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Fig. 9. PKL is a substrate of PTP-PEST (A) PTP-PEST^–/– cell lysates were incubated with GST-catalytic-domain fusions of either wild-type PTP-PEST or the C231S catalytically inactive `trapping' mutant. Pull-down assays were blotted with antibodies against phosphotyrosine (4G10), PKL or -actinin. `Trapped' proteins of 130, 95 and 70 kDa were observed. The PKL immunoblot shows that a significant amount of PKL is trapped, with only a modest amount being pulled down by the wild-type fusion protein. DLS, detergent soluble lysate. (B) GFP, GFP PKL or GFP-PKL triple YF mutant were expressed in PTP-PEST^–/– cells in the presence or absence of PTP-PEST. Cells were plated on fibronectin-coated dishes for 60 minutes and cell lysates were immunoprecipitated with the anti-GFP antibody. The precipitates were immunoblotted with anti-phosphotyrosine (4G10) and anti-GFP antibodies. The phosphorylation of PKL was reduced in the presence of PTP-PEST. (C) PTP-PEST suppresses PKL phosphorylation in the presence of constitutively active Rac. PTP-PEST^–/– cells expressing GFP or GFP PKL in the presence or absence of PTP-PEST and constitutively active Rac (V12 Rac) were plated for 60 minutes on fibronectin-coated dishes, cell lysates were then immunoblotted with anti-phosphotyrosine (4G10) and anti-GFP antibodies.

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