The Integral Inner Nuclear Membrane Protein MAN1 Physically Interacts with the R-Smad Proteins to Repress Signaling by the Transforming Growth Factor-β Superfamily of Cytokines

Abstract

Smad proteins are critical intracellular mediators of the transforming growth factor-␤, bone morphogenic proteins (BMPs), and activin signaling. Upon ligand binding, the receptor-associated R-Smads are phosphorylated by the active type I receptor serine/threonine kinases. The phosphorylated R-Smads then form heteromeric complexes with Smad4, translocate into the nucleus, and interact with various transcription factors to regulate the expression of downstream genes. Interaction of Smad proteins with cellular partners in the cytoplasm and nucleus is a critical mechanism by which the activities and expression of the Smad proteins are modulated. Here we report a novel step of regulation of the R-Smad function at the inner nuclear membrane through a physical interaction between the integral inner nuclear membrane protein MAN1 and R-Smads. MAN1, through the RNA recognition motif, associates with R-Smads but not Smad4 at the inner nuclear membrane in a ligand-independent manner. Overexpression of MAN1 results in inhibition of R-Smad phosphorylation, heterodimerization with Smad4 and nuclear translocation, and repression of transcriptional activation of the TGF␤, BMP2, and activin-responsive promoters. This repression of TGF␤, BMP2, and activin signaling is dependent on the MAN1-Smad interaction because a point mutation that disrupts this interaction abolishes the transcriptional repression by MAN1. Thus, MAN1 represents a new class of R-Smad regulators and defines a previously unrecognized regulatory step at the nuclear periphery.The transforming growth factor-␤ (TGF␤) 1 superfamily of cytokines, including TGF␤s, BMPs, and activins, plays important roles in the regulation of various aspects of mammalian embryogenesis and carcinogenesis. The signals initiated by these cytokines are transduced by their receptors and the downstream Smad proteins (1-5). Upon ligand binding, activin or TGF␤ receptor serine/threonine kinase phosphorylates and activates the type I receptor kinase, which then phosphorylates the downstream Smad proteins. The Smad proteins are critical mediators of TGF␤ superfamily signaling. Upon phosphorylation by the activated type I receptor kinases, the receptorassociated R-Smads (Smad2 and Smad3 for TGF␤ and activin; Smad1, Smad5, and Smad8 for BMPs) oligomerize with the common mediator Smad4, translocate into the nucleus where they interact with various transcription factors, bind to DNA, and regulate transcription of downstream genes.The expression and activity of the Smad proteins can be modulated by interaction with various cellular proteins at the plasma membrane or in the cytoplasm and nucleus (2, 6, 7). For example, Smad proteins can interact with various transcriptional co-activators on promoter DNAs to regulate the activation of TGF␤, activin, or BMP target genes (2, 6). The activity or intracellular localization of the Smads can be modulated through binding to adaptors molecules such as SARA (8), Hgs (9, 10), chaperones (11), microtubules (12), or various co-repressors such as Ski, SnoN, SNIP,. O...

Tissue-Specific Defects Are Caused by Loss of the Drosophila MAN1 LEM Domain Protein

“…LEM-D proteins interact with transcriptional repressors, such as germ cell-less and Bcl-2-associated transcription factor (Haraguchi et al 2004;Mansharamani and Wilson 2005). In addition, interactions with the downstream transcriptional effectors of multiple signal transduction pathways have been observed, such as Smads [receptor-regulated (R)-Smads], retinoblastoma protein, and b-catenin (Markiewicz et al 2002(Markiewicz et al , 2006Osada et al 2003;Raju et al 2003;Lin et al 2005;Pan et al 2005;Jiang et al 2008). These latter observations suggest that LEM-D proteins have a role in the regulation of gene expression.…”

Proteins that associate with lamins: Many faces, many functions

“…Mammalian MAN1 binds Receptor-Smads 2/3, downstream effectors of the TGFβ and bone morphogenic protein (BMP) signaling cascades [96]. MAN1 overexpression impaired Smad 2/3 phosphorylation, heterodimerization (with Smad 4), and nuclear translocation -events that are linked to an active pathway -and thus antagonizes signaling [97]. Given the ubiquitous expression of MAN1, the physiological relevance of MAN1 in TGFβ and …”

Unmasking the U2AF homology motif family: a bona fide protein–protein interaction motif in disguise

“…1): the inner nuclear membrane protein MAN1 (also called LEMD3) (Konde et al 2010) and the neurodegenerative disease-related Ataxin-1 (ATX1) (de Chiara et al 2009). An intramolecular ULM-UHM interaction within MAN1 is thought to regulate its association with SMAD2 (Konde et al 2010), which in turn represses TGF-β signaling for bone development (Hellemans et al 2004;Lin et al 2005;Pan et al 2005). A ULM sequence near the C terminus of ATX1 binds the UHMs of U2AF 65 (Lim et al 2006;de Chiara et al 2009) or SPF45 (Lim et al 2008;de Chiara et al 2009).…”