Urokinase-type Plasminogen Activator Stimulates the Ras/Extracellular Signal-regulated Kinase (ERK) Signaling Pathway and MCF-7 Cell Migration by a Mechanism That Requires Focal Adhesion Kinase, Src, and Shc. Rapid dissociation of GRB2/SOS-SHC complex is associated with the transient phosphorylation of ERK in urokinase-treated cells

Urokinase-type plasminogen activator (uPA) stimulates MCF-7 cell migration by binding to the UPA receptor and activating the Ras-extracellular signal-regulated kinase (Ras- ERK) signaling pathway. Studies presented here show that soluble uPA receptor and a peptide derived from the linker region between domains 1 and 2 of the uPA receptor also stimulate cellular migration via a mitogen-activated protein kinase/ERK kinase (MEK)-dependent pathway. Signaling proteins that function upstream of Ras in uPA- stimulated cells remain undefined. To address this problem, we transfected MCF-7 cells to express the noncatalytic carboxylterminal domain of focal adhesion kinase (FAK), FAK super(Y397F), kinase-defective c-Src, or Shc FFF, all of which express dominant-negative activity. In each case, ERK phosphorylation and cellular migration in response to uPA were blocked. Both activities were rescued by co-transfecting the cells to express constitutively active MEK1, indicating that FAK, c-Src, and Shc are upstream of MEK. Shc was tyrosine-phosphorylated in uPA- treated cells. The level of phosphorylated Shc was increased within 1 min and remained increased for at least 30 min. Sos co- immunoprecipitated with Shc in cells that were treated with uPA for 1-2.5 min, probably reflecting the formation of Shc-Grb2/Sos complex; however, by 10 min, co-immunoprecipitation of Sos with Shc was no longer observed. Rapid dissociation of Sos from Shc represents a possible mechanism for the transient phosphorylation of ERK in uPA-treated MCF-7 cells.