Echistatin inhibits pp125FAK autophosphorylation, paxillin phosphorylation and pp125FAK-paxillin interaction in fibronectin-adherent melanoma cells.
Echistatin, a snake-venom RGD-containing protein, was previously shown to disrupt cell-matrix adhesion by a mechanism that involves the reduction of pp125FAK tyrosine phosphorylation levels. The aim of this study was to establish the sequence of events downstream pp125FAK dephosphorylation that could be responsible for echistatin-induced disassembly of actin cytoskeleton and focal adhesions in fibronectin-adherent B16-BL6 melanoma cells. The results obtained show that echistatin induces a decrease of both autophosphorylation and kinase activity of pp125FAK. One hour of cell exposure to echistatin caused a 39% decrease of pp125FAK Tyr397 phosphorylation and a 31% reduction of pp125FAK autophosphorylation activity as measured by immune-complex kinase assay. Furthermore, 1 h of cell treatment by echistatin produced a 63% decrease of paxillin phosphorylation, as well as a reduction in the amount of paxillin bound to pp125FAK. Immunofluorescence analysis of echistatin treated cells showed the concomitant disappearance of both paxillin and pp125FAK from focal adhesions. The reduction of paxillin phosphorylation may represent a critical step in the pathway by which disintegrins exert their biological activity, including the inhibition of experimental metastasis in vivo.
Dipartimento di Biochimica e Biotecnologie Mediche, and CEOS - Dipartimento di Biologia e Patologia Cellulare e Molecolare, Università di Napoli Federico II, Napoli, Italy.
R
R
Della Morte
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C
Squillacioti
C
C
Garbi
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P
Derkinderen
M A
MA
Belisario
J A
JA
Girault
P
P
Di Natale
L
L
Nitsch
N
N
Staiano
D000818:Animals; D002448:Cell Adhesion / Q000187:drug effects / Q000502:physiology*; D015815:Cell Adhesion Molecules / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D005353:Fibronectins / Q000502:physiology*; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D036341:Intercellular Signaling Peptides and Proteins; D007700:Kinetics; D008546:Melanoma, Experimental; D051379:Mice; D051419:Paxillin; D010455:Peptides / Q000494:pharmacology*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D019030:Receptors, Vitronectin / Q000037:antagonists & inhibitors; D014407:Tumor Cells, Cultured
D015815:Cell Adhesion Molecules; D003598:Cytoskeletal Proteins; D005353:Fibronectins; D036341:Intercellular Signaling Peptides and Proteins; D051419:Paxillin; D010455:Peptides; D010750:Phosphoproteins; C496062:Pxn protein, mouse; D019030:Receptors, Vitronectin; C057986:echistatin; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496051:Ptk2 protein, mouse