Cytostatin, which is isolated from a microbial cultured broth as a low molecular weight inhibitor of cell adhesion to extracellular matrix (ECM), has anti-metastatic activity against
B16 melanoma cells in vivo. In this study, we examined a target of
cytostatin inhibiting cell adhesion to ECM.
Cytostatin inhibited
tyrosine phosphorylation of
focal adhesion kinase (FAK) and
paxillin upon B16 cell adhesion to
fibronectin. While the amount of FAK was not affected by
cytostatin, electrophoretically slow-migrating
paxillin appeared.
Alkaline phosphatase treatment diminished
cytostatin-induced slow-migrating
paxillin. Furthermore,
cytostatin increased intracellular
serine/
threonine-phosphorylated
proteins and was found to be a selective inhibitor of
protein phosphatase 2A (PP2A).
Cytostatin inhibited PP2A with an IC(50) of 0.09 microgram/ml in a non-competitive manner against a substrate,
p-nitrophenyl phosphate, but it had no apparent effect on other
protein phosphatases including PP1, PP2B and
alkaline phosphatase even at 100 microgram/ml. On the contrary, dephosphocytostatin, a
cytostatin analogue, without inhibitory effect on PP2A did not affect B16 cell adhesion including FAK and
paxillin. These results indicate that
cytostatin inhibits cell adhesion through modification of focal contact
proteins such as
paxillin by inhibiting a PP2A type
protein serine/threonine phosphatase. This is the first report that describes a
drug with anti-metastatic ability that inhibits PP2A selectively.