Evidence for a calpeptin-sensitive protein-tyrosine phosphatase upstream of the small GTPase Rho - A novel role for the calpain inhibitor calpeptin in the inhibition of protein-tyrosine phosphatases

Activation of the thiol protease calpain results in proteolysis of focal ad hesion-associated proteins and severing of cytoskeletal-integrin links. We employed a commonly used inhibitor of calpain, calpeptin, to examine a role for this protease in the reorganization of the cytoskeleton under a variet y of conditions. Calpeptin induced stress fiber formation in both forskolin -treated REF-52 fibroblasts and serum-starved Swiss 3T3 fibroblasts. Surpri singly, calpeptin was the only calpain inhibitor of several tested with the ability to induce these effects, suggesting that calpeptin may act on targ ets besides calpain. Here we show that calpeptin inhibits tyrosine phosphat ases, enhancing tyrosine phosphorylation particularly of paxillin, Calpepti n preferentially inhibits membrane-associated phosphatase activity. Consist ent with this observation, in vitro phosphatase assays using purified gluta thione S-transferase fusion proteins demonstrated a preference for the tran smembrane protein-tyrosine phosphatase-alpha over the cytosolic protein-tyr osine phosphatase-1B. Furthermore, unlike wide spectrum inhibitors of tyros ine phosphatases such as pervanadate, calpeptin appeared to inhibit a subse t of phosphatases. Calpeptin-induced assembly of stress fibers was inhibite d by botulinum toxin C3, indicating that calpeptin is acting on a phosphata se upstream of the small GTPase Rho, a protein that controls stress fiber a nd focal adhesion assembly. Not only does this work reveal that calpeptin i s an inhibitor of protein-tyrosine phosphatases, but it suggests that calpe ptin will be a valuable tool to identify the phosphatase activity upstream of Rho.