p125FAK tyrosine phosphorylation and focal adhesion assembly: studies with phosphotyrosine phosphatase inhibitors.
p125FAK is a major tyrosine kinase phosphorylated in response to integrin-dependent adhesion. In this study we use vanadate and phenylarsine oxide (PAO), known inhibitors of phosphotyrosine phosphatases (PTPases), as a tool to artificially modulate p125FAK phosphorylation in human endothelial and in Chinese hamster ovary cells. Vanadate treatment strongly upregulates in a dose-dependent manner the level of tyrosine phosphorylation of several proteins in adherent cells. PAO induces a more restricted profile of tyrosine-phosphorylated proteins, increasing primarily a broad band of 120-140 kDa. Maximal stimulation of p125FAK tyrosine phosphorylation is reached at 10 microM PAO. In contrast, in vanadate-treated cells the p125FAK tyrosine phosphorylation shows a biphasic curve, being increased at high doses of vanadate (100 microM) and downregulated at low doses (25 microM). Immunofluorescence analysis of cells treated with PTPase inhibitors showed a direct correlation between the level of p125FAK tyrosine phosphorylation and the assembly of focal adhesions and actin stress fibers. Downregulation of p125FAK tyrosine phosphorylation is observed by treating cells with cytochalasin D (CD), a drug known to rapidly disrupt the actin cytoskeleton. When PTPase inhibitors are added in combination to CD, the level of tyrosine phosphorylation of p125FAK remains high and focal adhesions and actin stress fibers are preserved from the CD-mediated disruption. Based on these data we suggest that assembly of actin cytoskeleton plays an important role in inhibiting PTPases involved in p125FAK tyrosine phosphorylation.
Defilippi
P
P
Dipartimento di Genetica, Biologia e Chimica Medica, Universita di Torino, Italy.
Retta
S F
SF
Olivo
C
C
Palmieri
M
M
Venturino
M
M
Silengo
L
L
Tarone
G
G
D000199:Actins / Q000502:physiology; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells / Q000166:cytology; D002448:Cell Adhesion / Q000502:physiology*; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D006224:Cricetinae; D015638:Cytochalasin D / Q000494:pharmacology; D003599:Cytoskeleton / Q000187:drug effects; D015536:Down-Regulation / Q000187:drug effects; D004727:Endothelium / Q000166:cytology; D005455:Fluorescent Antibody Technique; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D006801:Humans; D016023:Integrins / Q000378:metabolism; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011972:Receptor, Insulin / Q000378:metabolism*; D014471:Umbilical Veins / Q000166:cytology; D015854:Up-Regulation / Q000187:drug effects; D014638:Vanadates / Q000494:pharmacology
D000199:Actins; D015815:Cell Adhesion Molecules; D016023:Integrins; D015638:Cytochalasin D; D014638:Vanadates; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; D017027:Protein Tyrosine Phosphatases