While described in earlier studies , , , PI3K and smad activation differs in their subcellular location. to phosphorylate c-Raf, ultimately resulting in Erk activation. Activation of Erk was necessary for TGF- induced fibroblast replication. In addition, Erk phosphorylated the linker region of nuclear localized smads, resulting in increased half-life of C-terminal phospho-smad 2 and 3 and increased duration of smad target gene transcription. Together, these data show that in mesenchymal cell types the TGF-/PI3K/Pak2/Raf/MEK/Erk pathway regulates smad signaling, is critical for TGF–induced growth and is a part of an integrated signaling web made up of multiple interacting pathways rather than discrete smad/non-smad pathways. Introduction Transforming Growth Factor (TGF-) is the prototypic member AZD1208 of a family of structurally related cytokines that control a myriad of cellular functions. TGF- elicits its cellular responses by signaling through a receptor complex of serine/threonine kinase type I (TRI) and type II (TRII) receptors , . Ligand binding induced transmission transduction through this receptor complex results in receptor mediated (R-) smad2 and/or smad3 phosphorylation. This phosphorylation at the C-terminal SSXS motif of smad2/3 allows them to complex with the common mediator (Co-) smad4 , , translocate into the nucleus, and regulate target gene expression AZD1208 , . Although both mesenchymal and epithelial cells contain the canonical TGF-/smad signaling cascade, epithelial cell types are growth inhibited, whereas mesenchymal cells are growth stimulated by TGF- suggesting a fundamental mechanistic difference in TGF- signaling between cell types, supplimental to the smad signaling cascade. This has lead to the nomenclature of smad and non-smad or smad-dependant and impartial signaling cascades. There have been a number of these non-smad signaling pathways explained including Erk, Jnk, ROCK, and more recently, p21-activated kinase-2 (Pak2; C). In phenotypically normal cell lines (neither virally transformed nor cancer derived), TGF- regulation of Pak2 activity was found to be stimulated through cdc42/Rac1 and inhibited by Merlin/Erbin , . Pak2 is usually specifically activated by TGF- only in mesenchymal cells, as the result of phosphatidylinositol 3-kinase (PI3K) activation and may be associated with TGF- AZD1208 activation of Ras , , . Conversely, normal epithelial cells appear to inhibit Pak2 activation through an failure to activate PI3K and/or by directly inhibiting Pak2 through Merlin/Erbin . Functionally, PAKs regulate apoptosis, cell motility and cytoskeletal rearrangement . Relevant to this study, Paks have been implicated in mitogen activated protein kinase/extracellular transmission regulated kinase (MAPK/Erk) signaling cascades as potential MAP kinase kinase kinase kinases  by regulating the activity of both c-Raf and MEK1 , . Classically, with tyrosine kinase receptors, activation of Ras ,  results in activated Raf, which activates MEK1/2, followed by Erk activation. However, Ras independent mechanisms of Erk activation have been explained for both erythropoietin (Epo; ) and platelet derived growth factor (PDGF; ), suggesting different pathways lead to Erk activation. Although cross-talk between Erk and smad signaling was explained over a decade ago , , , the relationship and mechanism by which this occurs is still unknown. Within the linker region domains of smad2 and smad3 are several potential Erk phosphorylation sites , . However, these same sites have also been implicated in smad regulation by the cyclin dependent kinases, CDK8 and 9 . The phosphorylated linker region, has also been shown to both inhibit smad nuclear translocation and signaling , , C and enhance smad mediated transcriptional activity , , , two mutually exclusive functions. To address this controversy, in this study we further refine the mechanism for cell type specific TGF- activation of Erk. We show that via PI3K, Pak2 activation results in Erk activation in untransformed cells with endogenous levels of transmission transduction proteins. We also show that this activated AZD1208 Erk phosphorylates smads within their linker regions, resulting in the maintenance of smad mediated transcriptional activation, thus demonstrating integration of the Erk and smad pathways, both under the direct control of TGF-. Materials and Methods Cell Culture All cell lines used were managed in high glucose Dulbecco’s Modified Eagle Medium (DMEM; Invitrogen, Carlsbad, CA) and purchased from American Type Culture Collection repository (Mannassas, VA; NIH-3T3, CRL-1658; Mv1Lu, CCL-64; HEK-293A, CRL-1573; NMuMG, CRL-1636). The murine embryonic fibroblast cell collection, AKR-2B, was produced in DMEM supplemented with 5% Fetal Bovine Serum (FBS; PAA Labs Inc, Etobicoke, ON)), while NIH-3T3 cells were produced in DMEM supplemented with 10% Newborn Calf Serum (NBCS; Invitrogen, Carlsbad, CA). Pak2 flox/flox MEF parental cell collection and the Cre/Pak2 knockout derivative (kind gift of Dr. Jonathan Chernoff, Fox Chase Cancer Centre, OH) were managed CLG4B in DMEM supplemented with 10% FCS, as were Mv1Lu epithelial cells, while NMuMG growth media also contained 10 g/ml bovine Insulin (Sigma Biochemicial, St. Louis, MO) and 5 ng/ml EGF (Cell Signaling Technologies; Pickerington, ON). All buffer salts, bovine serum albumin (BSA) and acrylamide were purchased from ThermoFisher Biotechnology. Protein Analysis Mesenchymal cell lines were plated 24 h prior to serum depletion (0.1% NBCS/DMEM).