Competition between type I activin and BMP receptors for binding to ACVR2A regulates signaling to distinct Smad pathways.

BACKGROUND: Activins and bone morphogenetic proteins (BMPs) play critical, sometimes opposing roles, in multiple physiological and pathological processes and diseases. They signal to distinct Smad branches; activins signal mainly to Smad2/3, while BMPs activate mainly Smad1/5/8. This gives rise to the possibility that competition between the different type I receptors through which activin and BMP signal for common type II receptors can provide a mechanism for fine-tuning the cellular response to activin/BMP stimuli. Among the transforming growth factor-ß superfamily type II receptors, ACVR2A/B are highly promiscuous, due to their ability to interact with different type I receptors (e.g., ALK4 vs. ALK2/3/6) and with their respective ligands [activin A (ActA) vs. BMP9/2]. However, studies on complex formation between these full-length receptors situated at the plasma membrane, and especially on the potential competition between the different activin and BMP type I receptors for a common activin type II receptor, were lacking. RESULTS: We employed a combination of IgG-mediated patching-immobilization of several type I receptors in the absence or presence of ligands with fluorescence recovery after photobleaching (FRAP) measurements on the lateral diffusion of an activin type II receptor, ACVR2A, to demonstrate the principle of competition between type I receptors for ACVR2. Our results show that ACVR2A can form stable heteromeric complexes with ALK4 (an activin type I receptor), as well as with several BMP type I receptors (ALK2/3/6). Of note, ALK4 and the BMP type I receptors competed for binding ACVR2A. To assess the implications of this competition for signaling output, we first validated that in our cell model system (U2OS cells), ACVR2/ALK4 transduce ActA signaling to Smad2/3, while BMP9 signaling to Smad1/5/8 employ ACVR2/ALK2 or ACVR2/ALK3. By combining ligand stimulation with overexpression of a competing type I receptor, we showed that differential complex formation of distinct type I receptors with a common type II receptor balances the signaling to the two Smad branches. CONCLUSIONS: Different type I receptors that signal to distinct Smad pathways (Smad2/3 vs. Smad1/5/8) compete for binding to common activin type II receptors. This provides a novel mechanism to balance signaling between Smad2/3 and Smad1/5/8.

Autophagy is induced and modulated by cholesterol depletion through transcription of autophagy-related genes and attenuation of flux.

Perturbations to cellular homeostasis, including reduction of the cholesterol level, induce autophagy, a self-digestion process of cellular constituents through an autophagosomal-lysosomal pathway. In accord with its function as a membrane organizer and metabolic sentinel, the cellular response to cholesterol depletion comprises multiple phenomena, including the activation of transcriptional responses, accumulation of reactive oxygen species (ROS), and activation of stress-related signaling pathways. However, the molecular mechanisms by which cholesterol depletion regulates autophagy and the putative involvement of transcriptional responses, ROS and/or stress-related signaling in autophagy regulation in this biological context are not fully understood. Here, we find that cholesterol depletion regulates autophagy at three different levels. First, employing RNA-seq, we show that cholesterol depletion increases the expression of autophagy-related genes independent of ROS or JNK activity. Second, analysis of LC3 lipidation and intracellular localization, and of p62 levels and degradation kinetics, reveals that cholesterol depletion mediates autophagy induction while interfering with autophagic flux. Of note, only the latter depends on ROS accumulation and JNK activity. In view of the common use of cholesterol-reducing drugs as therapeutic agents, our findings have important implications for multiple cellular settings in which autophagy plays a prominent role.

Cholesterol depletion enhances TGF-ß Smad signaling by increasing c-Jun expression through a PKR-dependent mechanism.

Transforming growth factor-ß (TGF-ß) plays critical roles in numerous physiological and pathological responses. Cholesterol, a major plasma membrane component, can have pronounced effects on signaling responses. Cells continually monitor cholesterol content and activate multilayered transcriptional and translational signaling programs, following perturbations to cholesterol homeostasis (e.g., statins, the commonly used cholesterol-reducing drugs). However, the cross-talk of such programs with ligand-induced signaling responses (e.g., TGF-ß signaling) remained unknown. Here, we studied the effects of a mild reduction in free (membrane-associated) cholesterol on distinct components of TGF-ß-signaling pathways. Our findings reveal a new regulatory mechanism that enhances TGF-ß-signaling responses by acting downstream from receptor activation. Reduced cholesterol results in PKR-dependent eIF2α phosphorylation, which enhances c-Jun translation, leading in turn to higher levels of JNK-mediated c-Jun phosphorylation. Activated c-Jun enhances transcription and expression of Smad2/3. This leads to enhanced sensitivity to TGF-ß stimulation, due to increased Smad2/3 expression and phosphorylation. The phospho/total Smad2/3 ratio remains unchanged, indicating that the effect is not due to altered receptor activity. We propose that cholesterol depletion induces overactivation of PKR, JNK, and TGF-ß signaling, which together may contribute to the side effects of statins in diverse disease settings.

