Transforming growth factor-β (TGF-β) exerts its effects on cell proliferation, differentiation and migration in part through its modulation of extracellular matrix components, such as fibronectin and plasminogen activator inhibitor-1 (PAI-1). Although the SMAD family of proteins recently has been shown to be a key participant in TGF-β signaling, other signaling pathways have also been shown to be activated by TGF-β. We report here that c-Jun N-terminal kinase (JNK), a member of the MAP kinase family, is activated in response to TGF-β in the human fibrosarcoma HT1080-derived cell line BAHgpt. Stable expression of dominant-negative forms of JNK1 and MKK4, an upstream activator of JNK, results in loss of TGF-β-stimulated fibronectin mRNA and protein induction, while having little effect on TGF-β-induced levels of PAI-1. The human fibronectin promoter contains three CRE elements, one of which has been shown to bind a c-Jun-ATF-2 heterodimer. Utilizing a GAL4 fusion trans-reporting system, we demonstrate a decrease in transactivating potential of GAL4-c-Jun and GAL4-ATF-2 in dominant-negative JNK1-and MKK4-expressing cells. Finally, we show that TGF-β-induced fibronectin synthesis is independent of Smad4. These results demonstrate that TGF-β-mediated fibronectin induction requires activation of JNK which in turn modulates the activity of c-Jun and ATF-2 in a Smad4-independent manner.

TGFβ induces epithelial-mesenchymal transdifferentiation (EMT) accompanied by cellular differentiation and migration. Despite extensive transcriptomic profiling, identification of TGFβ-inducible, EMT-specific genes has met with limited success. Here, we identify a post-transcriptional pathway by which TGFβ modulates expression of EMT-specific proteins, and EMT itself. We show that heterogeneous nuclear ribonucleoprotein E1 (hnRNP E1) binds a structural, 33 nucleotides (nt) TGF beta-activated translation (BAT) element in the 3’-UTR of disabled-2 (Dab2) and interleukin-like EMT inducer (ILEI) transcripts, and repress their translation. TGFβ activation leads to phosphorylation at Ser43 of hnRNP E1 by protein kinase Bβ/Akt2, inducing its release from the BAT element and translational activation of Dab2 and ILEI mRNAs. Modulation of hnRNP E1 expression or its post-translational modification alters TGFβ-mediated reversal of translational silencing of the target transcripts and EMT. These results suggest the existence of a TGFβ-inducible post-transcriptional regulon that controls EMT during development and metastatic progression of tumors.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) comprise a family of RNA-binding proteins. The complexity and diversity associated with the hnRNPs render them multifunctional, involved not only in processing heterogeneous nuclear RNAs (hnRNAs) into mature mRNAs, but also acting as trans-factors in regulating gene expression. Heterogeneous nuclear ribonucleoprotein E1 (hnRNP E1), a subgroup of hnRNPs, is a KH-triple repeat containing RNA-binding protein. It is encoded by an intronless gene arising from hnRNP E2 through a retrotransposition event. hnRNP E1 is ubiquitously expressed and functions in regulating major steps of gene expression, including pre-mRNA processing, mRNA stability, and translation. Given its wide-ranging functions in the nucleus and cytoplasm and interaction with multiple proteins, we propose a posttranscriptional regulon model that explains hnRNP E1's widespread functional diversity.

Using a genetic complementation approach we have identi®ed disabled-2 (Dab2), a structural homolog of the Dab1 adaptor molecule, as a critical link between the transforming growth factor b (TGFb) receptors and the Smad family of proteins. Expression of wildtype Dab2 in a TGFb-signaling mutant restores TGFb-mediated Smad2 phosphorylation, Smad translocation to the nucleus and Smad-dependent transcriptional responses. TGFb stimulation triggers a transient increase in association of Dab2 with Smad2 and Smad3, which is mediated by a direct interaction between the N-terminal phosphotyrosine binding domain of Dab2 and the MH2 domain of Smad2. Dab2 associates with both the type I and type II TGFb receptors in vivo, suggesting that Dab2 is part of a multiprotein signaling complex. Together, these data indicate that Dab2 is an essential component of the TGFb signaling pathway, aiding in transmission of TGFb signaling from the TGFb receptors to the Smad family of transcriptional activators.

Bim, the Bcl-2 interacting mediator of cell death, is a member of the BH3-only family of pro-apoptotic proteins. Recent studies have demonstrated that the apoptotic activity of Bim can be regulated through a post-translational mechanism whereby ERK phosphorylation serves as a signal for Bim ubiquitination and proteasomal degradation.