Fig. 2: Alanine substitution of arginine 310 or treatment with furin inhibitors abrogates Activin-A hemicleavage by kallikrein 8. | Nature Communications

Fig. 2: Alanine substitution of arginine 310 or treatment with furin inhibitors abrogates Activin-A hemicleavage by kallikrein 8.

From: Kallikrein-8 mediates furin-independent Activin-A precursor processing to stimulate tumor growth in melanoma

Fig. 2

A Wild-type (WT) or R310A mutant proActivin-A (A110) analyzed (i) before or (ii and iii) after incubation with recombinant furin (rFurin) or rKLK8 for 5 hrs at pH 7. Where indicated, these SNs were first transiently acidified to pH 2 for 60 min. Results are representative of three experiments. B Effect of increasing concentrations of dec-RVKR-cmk (CMK) or D6R on the in vitro cleavage of Boc-VPR-AMC or Boc-RRVR-AMC by rKLK8 or rFurin, respectively. Data represent the mean of two experiments. C WT A110 treated as in (A), but with or without CMK or D6R. Data represent two experiments. D Sequence comparisons of the Activin-A precursor cleavage site with the KLK8 consensus motif in the MEROPS database. The KLK8 substrate specificity determined by proteomic identification of cleavage sites (PICS) or by a positional-scanning screening of a synthetic diverse tetrapeptide library (PSSCL) is shown below22. E Activin-A Western blot of SNs from B16-F1, FurKO#1 and DKO cells cultured for 48 hrs after transfection with WT or R310A mutant βA. Data represent three experiments. F Induction of CAGA-Luc in HepG2 reporter cells within 12 hrs after adding the SNs from the samples analyzed in (E). Where indicated, the HepG2 reporter cells were treated with 100 µM CMK. Data represent the means ± SD of four to five experiments (One-way ANOVA). G Activin-A Western blot of SNs from B16-F1 and FurKO#1 cells cultured for 48 hrs after transfection with WT-βA, R310A-βA, ΔR310, or RALE-βA. Asterisk, size-shifted RALE-mutant A30. Data represent three experiments. H Induction of CAGA-Luc in HepG2 reporter cells within 12 hrs after adding the SNs analyzed in (G). Where indicated, the HepG2 reporter cells were treated with 100 µM CMK. Data represent the means of two experiments. I Activin-A Western blot of wild-type or RALE mutant pro-Activin-A (A110) containing SNs that were transiently acidified to pH 2 for 60 min, followed by incubation for 5 hrs with rFurin or rKLK8. Results are representative of two experiments. *p < 0.05.

Back to article page