Identification of MAL B1 region sequences involved in nuclear import. (A) B1 region sequence, with deletions in blue and residues K234, K235, and K237 highlighted in red. (B) MALΔN B1 mutations affect nuclear import. Subcellular localization was scored as predominantly cytoplasmic, evenly distributed (c/n), or nuclear. Results are averages of two independent experiments (100 to 200 cells each). (C) Subcellular localization of the MALΔN B1 region deletion derivatives. F-actin (red) and transfected MALΔN (green) are shown. (D) Subcellular localization of MALΔN B1 region derivatives, as in panel C.

MAL and TCF contact the same hydrophobic groove on the SRF DNA-binding domain. (A) Schematic representation of SRF, with the DNA-binding domain in blue. Mutations and secondary structure elements in the C-terminal half of the DBD are shown below, with hydrophobic pocket residues in red. (B) SRF hydrophobic groove mutations affect complex formation. MALΔN (top), MALΔNΔQ (middle) or Elk-1 (bottom) were used in gel mobility shift assays with SRF.DBD (residues 120 to 265), and either ΔTCF SRE probe (top and middle) or WT SRE probe (bottom) (-, no SRF.DBD). (C) Hydrophobic pocket mutations differentially affect the MAL-SRF and Elk-1-SRF interactions.

Effects of the Q-box mutations in the MAL-SRF interaction and the subcellular localization of MAL. (A) Q-box sequence and mutations. Point mutations that affect SRF binding are shown in red. (B) Mutations in the Q-box decrease but do not abolish MAL-SRF complex formation. Gel mobility shift assays were performed using Flag-tagged MALΔN Q-box derivatives, SRF.DBD (residues 133 to 265), and the c-fos ΔTCF SRE probe (-, no MALΔN). Expression levels of the MAL derivatives were confirmed by immunoblotting (IB). αflag, anti-Flag antibody. (C) MAL(met) Q-box mutations affect nuclear import. MAL subcellular localization was scored as described in the legend to Fig. 2. (D) Q-box mutations do not affect SRF reporter activation.

Mutations in the SRF.DBD αI-helix inhibit MAL-SRF complex formation through their effect on DNA bending. (A) Secondary structure elements and mutations in the N-terminal half of the SRF DBD. Critical αI-helix residues are highlighted in red. N-ext., N-extension. (B) SRF αI-helix mutations disrupt MAL-SRF complex formation. Extracts from cells expressing MALΔN (top), MALΔNΔQ (middle), or Elk-1 (bottom) were used in gel mobility shift assays with the indicated SRF.DBD derivatives (residues 120 to 265) and either ΔTCF SRE probe (top and middle) or WT SRE probe (bottom) (-, no SRF.DBD). (C) Circular permutation analysis. (Top) Representative SRF mobility plots versus distance of the center of the SRF binding site from the probe end (for probes, see Materials and Methods). (Bottom) Estimated apparent bend angles for the different derivatives (two independent experiments).

Optimal MAL-SRF interaction requires DNA binding and distortion. (A) The SRF αI-helix mutation does not affect DNA bending in the MAL-SRF-DNA complex. Circular permutation assays were performed using wild-type SRF.DBD or its αI-helix derivative (residues 120 to 265) and either GST.MAL(214-298) or transiently expressed Elk-1 as indicated. The apparent bend angles for the ternary complexes are indicated above each panel. Note that MAL complex formation is specifically impaired on probes in which the SRF binding site is centered 16 base pairs from the fragment end (see Fig. 8). (B) Cognate DNA enhances MAL-SRF complex formation. Extracts containing HA-tagged MALΔN were incubated with myc-tagged SRF.DBD (residues 120 to 265) from wild-type SRF or SRF-M2 in the presence of either the wild-type c-fos SRE or its derivative SRE.M (sequences shown below). Following immunoprecipitation (IP) with anti-myc beads, MAL was detected by immunoblotting (IB) with anti-HA. (-, no SRF).

MAL and SRF residues involved in complex formation. (A) Alignment of the sequences of B1 and Q regions of mouse MAL, myocardin, and MAL16, with identities and conservative replacements indicated by asterisks, colons, and dots above the sequence. Conservation of the critical seven-residue MAL sequence (red) and Q-box-related sequence (gray) in bee (A.Mel) and fruit fly MAL (DMAL) is shown. (B) The MAL-binding surface of SRF. The SAP-1-SRF ternary complex (8) is shown: blue, SRF; green, DNA; yellow, SAP-1 B-box. (Left) Ribbon model, with secondary structure elements indicated. (Center) SRF and SAP-1 are shown in van der Waals and ribbon representations, respectively. SRF residues implicated in MAL interaction are shown in red (hydrophobic pocket), orange (β-strand residues also contacting DNA), and pink (critical αI-helix residues). (Right) Same as center but with DNA shown as van der Waals representation and SAP-1 aromatic side chains Y147 and F150 in backbone representation. Images were produced using Deep View/Swiss-Pdb Viewer version 3.7. (C) Potential sequence relationships between the critical MAL B1 region sequence and the β-strand segment of the SAP-1 B-box. For discussion, see text.

