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. 2017 Nov 2;7:14937. doi: 10.1038/s41598-017-14814-x

Figure 4.

Figure 4

TFIIA cleavage by Taspase1 regulates its subcellular localization and transcriptional activity. (A,B) A431 cells were transfected with indicated TFIIA-GFP variants together with the active Taspase1- (A) or inactive TaspaseT243V-mCherry (B) expression plasmid. Localization was analysed by fluorescence microscopy after 24 h. TFIIA-GFP is translocated from cytoplasm to nucleus after expression of active Taspase1 but not of inactive Taspase1T234V mutant. In contrast the cleavage-deficient mutant TFIIACSmut-GFP did not relocalize in presence of Taspase1. The export-deficient TFIIA variants did not alter their subcellular localization upon Taspase1 expression. Scale bars, 10 µm. (C) Proteolytic cleavage of TFIIA-GFP variants as shown by immunoblot analysis of whole-cell lysates. A431 cells were transfected with indicated TFIIA-GFP together the active Taspase1- (wt) or inactive Taspase1(T234V)- BFP expression plasmids. In contrast to TFIIA- and TFIIANESmut-GFP, the TFIIACSmut-GFP could not be cleaved by active wildtype (wt) Taspase1. The inactive Taspase1T234V mutant neither showed self-cleavage in two active subunits nor trans cleavage activity of TFIIA. Expression of proteins and cleavage products in cell lysates was visualized using α-GFP and α-Taspase1 Abs. α-GapDH served as loading control. *Uncleaved TFIIA-GFP, **cleaved TFIIA-GFP, fl: Taspase1-BFP full length protein, α/β: Taspase1 α-/β-subunit. Of note, the α-GFP antibody is as well detecting the related BFP and thus full-length Tasp-BFP and the β-subunit containing the fusion tag. α-Taspase1 Ab is recognizing the full-length (75 kDa) as well as the α-subunit (28 kDa) of Taspase1. Shown blots are cropped. Full-length blots are shown in Supplementary Fig. S6. (D) Expression of the cell cycle regulator p16INK4a is induced by impaired TFIIA export and proteolytic cleavage. A431 cells were co-transfected with either pGL3 basic or pGL3 basic-wt p16INK4a 5′-UTR reporter, pRLSV40 and the respective TFIIA variant or pc3-GFP as negative control. Relative light units (RLU) were measured 48 h later, and plotted after normalization for transfection efficiency (pRLSV40). Bars, means of triplicates used to calculate standard deviations. Results of one representative experiment are shown, n = 3. Significance, *p < 0.005.