NCK and PAK participate in the signaling pathway by which vascular endothelial growth factor stimulates the assembly of focal adhesions.
Vascular endothelial growth factor (VEGF)-induced endothelial cell migration is a key step in the angiogenic response and is mediated, in part, by an accelerated rate of focal adhesion complex assembly and disassembly. We investigated the signaling pathway by which VEGF regulates focal adhesion complex assembly by examining the signaling proteins involved. VEGF stimulated the tyrosine phosphorylation of the SH2 domain-containing signaling proteins NCK and CRK in human umbilical vein endothelial cells. The signaling pathways that couple the kinase insert domain-containing receptor to NCK and CRK is most likely mediated by another cellular protein, as NCK and CRK were tyrosine-phosphorylated in response to VEGF in cells expressing receptors mutated at each of several candidate SH2 domain-interacting cytosolic tyrosines. In the absence of VEGF treatment, NCK (but not CRK) associated with the p21 GTPase-activated kinase PAK. PAK catalytic activity was augmented after VEGF treatment; an association of PAK with 60- and 90-kDa tyrosine-phosphorylated proteins accompanied this. VEGF stimulated the recruitment of PAK to focal adhesions, and FAK immunoprecipitated with both NCK and PAK in VEGF-treated (but not untreated) human umbilical vein endothelial cells. Inhibition of NCK protein expression using antisense oligonucleotides led to the inhibition of both VEGF-induced focal adhesion assembly and VEGF-induced cell migration, demonstrating a necessary role of NCK in these cellular responses.
Stoletov
K V
KV
Departments of Medicine and Pathology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Ratcliffe
K E
KE
Spring
S C
SC
Terman
B I
BI
D048868:Adaptor Proteins, Signal Transducing; D001483:Base Sequence; D002478:Cells, Cultured; D017931:DNA Primers; D016228:Endothelial Growth Factors / Q000502:physiology*; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D004789:Enzyme Activation; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D006801:Humans; D008222:Lymphokines / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D015513:Oncogene Proteins / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism; D017978:Receptors, Growth Factor / Q000235:genetics / Q000378:metabolism; D040262:Receptors, Vascular Endothelial Growth Factor; D015398:Signal Transduction / Q000502:physiology*; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D054462:p21-Activated Kinases
D048868:Adaptor Proteins, Signal Transducing; D017931:DNA Primers; D016228:Endothelial Growth Factors; D008222:Lymphokines; C078332:Nck protein; D015513:Oncogene Proteins; D017978:Receptors, Growth Factor; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D011505:Protein-Tyrosine Kinases; D020794:Receptor Protein-Tyrosine Kinases; D040262:Receptors, Vascular Endothelial Growth Factor; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases