Phosphorylation of PTP-SL and STEP by PKA. (A) GST fusion proteins (1 μg) were phosphorylated in vitro by cPKA in the presence of γ-[³²P]ATP as indicated. (B) COS-7 cells were mock-transfected (pRK5 vector alone) or transfected with the pRK5 PTP-SL wild type or S231A, as indicated, followed by ³²P-labeling. Cells were left untreated (−) or were treated (+) with dibutyryl-cAMP, forskolin, or okadaic acid, as described in Materials and Methods. PTP-SL was precipitated from cell lysates with anti–PTP-SL antibody. All samples (A and B) were resolved by 10% SDS-PAGE under reducing conditions, and followed by autoradiography.

Effect of PTP-SL phosphorylation by PKA on the association with MAP kinases and their dephosphorylation. (A) GST-PTP-SL 147-288 wild type or S231A fusion proteins (1.5 μg) were left untreated (−) or were phosphorylated in vitro by cPKA (+) in the presence of cold ATP, as indicated. Rat-1 cell lysates (500 μg) were added, and the fusion proteins were precipitated with glutathione-Sepharose. The kinases were detected by immunoblot analysis with anti–ERK1/2 (top) or anti–p38α (bottom) antibodies. In lane 1, total lysate samples (20 μg) were loaded. Arrowheads indicate the migration of the kinases. (B) 293 cells were transfected with pRK5 GST-PTP-SL 147-549 (both panels); in the bottom panel, cells were cotransfected with pECE-HA-p38MAPK. After 48 h, cells were left untreated (−) or were treated with dibutyryl-cAMP, dibutyryl-cAMP plus H89, or forskolin, as indicated. The GST-PTP-SL fusion proteins were precipitated from the cell lysates with glutathione-Sepharose, and coprecipitated kinases were detected by immunoblot analysis with anti–ERK1/2 (top) or anti–HA (bottom) antibodies. (C) 293 cells were transfected with pRK5 GST (lane 2) or the pRK5 GST-PTP-SL 147-549 wild type or mutants, as indicated, and fusion proteins were precipitated as in B, followed by immunoblot with anti-ERK1/2 or anti-p38α antibodies. In lane 1, total lysate (20 μg) was loaded. All GST-PTP-SL proteins were equally expressed. (D) Tyrosine-phosphorylated HA-ERK2 or HA-p38α were precipitated with the anti–HA 12CA5 mAb from activated 293 cells, transfected with pCDNA3-HA-ERK2 (lanes 1–5) or pECE-HA-p38MAPK (lanes 6–10), and immune complexes were subjected to in vitro phosphatase assays during the indicated times (in minutes) in the presence of GST-PTP-SL 147-549 wild type (lanes 2, 3, 7, and 8) or S231E (lanes 4, 5, 9, and 10) (1 μg). In lanes 1 and 6, no fusion proteins were added, and samples were kept on ice. Tyrosine phosphorylation was detected by immunoblot with the anti-phosphotyrosine 4G10 mAb (top panels). Bottom panels show the equal presence of HA-ERK2 and HA-p38α in all lanes, after stripping of the filters and reprobing with the anti-HA 12CA5 mAb. Equal activities of GST-PTP-SL wild type and S231E towards pNPP were measured (not shown). All samples (A–D) were resolved by 10% SDS-PAGE under reducing conditions.

Transmembrane PTP-SL retains ERK2 and p38α outside of the nucleus. COS-7 cells were cotransfected with pcDNA3-HA-ERK2 or pECE-HA-p38MAPK, plus pRK5-PTP-SL 1-549 wild type or ΔKIM (Δ224-239) mutant, as indicated. 48 h after transfection, cells were costained and analyzed by immunofluorescence. HA-ERK2 and HA-p38α were stained with the mouse anti–HA mAb 12CA5 plus rhodamine-conjugated goat anti–mouse antibody (red, A, D, G, and J). PTP-SL was stained with rabbit polyclonal anti–PTP-SL antibody plus fluorescein isothiocyanate–conjugated goat anti–rabbit antibody (green, B, E, H, and K; subcellular localization of PTP-SL 1-549 corresponds to perinuclear areas in the cytoplasm). In C, F, I, and L, double color staining is shown; yellow areas correspond to colocalization of HA-ERK2 or HA-p38α, and PTP-SL.

(A) PTP-SL retains HA-ERK2 in the cytoplasm independently of its PTP domain. COS-7 cells were cotransfected with pcDNA3-HA-ERK2 plus pRK5 (mock), or plus pRK5 PTP-SL 147-549, 1-549, or 1-288 wild type or mutants, as indicated. Cells were costained and analyzed by immunofluorescence as in Fig. 3. PTP-SL 147-549 is located in the cytoplasm. Subcellular localization of PTP-SL 1-549 and 1-288 is identical (see Fig. 3). (B, C) Effect of PKA on the nuclear localization of HA-ERK2 and HA-p38α in the presence of PTP-SL. COS-7 cells were cotransfected with pcDNA3-HA-ERK2 (B) or pECE-HA-p38MAPK (C) plus pRK5-PTP-SL 1-549 wild type or mutants, as indicated. Similar results were obtained with the double mutant C480S/S231A and the single mutant S231A (not shown). In some points, cells were additionally transfected with pCαEV (cPKAα) (+), and were induced as described in Materials and Methods. Cells were left untreated (−) or were treated (+) with dibutyryl-cAMP or dibutyryl-cAMP plus H89, as indicated, and then were costained and analyzed by immunofluorescence. HA-ERK2 or HA-p38α nuclear localization is presented as the percentage of cells coexpressing PTP-SL and HA-ERK2, or PTP-SL and HA-p38α that showed the MAP kinase located into the nucleus. For each point, at least 100 double positive cells were scored. Bars represent the mean ± SD of at least two separate experiments.

Model of MAP kinase regulation by PKA and PTP-SL. Pools of MAP kinases are maintained in the cytoplasm and into the nucleus by the balance between activation stimuli and the PTP-SL (or other KIM-containing PTPs) inhibitory effects. Upon PKA activation, the association of PTP-SL with the MAP kinase is impaired, and MAP kinase tyrosine phosphorylation and nuclear translocation is favored. The putative regulatory role for serine/threonine phosphatases (PP) in the dephosphorylation of PTP-SL, is indicated (see details in the text).