Molecular basis for G-actin binding to RPEL motifs from the serum response factor coactivator MAL

SummaryPhactr proteins bind actin and protein phosphatase 1 (PP1), and are involved in processes ranging from angiogenesis to cell cycle regulation. Phactrs share a highly conserved RPEL domain with the myocardin-related transcription factor (MRTF) family, where actin binding to this domain regulates both the nuclear localization and the activity of these transcription coactivators. We show here that in contrast to MRTF-A, the RPEL domain is dispensable for the subcellular localization of Phactr4. Instead, we find the domain facilitating competitive binding of monomeric actin and PP1 to Phactr4. Binding of actin to Phactr4 influences the activity of PP1 and the phosphorylation status of one of its downstream targets, cofilin. Consequently, at low actin monomer levels, Phactr4 guides PP1 to dephosphorylate cofilin. This active form of cofilin is then able to sever and depolymerize actin filaments and thus restore the actin monomer pool. Accordingly, our data discloses the central role of Phactr4 in a feedback loop, where actin monomers regulate their own number via the activation of a key regulator of actin dynamics. Depending on the protein context, the RPEL domain can thus elicit mechanistically different responses to maintain the cellular actin balance.

Section: Discussionmentioning

confidence: 99%

Actin-regulated feedback loop based on Phactr4, PP1 and cofilin maintains the actin monomer pool

Huet

Rajakylä

Viita

et al. 2013

“…Upon dissociation from G-actin, MAL/MRTF-A upregulates a Rhofamily dependent, but MAPK-independent, subset of SRF target genes (Gineitis and Treisman, 2001;Vartiainen et al, 2007). These genes and their promoters respond differentially to actin binding drugs: treatment with cytochalasin D activates transcription by releasing MAL from G-actin, whereas latrunculin B stabilises the G-actin-MAL complex and inhibits gene expression (Posern et al, 2002;Posern et al, 2004;Vartiainen et al, 2007;Mouilleron et al, 2008).…”

Section: Introductionmentioning

confidence: 99%

“…Serum-induced gene expression is directly controlled by monomeric G-actin, which forms a repressive complex with MRTFs (Posern et al, 2002;Miralles et al, 2003;Posern et al, 2004;Vartiainen et al, 2007;Mouilleron et al, 2008). Upon dissociation from G-actin, MAL/MRTF-A upregulates a Rhofamily dependent, but MAPK-independent, subset of SRF target genes (Gineitis and Treisman, 2001;Vartiainen et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

MAL/MRTF-A controls migration of non-invasive cells by upregulation of cytoskeleton-associated proteins

Leitner

Shaposhnikov

Mengel

et al. 2011

SummaryMonomeric actin regulates gene expression through serum response factor (SRF) by inhibiting its transcriptional coactivator myocardinrelated transcription factor (MAL/MRTF). Many affected genes encode cytoskeletal components. We have analysed the migratory effects of actin-MAL signalling and of new target genes in non-invasive highly adherent cells. Expression of active MAL impaired migration of both fibroblasts and epithelial cells, whereas dominant-negative constructs and partial knockdown of MAL/MRTF enhanced motility. Knockdown of three newly characterised G-actin-regulated MAL targets, integrin a5, plakophilin 2 (Pkp2) and FHL1, enhanced cell migration. All three were upregulated by external stimulation through actin-MAL-SRF signalling, and MAL and SRF were inducibly recruited to cis-regulatory elements of the integrin a5 and Pkp2 genes. Finally, the reduced migration of epithelial cells stably expressing MAL was partially reversed by knockdown of Pkp2 and FHL1. We conclude that the actin-MAL pathway promotes adhesive gene expression, including integrin a5, Pkp2 and FHL1, and that this is anti-motile for non-invasive cells harbouring high basal activity.

“…Perhaps the best understood of these functions is the role of G-actin in control of nucleocytoplasmic shuttling and transcriptional activation by the MRTF family of SRF transcriptional coactivators (Miralles et al, 2003;Vartiainen et al, 2007). Signal-induced changes in the availability of Gactin regulate the assembly of different G-actin complexes on the MRTF regulatory domain, which contains three G-actin binding RPEL motifs (Mouilleron et al, 2008;Hirano and Matsuura, 2011;Mouilleron et al, 2011).…”

Section: Introductionmentioning

confidence: 99%

G-actin regulates the shuttling and PP1 binding of the RPEL protein Phactr1 to control actomyosin assembly

Wiezlak

Diring²,

Abella

et al. 2012

Self Cite

111

SummaryThe Phactr family of PP1-binding proteins is implicated in human diseases including Parkinson's, cancer and myocardial infarction. Each Phactr protein contains four G-actin binding RPEL motifs, including an N-terminal motif, abutting a basic element, and a Cterminal triple RPEL repeat, which overlaps a conserved C-terminus required for interaction with PP1. RPEL motifs are also found in the regulatory domains of the MRTF transcriptional coactivators, where they control MRTF subcellular localisation and activity by sensing signal-induced changes in G-actin concentration. However, whether G-actin binding controls Phactr protein function -and its relation to signalling -has not been investigated. Here, we show that Rho-actin signalling induced by serum stimulation promotes the nuclear accumulation of Phactr1, but not other Phactr family members. Actin binding by the three Phactr1 C-terminal RPEL motifs is required for Phactr1 cytoplasmic localisation in resting cells. Phactr1 nuclear accumulation is importin a-b dependent. G-actin and importin a-b bind competitively to nuclear import signals associated with the N-and C-terminal RPEL motifs. All four motifs are required for the inhibition of serum-induced Phactr1 nuclear accumulation when G-actin is elevated. G-actin and PP1 bind competitively to the Phactr1 C-terminal region, and Phactr1 C-terminal RPEL mutants that cannot bind G-actin induce aberrant actomyosin structures dependent on their nuclear accumulation and on PP1 binding. In CHL-1 melanoma cells, Phactr1 exhibits actin-regulated subcellular localisation and is required for stress fibre assembly, motility and invasiveness. These data support a role for Phactr1 in actomyosin assembly and suggest that Phactr1 G-actin sensing allows its coordination with F-actin availability.