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First published online 22 December 2004
doi: 10.1242/jcs.01618

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Profilin regulates the activity of p42^POP, a novel Myb-related transcription factor

Cell Biology, Zoological Institute, Technical University of Braunschweig, 38092 Braunschweig, Germany

View larger version (46K): [in a new window]   Fig. 1. Deduced amino acid sequence and molecular anatomy of p42POP. The various motifs identified by the program PSORTII are indicated. The sequence of POP1, identified in the yeast two-hybrid analysis as a profilin ligand, is underlined.

View larger version (57K): [in a new window]   Fig. 2. p42POP is a widely produced nuclear protein with functional NLS and NES. (A) Expression pattern of p42POP obtained by quantitative PCR and subsequent Southern-blot analysis with standardized samples. p42POP is produced in all adult mouse tissues tested, with high expression levels in brain and testis, and is already present at embryonic stage E7. (B,C) Subcellular localization of BiPro-tagged p42POP (B) and POP6 (C) in PtK2 cells transfected with the appropriate constructs, as revealed by immunofluorescence with anti-BiPro antibody. (D) Compiled data for all constructs tested in analogous assays, using either BiPro- or GFP-tagged probes. Both sets of experiments gave identical results. Whereas p42POP was seen exclusively in the nucleus, deletion of the C-terminal NLS resulted in an exclusively cytoplasmic location of the appropriate fragments POP6 and POP9. Bar, 10 µm.

View larger version (28K): [in a new window]   Fig. 3. The proline-rich domain of p42POP mediates profilin binding. (A) Yeast two-hybrid analysis with profilins 1 and 2a (PFN1/2a) as a bait and different p42POP fragments as prey. Only fragments containing the proline clusters gave a positive reaction (+). (B) Autoradiograms obtained after SDS-PAGE of pellets (P) and supernatants (S) derived from pull-down assays with profilin-1-coupled Sepharose (PFN1) or BSA-coupled Sepharose (control). Both types of Sepharose bead were incubated with in-vitro-translated, radioactively labelled full-length p42POP or the N-terminal (N-POP) or C-terminal (C-POP) fragment. Only C-POP was precipitated by PFN1 beads.

View larger version (36K): [in a new window]   Fig. 4. p42POP can dimerize via the leucine zipper but profilin binding is restricted to the monomer. (A) Yeast two-hybrid analysis with full-length p42POP as a bait and various p42POP fragments as prey. Only fragments containing the leucine-zipper motif give a positive reaction (+), indicating dimerization. (B) Immunoblots derived from p42POP pull-down assays. Crude extracts of E. coli, transformed with plasmids coding for GFP-tagged POP10, POP12 or POP13, respectively, displayed the respective fragments in immunoblots with a GFP antibody (e). They were then incubated with glutathione-Sepharose, in the presence (+) or absence (-) of extracts expressing the GST-tagged POP14 or GST only (c, control). After centrifugation, pellets were subjected to SDS-PAGE and immunoblotting with the anti-GFP antibody. Only POP10 and POP12, both harbouring the leucine zipper, were precipitated. (C) Liquid yeast two-hybrid assays to analyse the importance of an intact leucine zipper for p42POP dimerization. Full-length p42POP was incubated with samples containing C-POP and C-POP variants containing single or double point mutations within the leucine zipper, as indicated. ß-Galactosidase activity was monitored for quantitation of the interaction. Bars indicate the standard deviations of four independent experiments. Replacement of two leucines within the leucine zipper with either alanine or proline abolishes dimerization. (D) Dimerization of FLAG/C-POP and failure of the mutant FLAG/C-POP-LLPP to dimerize as demonstrated by chemical cross-linking. The recombinant fragments were exposed to the cross-linker EDC/NHS followed by SDS-PAGE and immunoblotting with an anti-FLAG antibody. Whereas C-POP is revealed as a monomer (25 kDa) and a dimer (50 kDa), POP-LLPP cannot form dimers. (E) Reactivity of profilin 1 with monomeric or dimeric POP variants. Profilin 1 was incubated with the cross-linker EDC/NHS in the absence (lane a) or the presence of either FLAG/C-POP (lanes b,b') or FLAG/C-POP-LLPP (lanes c,c'). Samples were subjected to SDS-PAGE and immunoblotting with monoclonal antibodies against profilin 1 (a-PFN1) or FLAG (a-FLAG), respectively. Free profilin is seen at a position corresponding to 14 kDa. In the samples containing C-POP (b) or C-POP-LLPP (c), it is also present in the band at 39 kDa, which corresponds to the heterodimer of profilin with either C-POP or C-POP-LLPP (arrow). The immunoblot with the FLAG antibody reveals that only C-POP, but not C-POP-LLPP, has formed dimers (at 50 kDa, b',c', arrowhead) but profilin is not found complexed to C-POP dimers (b,b'). Hence, profilin binds only to the monomeric p42POP.

