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Fig. 9. Profilin modulates p42^POP transcriptional activity, mediated by its polyproline binding site. (A) Extracts of E. coli expressing wild-type profilin (PFN1) or a profilin variant with a single point mutation in the polyproline-binding site (PFN1-S138D) were incubated with polyproline-Sepharose. Sedimented material was subjected to SDS-PAGE and immunoblotting with the monoclonal anti-profilin antibody. Although both proteins are present in the bacterial lysates (extract), only PFN1 is precipitated by polyproline-Sepharose (PLP), demonstrating that PFN138D is defective in polyproline binding. (B) Luciferase assays with HeLa cells transfected with pGL3-mreA-Luc, BiPro-p42^POP and either cDNAs encoding FLAG-tagged profilin or the empty reporter plasmid (control). Analogous to the results shown in Fig. 8B, the presence of p42^POP (+) consistently induced a strong repression of the basic transcriptional activity (left, -). When profilin was expressed with p42^POP, this repression was markedly reduced (centre). However, when the profilin mutant PFN1-S138D was present, no such rescue effect was seen (right). (C) Immunoblots with antibodies against the BiPro or FLAG tag obtained from HeLa-cell extracts used in B. Comparable protein expression is seen for all three analyses. Hence, profilin markedly alters the transcriptional activity of p42^POP and this effect depends strongly on an intact polyproline-binding site.

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