Differential regulation of translation and endocytosis of alternatively spliced forms of the type II bone morphogenetic protein (BMP) receptor.

The expression and function of transforming growth factor-ß superfamily receptors are regulated by multiple molecular mechanisms. The type II BMP receptor (BMPRII) is expressed as two alternatively spliced forms, a long and a short form (BMPRII-LF and -SF, respectively), which differ by an ∼500 amino acid C-terminal extension, unique among TGF-ß superfamily receptors. Whereas this extension was proposed to modulate BMPRII signaling output, its contribution to the regulation of receptor expression was not addressed. To map regulatory determinants of BMPRII expression, we compared synthesis, degradation, distribution, and endocytic trafficking of BMPRII isoforms and mutants. We identified translational regulation of BMPRII expression and the contribution of a 3' terminal coding sequence to this process. BMPRII-LF and -SF differed also in their steady-state levels, kinetics of degradation, intracellular distribution, and internalization rates. A single dileucine signal in the C-terminal extension of BMPRII-LF accounted for its faster clathrin-mediated endocytosis relative to BMPRII-SF, accompanied by mildly faster degradation. Higher expression of BMPRII-SF at the plasma membrane resulted in enhanced activation of Smad signaling, stressing the potential importance of the multilayered regulation of BMPRII expression at the plasma membrane.

Dab2 inhibits the cholesterol-dependent activation of JNK by TGF-ß.

Transforming growth factor-ß (TGF-ß) ligands activate Smad-mediated and noncanonical signaling pathways in a cell context-dependent manner. Localization of signaling receptors to distinct membrane domains is a potential source of signaling output diversity. The tumor suppressor/endocytic adaptor protein disabled-2 (Dab2) was proposed as a modulator of TGF-ß signaling. However, the molecular mechanism(s) involved in the regulation of TGF-ß signaling by Dab2 were not known. Here we investigate these issues by combining biophysical studies of the lateral mobility and endocytosis of the type I TGF-ß receptor (TßRI) with TGF-ß phosphoprotein signaling assays. Our findings demonstrate that Dab2 interacts with TßRI to restrict its lateral diffusion at the plasma membrane and enhance its clathrin-mediated endocytosis. Small interfering RNA-mediated knockdown of Dab2 or Dab2 overexpression shows that Dab2 negatively regulates TGF-ß-induced c-Jun N-terminal kinase (JNK) activation, whereas activation of the Smad pathway is unaffected. Moreover, activation of JNK by TGF-ß in the absence of Dab2 is disrupted by cholesterol depletion. These data support a model in which Dab2 regulates the domain localization of TßRI in the membrane, balancing TGF-ß signaling via the Smad and JNK pathways.

Coated pit-mediated endocytosis of the type I transforming growth factor-ß (TGF-ß) receptor depends on a di-leucine family signal and is not required for signaling.

The roles of transforming growth factor-ß (TGF-ß) receptor endocytosis in signaling have been investigated in numerous studies, mainly through the use of endocytosis inhibitory treatments, yielding conflicting results. Two potential sources for these discrepancies were the pleiotropic effects of a general blockade of specific internalization pathways and the scarce information on the regulation of the endocytosis of the signal-transducing type I TGF-ß receptor (TßRI). Here, we employed extracellularly tagged myc-TßRI (wild type, truncation mutants, and a series of endocytosis-defective and endocytosis-enhanced mutants) to directly investigate the relationship between TßRI endocytosis and signaling. Our findings indicate that TßRI is targeted for constitutive clathrin-mediated endocytosis via a di-leucine (Leu(180)-Ile(181)) signal and an acidic cluster motif. Using Smad-dependent transcriptional activation assays and following Smad2/3 nuclear translocation in response to TGF-ß stimulation, we show that TßRI endocytosis is dispensable for TGF-ß signaling and may play a role in signal termination. Alanine replacement of Leu(180)-Ile(181) led to partial constitutive activation of TßRI, resulting in part from its retention at the plasma membrane and in part from potential alterations of TßRI regulatory interactions in the vicinity of the mutated residues.

