Transforming Growth Factor β Activation of c-Abl Is Independent of Receptor Internalization and Regulated by Phosphatidylinositol 3-Kinase and PAK2 in Mesenchymal Cultures

Mark C Wilkes,Edward B Leof

doi:10.1074/jbc.m603721200

Abstract

Transforming growth factor beta (TGF-beta) modulates a number of cellular phenotypes as divergent as growth stimulation and growth inhibition. Although the Smad pathway is critical for many of these responses, recent evidence indicates that Smad-independent pathways may also have a critical role. One such protein previously shown to regulate TGF-beta action independent of the Smad proteins is the c-Abl nonreceptor tyrosine kinase. In the current study we determined that TGF-beta receptor signaling activates c-Abl kinase activity in a subset of fibroblast but not epithelial cultures. This cell type-specific response occurs in a membrane-proximal locale independent of receptor internalization and upstream of dynamin action. Although c-Abl activation by TGF-beta is independent of Smad2 or Smad3, it is prevented by inhibitors of phosphatidylinositol 3-kinase or PAK2. Thus, c-Abl represents a target downstream of phosphatidylinositol 3-kinase-activated PAK2, which differentiates TGF-beta signaling in fibroblasts and epithelial cell lines and integrates serine/threonine receptor kinases with tyrosine kinase pathways.

Highlights

Signaling pathways for TGF-␤ are routinely classified as being either Smad-dependent or Smad-independent
phosphatidylinositol 3-kinase (PI3K) was required for both p21-activated kinase 2 (PAK2) and Akt activation, TGF-␤ morphologic transformation and monolayer proliferation resulted from signals emanating downstream of PAK2
The medium was replaced with 2% fetal bovine serum (FBS)/DMEM for 48 h, and Smad-independent TGF-␤ signaling target that reflected a spefollowing a 24-h incubation in 0.1% FBS/DMEM cultures were cific cell tropism of activation and (ii) whether c-Abl was actistimulated in 2 ml of serum-free DMEM alone or containing 10 vated in a similar and/or parallel pathway to PI3K and PAK2

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—Cells were obtained from ATCC (Rockville, MD) and grown in high glucose DMEM (Invitrogen) supplemented with 10% fetal bovine serum (FBS; Biosource International, Camarillo, CA). The medium was replaced with 2% FBS/DMEM for 48 h, and Smad-independent TGF-␤ signaling target that reflected a spefollowing a 24-h incubation in 0.1% FBS/DMEM cultures were cific cell tropism of activation and (ii) whether c-Abl was actistimulated in 2 ml of serum-free DMEM alone or containing 10 vated in a similar and/or parallel pathway to PI3K and PAK2. There (IMR90) fibroblasts; no effect was observed in mink (Mv1Lu), canine (MDCK), or human (HeLa) epithelial cultures This cell tropism is identical to what we have reported for PI3K and PAK2 activation [14, 21] and suggests that PI3K, PAK2, and c-Abl might be targets within the same TGF-␤ signaling pathway (see below).

Smad Phosphorylation and Defines

Findings

DISCUSSION