2 (N-19) antibodies. Levels of proteins in total cell lysates (TCL) were determined with anti-Cbl (TL), anti-Pyk2 (N-19) and anti-Src (Ab1) antibodies. (C) Pyk2 or PKM were overexpressed in HEK 293T cells and equal amounts of cell lysates were subjected to glutathione S-transferase (GST) pulldown with GST fusion proteins of the amino terminus of Cbl spanning the SH2 and RING finger domains (GST-Cbl NT) or the carboxyl terminus of Cbl containing the proline-rich sequences; the acidic box and the leucine zipper domain (GST-Cbl CT) and the western blot (WB) was probed with anti-Pyk2 (600) antibodies.

View larger version (29K): [in a new window]   Fig. 2. Adaptor protein ArgBP2 links Pyk2 and Cbl in a complex. (A) Pyk2 and Cbl were co-expressed together with FLAG-tagged ArgBP2 (88 kDa), CAP (90 kDa) or CMS (85 kDa) as indicated. Immunoprecipitation (IP) was performed with anti-FLAG (M2) or anti-Cbl (RF) antibodies. Western blotting (WB) was done as indicated with anti-Cbl (TL), anti-Pyk2 (N-19) and anti-FLAG (M5) antibodies. (B) Pyk2 and Cbl were co-expressed in HEK 293T cells with increasing amounts of ArgBP20 Cbl, Pyk2 or FLAG-tagged ArgBP2 were immunoprecipitated (IP) with anti-Cbl (RF), anti-Pyk2 (600) or anti-FLAG (M2) antibodies, respectively. Western blotting (WB) was performed as in A). (C) Glutathione S-transferase (GST) alone or GST fusion proteins of each of the three SH3 domains of ArgBP2 (SH3A, SH3B, SH3C) were incubated with equal amounts of lysates from HEK 293T cells overexpressing Pyk2. GST-ArgBP2-SH3A was also incubated with lysates expressing a kinase inactive mutant of Pyk2 (PKM) or a double mutant of PKM mutated at two prolines in major proline-rich motifs (PKM P717/859A). Western blotting (WB) was performed with anti-Pyk2 (N-19) antibodies. Levels of GST fusion proteins were examined by Ponceau staining. (D) PKM, PKM-P717/859A or Pyk2 were coexpressed with or without Cbl and cell lysates were subjected to immunoprecipitation (IP) with antibodies against Pyk2 (600). Western blotting (WB) was performed with anti-Cbl (RF) and anti-Pyk2 (N-19) antibodies. TCL, total cell lysate.

View larger version (51K): [in a new window]   Fig. 3. Cellular localization of Pyk2, ArgBP2 and Cbl in PC12 cells. (A) PC12 cells stably expressing PDGF ß-receptors were grown on collagen I-coated coverslips and subjected to immunofluorescence with anti-Pyk2 (N-19), anti-Cbl antibodies (C-15) and anti-ArgBP2 antibodies. In the upper panel, Pyk2 is visualized in green by Alexa 488-labeled secondary donkey anti-goat antibodies (left), Cbl (middle) and ArgBP2 (right) are stained red with TRITC-labeled swine anti-rabbit secondary antibodies. In the lower panel, F-actin is stained blue by Alexa 305-labeled phalloidin (left), the merged picture between Pyk2, Cbl and F-actin is in the middle and the superimposed image of ArgBP2 and F-actin is on the right. Left and middle panels are from one experiment and the right panel from a separate experiment. (B) PC12 cells stably expressing PDGF ß-receptors were grown on collagen I-coated coverslips and subjected to immunofluorescence after 48 hours of PDGF-BB (50 ng/ml) stimulation. F-actin is stained with Alexa 305-labeled phalloidin (upper panel) or FITC-phalloidin (lower panel). Immunofluorescence was performed using goat polyclonal anti-Pyk2 (N-19), rabbit polyclonal anti-Cbl (C-15) and anti-ArgBP2 as indicated. Pyk2 was visualized by Alexa 488-labeled donkey anti-goat antibodies and Cbl (upper panel) and ArgBP2 (lower panel) by TRITC-labeled swine anti-rabbit antibodies. Upper and lower panels are form two different experiments.

View larger version (42K): [in a new window]   Fig. 4. A Pyk2-ArgBP2-Cbl complex is recruited to lipid rafts following growth factor stimulation. (A) Serum-starved PC12-Myc-ArgBP2 cells were mock-stimulated or treated for 10 minutes or 2 hours with 50 ng/ml NGF and homogenized lysates were subjected to sucrose gradient centrifugation as described in the Materials and Methods. Immunoprecipitates of Cbl, Pyk2, Myc-ArgBP2, Crk and flotillin-1 from lipid rafts and Triton X-100 soluble fractions were subjected to western blot with the following antibodies: anti-Cbl (RF), anti-Pyk2 (N-19), anti-Myc (9E10), anti-Crk and anti-flotillin-1. (B) HEK 293T cells were transfected with Pyk2 or PKM together with Cbl and FLAG-ArgBP2, serum-starved and stimulated with a mixture of 50 ng/ml EGF and 50 ng/ml PDGF-BB for 20 minutes. Immunoprecipitates of Cbl, Pyk2 and ArgBP2 from lipid rafts and Triton X-100-soluble fractions were analyzed with antibodies against Cbl (RF), Pyk2 (N-19), FLAG(M2). Fyn was detected in total cell lysates. (C) Lysates of HEK 293T cells overexpressing FLAG-CAP, FLAG-ArgBP2 or nArgBP2 were subjected to GST pulldown with GST alone or GST-flotillin-1. Western blotting was done with anti-FLAG and anti-nArgBP2 antibodies. (D) HEK 293T cells overexpressing Pyk2, Cbl and Myc-ArgBP2-SoHo were serum-starved and stimulated with a mixture of 50 ng/ml EGF and 50 ng/ml PDGF-BB for 20 minutes. Immunoprecipitates of Cbl (RF), Pyk2 (600), Myc-ArgBP2-SoHo (9E10) and flotillin-1 from lipid rafts and Triton X-100 soluble fractions are shown. Western blotting was performed with antibodies against Cbl (RF), Pyk2 (N-19), Myc (9E10) and Flotillin-1.