View larger version (74K): [in a new window]   Fig. 5. p42POP is a Myb-like transcription factor. (A) Comparison of the p42POP N-terminus with DBDs of Myb-related proteins. Bold characters indicate conserved residues indicative of the Myb repeat consensus. Sequences are from mouse (MM c-Myb: AAB59713, human (HS A-Myb: CAA57552, potato (MybSt1: AAB32591, Arabidopsis (AT MybL2: S71283), maize (ZM MYB1: S04898; ZM MYB38: S04899), barley (HV MYB1: P20026) and Drosophila (Zeste: P09956). (B) DNA-binding ability of p42POP as revealed by EMSA. In-vitro-translated p42POP was seen to bind to double-stranded [32P]-labelled mreA oligodeoxynucleotides [left, without (-) and with (+) p42POP]. (centre) Competition of complex formation by an identical unlabelled probe. (right) Control with a random DNA sequence. (C) The N-terminal fragment of p42POP (N-POP) is sufficient for complex formation with mreA. Purified recombinant N-POP and the mutants N-POP-S25D and N-POP-R83E/W84P were incubated with the mreA sequence as described in (B). Whereas N-POP displays the electrophoretic shift indicating binding, single and double point mutations within the Myb-like repeat (asterisks in A) abolish this interaction. Arrows indicate the positions of the complexes.

View larger version (58K): [in a new window]   Fig. 6. POP6 and endogenous profilin localize to cytoplasmic membranes in PtK2 cells in a mitochondrial targeting assay. Cells were transiently transfected with vectors coding for a fusion protein containing a mitochondrial outer membrane fragment (MOM) and either BiPro-POP6 or BiPro-E-cadherin (control). Staining was performed with MitoTrackerTM, a monoclonal antibody against the BiPro tag and a polyclonal anti-profilin antibody. Double-fluorescence images show that both MOM fusion proteins target to mitochondrial membranes (A,A',C,C') but only POP6 recruits endogenous profilin to this position (B,B'), whereas E-cadherin fails to do so (D,D'). Bar, 10 µm.

View larger version (51K): [in a new window]   Fig. 7. p42POP localizes with profilin 1 and recruits profilin 1 into the nucleus. Fluorescence images obtained as optical sections by confocal laser-scanning microscopy revealed that GFP-p42POP (A) and profilin 1, seen by immunostaining with a monoclonal antibody (A'), are both localized to the nucleus, in a fine granular pattern with some overlap (A''). Bar, 10 µm. (B) Quantitation of nuclear profilin (PFN1) in PtK2 cells transiently transfected with GFP-fused p42POP, POP4 or POP6. The bars display the relative fluorescences of the profilin-specific signals obtained for cells transfected with p42POP, POP4 or POP6 compared with values obtained with untransfected cells on the same coverslip (defined as 1.0). Values were obtained for 150 cells and are shown with standard errors compared by variance analysis (Student-Newman-Keuls; P<0.05). Expression of p42POP raised the nuclear profilin level by 17% in comparison with untransfected cells, whereas neither POP4 (which lacks the proline clusters) nor POP6 (which does not target to the nucleus) (cf. Fig. 2) showed this increase in nuclear profilin.

View larger version (26K): [in a new window]   Fig. 8. p42POP modulates transcriptional activity. HeLa cells were transfected with the reporter plasmid pGL3-mreA-Luc and a cDNA encoding c-Myb, BiPro-p42POP or BiPro-POP4. (A) Immunoblot of cell extracts with the anti-BiPro antibody after transfection reveals comparable expression of p42POP and the POP4 fragment. (B) Luciferase activity in extracts of cells transfected with the reporter plasmid only (control) or with the reporter plus c-Myb, BiPro-p42POP or BiPro-POP4. Whereas c-Myb acts as a transcriptional activator in this assay, p42POP causes a decrease in transcription to less than 15% of the control value. By contrast, POP4, lacking the C-terminal modules, induces a threefold increase in transcription.

View larger version (29K): [in a new window]   Fig. 9. Profilin modulates p42POP 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-p42POP and either cDNAs encoding FLAG-tagged profilin or the empty reporter plasmid (control). Analogous to the results shown in Fig. 8B, the presence of p42POP (+) consistently induced a strong repression of the basic transcriptional activity (left, -). When profilin was expressed with p42POP, 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 p42POP and this effect depends strongly on an intact polyproline-binding site.

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