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/Sps-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(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.
Nguyen
D H
DH
Departments of Biochemistry and Molecular Biology, Pathology, and Microbiology, University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA.
Webb
D J
DJ
Catling
A D
AD
Song
Q
Q
Dhakephalkar
A
A
Weber
M J
MJ
Ravichandran
K S
KS
Gonias
S L
SL
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D002465:Cell Movement / Q000502:physiology*; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D051380:GRB2 Adaptor Protein; D006801:Humans; D020935:MAP Kinase Signaling System / Q000502:physiology*; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000502:physiology; D011506:Proteins / Q000378:metabolism; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014568:Urokinase-Type Plasminogen Activator / Q000502:physiology*
D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D011506:Proteins; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; D016392:Proto-Oncogene Proteins pp60(c-src); D014568:Urokinase-Type Plasminogen Activator