View larger version (45K): [in a new window]   Fig. 5. Co-expression of Pyk2, Cbl and ArgBP2 induces membrane ruffles and lamellipodia formation in PC12 cells, a process dependent on intact lipid rafts. (A) Pyk2, Cbl and ArgBP2 were transiently transfected into PC12-PDGFR cells. Cells were stimulated with 100 ng/ml PDGF-BB for 48 hours and subjected to immunofluorescence with goat polyclonal anti-Pyk2 (N-19), mouse monoclonal anti-Cbl (TL) and rabbit polyclonal anti-ArgBP2 antibodies. Pyk2 was stained with Alexa 488-labeled donkey anti-goat, Cbl with AMCA-labeled goat anti-mouse and ArgBP2 with TRITC-labeled swine anti-rabbit antibodies. Shown is a cell with developed lamellipodia with the focus on two different layers of the cell, as indicated by the cell drawings. The merged pictures from the three channels are also shown. (B) PC12-Myc-ArgBP2 cells, grown in the presence of 50 ng/ml NGF for 78 hours after transfection with Pyk2 and Cbl, were mock-treated (mock) or treated with 4 µM lovastatin (lovast) for 24 hours and 0.2 M methyl-ß-cyclodextrin (MBCD) for 4 hours at 37°C. Transfected cells were detected by immunofluorescence with antibodies against Pyk2 (N-19) and Cbl (C-15), which were visualized with donkey anti-goat-Alexa 488 and TRITC-labeled swine anti-rabbit antibodies. Cells showing neurites in the absence (mock) or presence of lipid raft inhibitors (MBCD/lovast) were quantified and the percentages of cells with neurites were calculated as described in Materials and Methods. Moreover, the percentage of cells showing lamellipodia at growth cones out of those cells with neurites was also determined for both untreated (mock) and treated (MBCD/lovast) cells. Error bars represent the standard error of the mean.

View larger version (29K): [in a new window]   Fig. 6. Binding of Crk and p85/PI 3-kinase to phosphorylated Cbl mediates lamellipodia formation in PC12 cells. (A) PC12 cells stably expressing the PDGFR ß-receptor and overexpressing Pyk2 and Cbl-CT (proline-rich regions, acidic box and leucine zipper domain) were analyzed for the formation of lamellipodia following stimulation with PDGF-BB for 48 hours. Cbl was detected with anti-Cbl (C-15) and Pyk2 with anti-Pyk2 (NT) antibodies. Cbl was visualized with swine anti-rabbit TRITC secondary antibodies, Pyk2 with donkey anti-goat Alexa 488 and F-actin with AMCA-labeled phalloidin. The boxed regions are enlarged in the panels below to show lamellipodia. (B) PC12-PDGFR cells were transiently transfected with Pyk2 and wild-type Cbl or Cbl mutants (Cbl-Y700F, Cbl-Y700/774F or Cbl-Y700,731,774F=Cbl-3YF) and stimulated with 100 ng/ml PDGF-BB for 48 hours. Cells were then subjected to immunofluorescence with goat polyclonal anti-Pyk2 (N-19) and rabbit polyclonal anti-Cbl (C-15) antibodies followed by incubation with Alexa-488-conjugated donkey anti-goat and TRITC-labeled swine anti-rabbit antibodies. Cells overexpressing Pyk2 and Cbl or Cbl mutants were quantified for the presence of neurite lamellipodia. Percentages for each construct relative to the wild-type control were determined as described in Materials and Methods. The error bars represent the standard error of the mean. (C) PC12-PDGFR cells overexpressing Pyk2 and Cbl were stimulated with 100 ng/ml PDGF-BB for 48 hours and treated with 100 mM LY294002 (a PI 3-kinase inhibitor) for 30 minutes, 4 hours or 6 hours. Cells overexpressing Pyk2 and Cbl were visualized by immunofluorescence as in B. Transfected cells showing neurite lamellipodia were quantified for each time period and compared to untreated cells. Percentages were determined as described in Materials and Methods. The error bars represent the standard error of the mean. (D) PC12-PDGFR cells overexpressing Pyk2, Cbl and wild-type CrkII, CrkII-SH2M or CrkII-SH3M were stimulated for 48 hours with PDGF-BB and quantified for the presence of neurite lamellipodia. Percentages for each construct were determined as in Materials and Methods. The error bars represent the standard error of the mean. (E) Model of interactions between Cbl, ArgBP2 and Pyk2 in lipid rafts and of how growth factor signals might regulate lamellipodia formation via this complex. ArgBP2 associates via its SH3 domains with proline-rich motifs in Pyk2 (via SH3A) and Cbl (via SH3B and SH3C) and is able to recruit Pyk2 and Cbl to lipid rafts via its SoHo domain. Growth factor stimulation promotes Pyk2 activation and its autophosphorylation on Y402. This leads to activation of Src family kinases, which are able to phosphorylate tyrosines in the carboxyl termini of Pyk2 and Cbl (Y700, Y731 and Y774). This in turn allows Pyk2 and Cbl to recruit several effectors involved in regulating the cytoskeleton. In particular, Cbl recruits Crk and the p85 subunit of PI 3-kinase, which are involved in Rac-dependent lamellipodia formation in the neurite growth cone.

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