RPEL Motifs Link the Serum Response Factor Cofactor MAL but Not Myocardin to Rho Signaling via Actin Binding

Abstract

Myocardin (MC) family proteins are transcriptional coactivators for serum response factor (SRF). Each family member possesses a conserved N-terminal region containing three RPEL motifs (the “RPEL domain”). MAL/MKL1/myocardin-related transcription factor A is cytoplasmic, accumulating in the nucleus upon activation of Rho GTPase signaling, which alters interactions between G-actin and the RPEL domain. We demonstrate that MC, which is nuclear, does not shuttle through the cytoplasm and that the contrasting nucleocytoplasmic shuttling properties of MAL and MC are defined by their RPEL domains. We show that the MAL RPEL domain binds actin more avidly than that of MC and that the RPEL motif itself is an actin-binding element. RPEL1 and RPEL2 of MC bind actin weakly compared with those of MAL, while RPEL3 is of comparable and low affinity in the two proteins. Actin binding by all three motifs is required for MAL regulation. The differing behaviors of MAL and MC are specified by the RPEL1-RPEL2 unit, while RPEL3 can be exchanged between them. We propose that differential actin occupancy of multiple RPEL motifs regulates nucleocytoplasmic transport and activity of MAL.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://mcb.asm.org/ .

ACKNOWLEDGMENTS

We thank Nicola O'Reilly and the peptide synthesis lab for outstanding peptide synthesis, Maureen Biggerstaff and Rachel Coulthard for assistance with fluorescence anisotropy measurements and data analysis, Stéphane Mouilleron for helpful discussions, and Rob Nicolas for continuous support. We thank colleagues of the lab and Caroline Hill, Michael Way, Neil McDonald, and laboratory members for technical help, discussions, and comments on the manuscript. We thank the London Research Institute Light Microscopy Facility for assistance with life cell imaging.

This work was funded by Cancer Research UK. M.K.V. is supported by an EMBO long-term fellowship, and S.G., a fellow of the Studienstiftung des deutschen Volkes, by a Boehringer Ingelheim Fonds predoctoral scholarship.

The authors have no conflicting financial interests.