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. Author manuscript; available in PMC: 2014 Feb 13.
Published in final edited form as: J Biol Chem. 2007 Jun 25;282(33):24381–24387. doi: 10.1074/jbc.M701499200

FIGURE 1. PTPL1 mediates dephosphorylation of phosphotyrosine 55 of TRIP6 in vitro and inhibits LPA-induced tyrosine phosphorylation of TRIP6 in cells.

FIGURE 1

A, PTPL1 dephosphorylates c-Src-phosphorylated TRIP6 in a dose- and time-dependent manner in vitro. 1 μg of purified recombinant GST-TRIP6 was phosphorylated by c-Src in vitro. After extensive washing to remove c-Src, phosphorylated GST-TRIP6 was incubated with 0.1 or 0.5 μg of the catalytic domain of PTPL1 (PTPL1-CD) for 30 min (left panel) or with 0.5 μg of PTPL1-CD for 10 or 30 min (right panel). After SDS-PAGE, the immunoblot was probed with an anti-phosphotyrosine antibody (PY20H) to detect phosphorylated TRIP6. The immunoblot was then stripped and reprobed with a TRIP6-specific antibody. B, overexpression of PTPL1, but not a PTPL1-ΔCD mutant that lacks the catalytic domain of phosphatase, eliminates LPA-induced tyrosine phosphorylation of TRIP6 in SYF + c-Src cells. SYF + c-Src cells were transiently transfected with pEGFP, pEGFP-PTPL1, or pEGFP-PTPL1-ΔCD. Cells were starved in 0.1% fatty acid-free BSA-containing medium for 8 h followed by incubation with 2 μM LPA for 15 min. The endogenous TRIP6 was immunoprecipitated with a TRIP6-specific monoclonal antibody or a control mouse IgG. After SDS-PAGE, the immunoblot was probed with an anti-phosphotyrosine antibody to detect tyrosine-phosphorylated TRIP6 and then was stripped and reprobed with a rabbit anti-TRIP6 antibody. The right panel shows the expression of endogenous TRIP6, endogenous PTP-BL, GFP-PTPL1, GFP-PTPL1-ΔCD, and GFP in the whole cell lysates. C, inhibition of PTPL1 expression enhances LPA-induced tyrosine phosphorylation of TRIP6 in SYF + c-Src cells. SYF + c-Src cells were transiently transfected with a control pSUPER-siScramble vector expressing a control siRNA or pSUPER-si(m)PTPL1 vector expressing an siRNA of PTP-BL, a mouse homologue of PTPL1. LPA-induced tyrosine phosphorylation of TRIP6 was determined as described in panel B. The bottom two panels show the expression of endogenous PTP-BL and TRIP6 in the whole cell lysates, respectively. Data shown in each figure are representative of three to four independent experiments.