The B1 region of MAL is necessary and sufficient for SRF interaction. (A) Peptide competition studies. (Left) MAL peptide sequences. (Right) Competition assays. (Top) Binding reaction mixtures contained MALΔN extract, SRF.DBD (residues 133 to 265), and c-fos wild-type SRE with 4 μM or 20 μM MAL B1 peptide as indicated (-, no MAL B1 peptide). (Bottom) Peptide titrations using 1.3, 3.8, 11.3, and 34 μM peptide. Only the SRF.DBD-MAL complex band is shown. (B) Complex formation between MAL B1 and B1Q peptides and SRF.DBD (residues 120 to 265). The sequences of B1Q peptides are shown at the top. Binding reactions with SRF.DBD and c-fos ΔTCF probe with B1 peptides (0.16, 0.32, 0.63, 1.25, 2.5, and 5 μM) or B1Q peptides (wild-type or 3×A peptide, 0.014, 0.04, 0.12, 0.37, 1.11, and 3.33 μM; Y238A peptide, 0.37, 1.11, and 3.33 μM) are shown. (C) Complex formation between purified GST.MAL(214-298) and recombinant SRF.DBD (residues 132 to 223). (D) The B1 region mediates protein-protein interaction with SRF. Mutations in GST.MAL(214-298) have similar effects on complex formation with SRF.DBD (residues 120 to 265) in mobility shift assays (left) and in pulldown assays (right). IB, immunoblotting.

Identification of MAL B1 region residues critical for SRF binding. (A) Conserved motifs in MAL are shown as bars, with RPEL motifs in red. Amino acids are numbered, with the first in-frame methionine (MALmet isoform) as +1; intact NIH 3T3 MAL initiates translation at Leu-92 (23). The MALΔN truncation to position +81 is indicated. The B1 region is shown in blue, and its sequence and deletion mutations are shown. The seven-residue critical sequence identified by the alanine scan is highlighted in red. (B) Deletion and alanine-scanning mutations in the MAL B1 region affect complex formation. Gel mobility shift assays contained Flag-tagged MALΔN B1 region derivatives, SRF.DBD (residues 133 to 265), and the c-fos ΔTCF SRE probe (-, no MALΔN). At the bottom, quantitation of MALΔN expression by immunoblotting (IB) is shown. αflag, anti-Flag antibody. (C) Analysis of interactions by residues K237, Y238, H239, and Y241. Gel mobility shift assays and immunoblotting as in panel B. (D) Activation of the SRF-dependent reporter 3DA.luc by B1 region derivatives. Deletion and alanine scan derivatives (left) and other MALΔN derivatives (right) are shown. (E) Titration of MALΔN derivatives. Reporter assays as in panel D with 6 ng, 18 ng, 55 ng, 166 ng, and 500 ng MALΔN plasmid inputs are shown. (F) Activation of endogenous sm α-actin and egr-1 expression by MALΔN derivatives. Gene expression was scored by immunofluorescence in 100 to 200 transfected cells per experiment (two independent experiments). FBS, fetal bovine serum. (G) Potentiation of cytochalasin D-induced SRF reporter activity by intact MAL requires integrity of the B1 region. Reporter assays were as in panel D. Cells were treated with 0.5 μM cytochalasin D (CD) as indicated.

MAL makes direct DNA contacts that are required for complex formation. (A) DNase I footprinting of the MAL-SRF complex. Reaction mixtures contained c-fos DNA, SRF.DBD (residues 132 to 223) (+) as indicated, and increasing amounts (0.8, 2.5, 7.6, and 22.8 ng) of WT or H239A GST.MAL(214-298) proteins. The probe DNA sequences are aligned with the AG marker ladders (line, SRE; open box, SRF core consensus binding site). (B) Complex formation between recombinant SRF.DBD (residues 132 to 223) and transiently expressed MALΔN (top) or MALΔNΔQ (bottom) on nested DNA probes with ends at the indicated positions relative to the SRE dyad (see panel C). Only the MAL-SRF-DNA complex is shown. Similar results were obtained with GST.MAL (214-298) (data not shown). (C) Summary of the DNase I footprinting data. The c-fos SRE region is shown, with black dots representing phosphodiester bonds where DNase I cleavage was readily detectable on naked DNA. For simplicity, the footprint with SRF alone is indicated by the solid lines. The colored circles and arrowheads show protection from and enhancement of DNase I cleavage, respectively. Red symbols, GST.MAL(214-298); blue symbols, peptide J. The red arrowhead within a gray bar indicates where a sequencing compression precludes conclusive interpretation of top strand changes at the 3′ side of the SRE. Within the SRE region, changes were visible only after prolonged exposure of the autoradiogram. The start positions and direction of the nested band shift probes used in panel B are shown at the bottom. (D) Footprint analysis of MAL B1 region peptides (Fig. 4A), presented as in panel A. Reaction mixtures contained 7.6 ng GST.MAL(214-298), 1.7 μM peptide B1Q, 8.5 μM peptides A and M, and 85 μM peptide J.