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First published online 27 September 2005
doi: 10.1242/jcs.02590

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p190 Rho-GTPase activating protein associates with plexins and it is required for semaphorin signalling

Davide Barberis^1,*, Andrea Casazza^1,*, Raffaella Sordella², Simona Corso¹, Stefania Artigiani¹, Jeff Settleman², Paolo M. Comoglio¹ and Luca Tamagnone^1,

¹ Institute for Cancer Research and Treatment (IRCC), University of Turin Medical School, Candiolo, Torino 10060, Italy
² Harvard Medical School, MGH Cancer Center, 149 13th Street, Charlestown, MA 02129, USA

View larger version (35K): [in a new window]   Fig. 1. Downregulation of RhoA-GTP in response to PLXNB1 activation. (A) NIH-3T3 fibroblasts either expressing PLXNB1 or mock transfected, were treated with the ligand Sema4D (5 nM) for the indicated times. RhoA-GTP levels were measured by pull-down with rhotekin-RBD-coated beads, followed by immunoblotting with anti-RhoA antibodies. Expression levels of RhoA and of the exogenous plexin in input lysates are shown below. The amount of pulled-down GTP-RhoA relative to total RhoA levels in each condition is reported at the bottom, based on band intensity quantification. (B) Measurement of RhoA-GTP levels by rhotekin-RBD pull-down, as above, in SKBR3 cells expressing endogenous PLXNB1 and treated with 10 nM Sema4D for the indicated times. Relative amounts of active RhoA were measured as above and are shown at the bottom.

View larger version (37K): [in a new window]   Fig. 2. p190 Rho-GAP is required to mediate plexin signalling in fibroblasts. (A) Immunoblots of total cell lysates of NIH-3T3 fibroblasts expressing PLXNB1 and engineered with siRNAs targeted to p190 transcript or to an unrelated sequence. The filter was probed with specific antibodies directed against p190, PLXNB1 and actin. The endogenous levels of p190 are greatly reduced in NIH-3T3 fibroblasts by expression of targeted siRNA, while plexin expression is unaffected. (B) NIH-3T3 fibroblasts expressing PLXNB1 (or PLXNB1/A1) and engineered with p190-targeted siRNAs (or controls) were grown on glass coverslips and subjected to treatment with 5 mM Sema4D for 15 minutes. Cell were then analysed by immunofluorescence with EC-6.9 antibodies directed against the extracellular domain of PLXNB1. Scale bar: 20 µm. The average fraction of collapsed cells was determined in each condition (Barberis et al., 2004) and it is shown on the right. Plexin-dependent collapse response is greatly impaired in the absence of p190. (C) The attachment of the same cells as above to fibronectin-coated wells was assayed, in the presence or absence of 5 nM Sema4D. After 30 and 120 minutes, adherent cells were fixed and stained with crystal violet. Cell adhesion was eventually quantified by eluting the dye and measuring the absorbance at 595 nm. (D) In a similar experiment as described in C, we scored the average fraction of fibroblasts spread on fibronectin, after 1 hour incubation with or without the indicated amounts of Sema4D (see Barberis et al., 2004).

View larger version (46K): [in a new window]   Fig. 3. Re-expression of p190 Rho-GAP in gene-deficient cells restores the functional response to plexin activation. (A) Fibroblasts derived from wild-type or from p190–/– mouse embryos (Brouns et al., 2000) were engineered to express PLXNB1 (see B for protein expression analysis) and then treated with 10 mM Sema4D for 1 hour to test their collapse response (in analogy to Fig. 2B). Scale bar: 20 µm. (B) PLXNB1-expressing p190–/– knockout fibroblasts were engineered to express exogenous p190 or its inactive mutant p190RA (both conjugated with GFP). Protein expression levels were analysed by immunoblotting with specific antibodies. Hsp90 expression was determined as loading reference. (C) Merged fluorescence images of the cells described in B after treatment with 10 nM Sema4D for 30 minutes to induce cellular collapse: the red channel reveals PLXNB1, detected with specific antibodies (EC-6.9), while the green channel shows p190-GFP. Co-expression of PLXNB1 with p190, but not with its inactive mutant, is required and sufficient to rescue the functional response to Sema4D in these cells. The asterisk marks a cell expressing PLXNB1 but not p190, which is insensitive to the ligand. Scale bar: 20 µm. The fraction of GFP-positive collapsed cells in each condition was counted and it is shown at the bottom. (D) p190-deficient fibroblasts expressing PLXNB1, and further transfected with p190-GFP or its inactive mutant p190RA-GFP (the same shown in A), were grown on fibronectin-coated coverslips and treated with 5 nM Sema4D for 5 minutes. Cells were then fixed and focal adhesions revealed in the red channel with anti-paxillin antibodies. After this short stimulation, very few cells underwent collapse [consistent with that described in 3T3 fibroblasts (see Barberis et al., 2004)], however, focal adhesions were disassembled in most of the cells expressing functionally competent p190-RhoGAP (identified by GFP expression). In contrast, semaphorin stimulation had no effect in cells lacking p190 or expressing the mutant devoid of RhoGAP activity (p190RA). Scale bar: 20 µm. The fraction of GFP-positive cells containing focal adhesion was counted and it is shown at the bottom. (E) Affinity purification of RhoA-GTP (by rhotekin-RBD pull-down) from equal amounts of protein lysates of p190–/– fibroblasts expressing PLXNB1 and the indicated exogenous p190 proteins, after treatment with 10 nM Sema4D for 15 minutes. Plexin-mediated Rho inactivation is abrogated in the absence of p190, but it is rescued by expression of exogenous p190-GFP in the gene-deficient cells. The mutated form of p190 (p190RA), lacking GAP activity, is unable to rescue the function. The effect was quantified by measuring the relative amounts of active RhoA in each condition (band intensity of RhoA-GTP versus total RhoA) and it is shown at the bottom.