Different domains regulate homomeric and heteromeric complex formation among type I and type II transforming growth factor-beta receptors.

Transforming growth factor-beta (TGF-beta) binds to and signals via two serine-threonine kinase receptors, type I (TbetaRI) and type II (TbetaRII). The oligomerization of TGF-beta receptors modulates ligand binding and receptor trafficking and may contribute to signal diversification. However, numerous features of the molecular domains that determine the homo- and hetero-oligomerization of full-length receptors at the cell surface and the mode of these interactions remain unclear. Here, we address these questions through computerized immunofluorescence co-patching and patch/fluorescence recovery after photobleaching measurements of different combinations of epitope-tagged receptors and their mutants in live cells. We show that TbetaRI and TbetaRII are present on the plasma membrane both as monomers and homo- and hetero-oligomers. The homodimerization of TbetaRII depends on a cytoplasmic juxtamembrane region (amino acid residues 200-220). In contrast, the cytoplasmic domain of TbetaRI is dispensable for its homodimerization. TbetaRI.TbetaRII hetero-oligomerization depends on the cytoplasmic domain of TbetaRI and on a C-terminal region of TbetaRII (residues 419-565). TGF-beta1 elevates TbetaRII homodimerization to some degree and strongly enhances TbetaRI.TbetaRII heteromeric complex formation. Both ligand-induced effects depend on the region encompassed between residues 200-242 of TbetaRII. Furthermore, the kinase activity of TbetaRI is also necessary for the latter effect. All forms of the homo- and hetero-oligomers, whether constitutively present on the membrane or formed upon TGF-beta1 stimulation, were stable in the time-scale of our patch/FRAP measurements. We suggest that the different forms of receptor oligomerization may serve as a basis for the heterogeneity of TGF-beta signaling responses.

Mutant p53 attenuates the SMAD-dependent transforming growth factor beta1 (TGF-beta1) signaling pathway by repressing the expression of TGF-beta receptor type II.

Both transforming growth factor beta (TGF-beta) and p53 have been shown to control normal cell growth. Acquired mutations either in the TGF-beta signaling pathway or in the p53 protein were shown to induce malignant transformation. Recently, cross talk between wild-type p53 and the TGF-beta pathway was observed. The notion that mutant p53 interferes with the wild-type p53-induced pathway and acts by a "gain-of-function" mechanism prompted us to investigate the effect of mutant p53 on the TGF-beta-induced pathway. In this study, we show that cells expressing mutant p53 lost their sensitivity to TGF-beta1, as observed by less cell migration and a reduction in wound healing. We found that mutant p53 attenuates TGF-beta1 signaling. This was exhibited by a reduction in SMAD2/3 phosphorylation and an inhibition of both the formation of SMAD2/SMAD4 complexes and the translocation of SMAD4 to the cell nucleus. Furthermore, we found that mutant p53 attenuates the TGF-beta1-induced transcription activity of SMAD2/3 proteins. In searching for the mechanism that underlies this attenuation, we found that mutant p53 reduces the expression of TGF-beta receptor type II. These data provide important insights into the molecular mechanisms that underlie mutant p53 "gain of function" pertaining to the TGF-beta signaling pathway.

A unique element in the cytoplasmic tail of the type II transforming growth factor-beta receptor controls basolateral delivery.

Transforming growth factor (TGF)-beta receptors stimulate diverse signaling processes that control a wide range of biological responses. In polarized epithelia, the TGFbeta type II receptor (T2R) is localized at the basolateral membranes. Sequential cytoplasmic truncations resulted in receptor missorting to apical surfaces, and they indicated an essential targeting element(s) near the receptor's C terminus. Point mutations in the full-length receptor confirmed this prediction, and a unique basolateral-targeting region was elucidated between residues 529 and 538 (LTAxxVAxxR) that was distinct, but colocalized within a clinically significant signaling domain essential for TGFbeta-dependent activation of the Smad2/3 cascade. Transfer of a terminal 84 amino-acid fragment, containing the LTAxxVAxxR element, to the apically sorted influenza hemagglutinin (HA) protein was dominant and directed basolateral HA expression. Although delivery to the basolateral surfaces was direct and independent of any detectable transient apical localization, fluorescence recovery after photobleaching demonstrated similar mobility for the wild-type receptor and a missorted mutant lacking the targeting motif. This latter finding excludes the possibility that the domain acts as a cell membrane retention signal, and it supports the hypothesis that T2R sorting occurs from an intracellular compartment.