Effect of cancer-associated mutations in the PlexinB1 gene

Abstract

Background: Semaphorins act as chemotactic cues for cell movement via their transmembrane receptors, plexins. Somatic missense mutations in the plexinB1 gene coupled with overexpression of the protein frequently occur in prostate tumours, indicating a role for plexinB1 in the pathogenesis of prostate cancer.

Results: Two specific mutations found in prostate cancer enhance RhoD binding and one other mutation results in loss of inhibition of Rac-dependent Pak1 phosphorylation and lamellipodia formation and in impairment of trafficking of plexinB1 to the membrane. None of the three characterised mutations affect PDZRhoGEF binding, RhoA activity, the interaction of plexinB1 with the oncogenes ErbB2 or c-Met or ErbB2 phosphorylation. The mutations have the net effect of increasing cell motility by blocking plexinB1-mediated inhibition of Rac while enhancing the interaction with RhoD, an anti-migratory factor.

Conclusions: PlexinB1 mutations block plexinB1-mediated signalling pathways that inhibit cell motility.

Figure 1

Mutation of plexinB1 enhances RhoD binding. A).GST-cyto-plexinB1 (WT and mutant) fusion proteins were used in GST pulldown assays with lysates of HEK293 cells expressing constitutively active (G26V) or dominant negative (T31K) RhoD-myc. B). Input of RhoD(G26V)-myc, RhoD(T31K)-myc and WT and mutant GST-cyto-plexinB1 fusion proteins. C). Expression of RhoDGTP decreases cell collapse. COS-7 cells co-transfected with plexinB1, Rnd and RhoD(G26V)-myc, or RhoD(T31K)-myc were stimulated with sema4D for 5mins, fixed and stained for plexinB1 (FITC) and myc (TRITC) and the % cell collapse scored. i).% cell collapse +/- SE, * p < 0.001 versus RhoD(T31K) 2 tailed ttest. ii) representative non-collapsed cell. iii) representative collapsed cell.

Figure 2

The effect of plexinB1 mutations on Rac signalling. A). Mutation of plexinB1 impairs inhibition of Pak1 phosphorylation. The phosphorylation status of Pak1 in COS-7 cells transfected with plexinB1 (WT or mutant), RacL61 and Pak1 was monitored by western blotting using anti- phospho199/204-Pak1 antibody. B). Mutation of plexinB1 impairs inhibition of cell spreading. COS-7 cells transfected with RacL61 and plexinB1 (WT or mutants) were plated on fibronectin for 2 h and those with a large surface area and discernible lamellipodia ('spread' phenotype) scored. Bars represent means +/- SE, *p < 0.05 versus RacL61 + WT, one tail t-test. C). Mutation in plexinB1 inhibits Rac-dependent trafficking of plexinB1 to the cell surface. COS-7 cells co-transfected with plexinB1 (WT or mutant) and RacL61 were treated with Sema4D-AP fusion protein and cell surface binding of Sema4D-AP assessed. i). Sema4D-AP cell surface binding detected with BCIP/NBT solution, representative of 3 independent experiments, ii). Western blot to show expression of VSV-plexinB1 and myc-RacL61 in transfected cells. iii). Bound alkaline phosphatase activity in transfected cells. Bars represent means +/- SE. *p < 0.01 versus vector without RacL61; **p < 0.01 versus WT with RacL61, one tail t-test. D). Mutation of plexinB1 does not affect Sema4D binding. HEK293T cells were cotransfected with VSV-plexinB1 (WT or mutant) and Sema4D-Fc. The plexinB1 protein in the cell lysate was immunoprecipitated with anti-Fc antibody.

Figure 3

Effect of plexinB1 mutation on RhoA activation. A). Mutation of plexinB1 does not affect PDZRhoGEF (PRG) binding. Lysates of HEK293 cells co-expressing VSV-plexinB1 and PDZRhoGEF were immunoprecipitated with anti-VSV and bound proteins detected by western blotting. B). Mutation of plexinB1 does not affect RhoA activation. Lysates of HEK293 cells co-expressing VSV-plexinB1, FLAG-PDZRhoGEF and myc-RhoA were immunoprecipitated with GST-Rhotekin and RhoGTP detected by western blotting with anti-myc. C). COS-7 cells seeded onto coverslips were either transfected with plexinB1 (iii and iv) or vector (i and ii). Fixed cells were stained with anti-plexinB1 (ii and iv) or for actin (i and iii). Panel i and ii, and iii and iv, show the same field. Scale bar = 20 μm. D). The number of cells possessing more than three straight actin bundles of at least 5 μm long was counted and expressed as a percentage of the total number of cells in the field. Bars represent means +/- SE. D). Sema4D in conditioned medium. Conditioned medium was collected from Cos7 cells transfected with vector or Sema4D-Fc and the protein detected by immunoblotting with anti-Sema4D antibody.

Figure 4

Mutation of plexinB1 does not affect interaction of plexinB1 with ErbB2 or c-Met. Lysates of HEK293 cells co-expressing ErbB2(A) or c-Met (B) and VSV-plexinB1 (WT or mutant) were immunoprecipitated with anti-VSV and bound proteins detected with anti-ErbB2 (A) or anti c-Met (B). Representative of 3 independent experiments. C). HEK293 cells transfected with plexinB1(WT or mutant) or vector were treated with control or sema4D conditioned medium or with EGF(500 ng/ml) for 20 mins and the phosphorylation of ErbB2 detected by immunoblotting.