View larger version (51K): [in a new window]   Fig. 4. p190 is required for multiple functional responses mediated by Sema4D in epithelial cells expressing endogenous PLXNB1. (A) Selective downregulation of p190 protein in SKBR3 cells by siRNA-mediated technology, demonstrated by immunoblotting with specific antibodies. The expression of endogenous PLXNB1 is unchanged. (B) Focal complex disassembly (revealed by vinculin immunostaining) and cellular collapse mediated by 1 hour treatment with 10 nM Sema4D was abrogated in p190-depleted SKBR3 cells. The functional response was unaffected by siRNAs targeting an unrelated sequence. The micrographs show results representative of at least three independent experiments. Scale bar: 40 µm. (C) Chemotactic migration of SKBR3 treated with siRNAs (as above) was assessed in Transwell® inserts, in the presence of 0.2 nM heregulin-ß1 (HRG), with or without 5 nM Sema4D. After 6 hours, the cells that had migrated across the porous membrane were stained with Crystal Violet. Cell migration was quantified by eluting the dye and measuring absorbance at 595 nm. Results shown are the average of two independent experiments, performed in duplicate. The expected inhibition of directional cell migration is lost in p190-deficient cells.

View larger version (54K): [in a new window]   Fig. 5. p190 is required for semaphorin-induced repulsion of primary endothelial cells. (A) MDA-MB435 mammary carcinoma cells were engineered to express myc-tagged Sema3F, Sema3A, Sema4D or mock transfected. The secretion of the semaphorins in the conditioned medium as demonstrated by immunoblotting with anti-myc antibodies. (B) HUVEC cells were engineered to express siRNAs targeted to p190 transcript or to an unrelated sequence. The expression levels of p190 in cell lysates were then analysed by immunoblotting with specific antibodies. (C) siRNA-expressing HUVECs analysed as in B were grown to confluence on glass coverslips. 6x103 MDA-MB435 tumour cells engineered to express the indicated semaphorins (as shown in A) were then seeded onto the monolayer of endothelial cells. After 48 hours of co-culture, the cells were fixed and analysed by immunofluorescence with anti-myc antibodies to reveal semaphorin-expressing cells. HUVECs were identified by GFP expression (associated with siRNA-expression vectors). Scale bar: 200 µm. In other experiments, the endothelial cell monolayers were revealed by immunocytochemistry with anti-CD31 antibodies (supplementary material Fig. S3). (D) HUVEC-free areas were identified by GFP expression or CD31 positivity (as described above) and measured by ImageQuant software. At least three independent low magnification fields were analysed, by two separate investigators, for each experimental point. The table shows average values.

View larger version (26K): [in a new window]   Fig. 6. p190 is required for Sema4D-induced neurite outgrowth. PC12 neuroblasts were grown for 48 hours in the presence of 50 ng/ml NGF, with or without 1 nM Sema4D, and then fixed and photographed. Scale bar: 20 µm. The bar chart shows the mean percentage of cells with neurites (extending for at least one cell diameter) counted in three separate fields. Results are representative of two independent experiments. As discussed in the text, PC12 neuroblasts displayed minimal (if any) functional response to treatment Sema4D only (not shown). However, Sema4D-dependent synergism with NGF-induced neuritogenesis is abrogated in cells depleted of p190 by targeted expression of siRNAs.

View larger version (35K): [in a new window]   Fig. 7. p190 is recruited to plexins, and its GAP activity is induced upon receptor activation. (A) p190-GFP and either PLXNB1 or PLXNA1 (VSV-tagged) specifically co-precipitate from lysates of co-transfected 293T cells, whereas a truncated form of PLXNB1 lacking the cytoplasmic domain (PlexinB1-IC) does not associate with p190. Immunoprecipitations were performed using either anti-tag antibodies (GFP for p190 and anti-VSV for plexins, respectively) or non-related serum (nrs). Western blots were probed with the indicated anti-tag or protein-specific antibodies. Note that PLXNB1 is larger than other plexins (approx. 300 kDa), while PlexinB1-IC and PLXNA1 are almost identical in size (approx. 220 kDa). (B) Co-immunopurification of endogenous p190 with PLXNB1 transfected in 293T cells is induced after 5 minutes stimulation with 5 nM Sema4D. The expression levels of p190 and PLXNB1 in cell lysates are shown at the bottom. (C) Functionally active p190 was pulled-down (by means of constitutive active RhoQ63L-GST coated beads) from lysates of 3T3 fibroblasts expressing PLXNB1 and treated with 5 nM Sema4D for the indicated times. Immunoblotting with specific antibodies was used to detect p190 in pull-downs and in total lysates (included as loading controls). As measured by band density quantification, the level of activated p190 transiently increases upon plexin activation.