A novel regulatory mechanism of MAP kinases activation and nuclear translocation mediated by PKA and the PTP-SL tyrosine phosphatase.
The PKA phosphorylation site on PTP-SL was identified as the Ser(231) residue, located within the KIM.Furthermore, treatment of COS-7 cells with PKA activators, or overexpression of the Calpha catalytic subunit of PKA, inhibited the cytoplasmic retention of ERK2 and p38alpha by wild-type PTP-SL, but not by a PTP-SL S231A mutant.These findings support the existence of a novel mechanism by which PKA may regulate the activation and translocation to the nucleus of MAP kinases.
Affiliation: Instituto de Investigaciones Citológicas, 46010 Valencia, Spain.
Protein tyrosine phosphatase PTP-SL retains mitogen-activated protein (MAP) kinases in the cytoplasm in an inactive form by association through a kinase interaction motif (KIM) and tyrosine dephosphorylation. The related tyrosine phosphatases PTP-SL and STEP were phosphorylated by the cAMP-dependent protein kinase A (PKA). The PKA phosphorylation site on PTP-SL was identified as the Ser(231) residue, located within the KIM. Upon phosphorylation of Ser(231), PTP-SL binding and tyrosine dephosphorylation of the MAP kinases extracellular signal-regulated kinase (ERK)1/2 and p38alpha were impaired. Furthermore, treatment of COS-7 cells with PKA activators, or overexpression of the Calpha catalytic subunit of PKA, inhibited the cytoplasmic retention of ERK2 and p38alpha by wild-type PTP-SL, but not by a PTP-SL S231A mutant. These findings support the existence of a novel mechanism by which PKA may regulate the activation and translocation to the nucleus of MAP kinases.
- Cell Nucleus/enzymology*/metabolism*
- Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors/chemistry/genetics/metabolism*
- Mitogen-Activated Protein Kinases/genetics/metabolism*
- Nerve Tissue Proteins/chemistry/genetics/metabolism*
- Protein Tyrosine Phosphatases/chemistry/genetics/metabolism*
- Amino Acid Motifs
- Biological Transport
- COS Cells
- Cell Line
- Enzyme Activation
- Intracellular Signaling Peptides and Proteins
- Mitogen-Activated Protein Kinase 1/genetics/metabolism
- Mitogen-Activated Protein Kinase 3
- Models, Biological
- Protein Tyrosine Phosphatases, Non-Receptor
- Receptor-Like Protein Tyrosine Phosphatases, Class 7
- Recombinant Fusion Proteins/chemistry/metabolism
- Signal Transduction
- p38 Mitogen-Activated Protein Kinases
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Figure 2: 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.
For PKA in vitro kinase assays, GST fusion proteins (1 μg) were incubated at room temperature during 1 h with 0.5 U/μl of cPKA in the presence of 2 μCi of γ-[32P]ATP, 10 μM ATP, and 8 mM MgCl2 (20 μl final volume). The reactions were stopped by adding SDS sample buffer and boiling, followed by SDS-PAGE and autoradiography. For in vitro association assays (see Fig. 2 A), GST fusion proteins were phosphorylated with cPKA as above, in the presence of 200 μM cold ATP, and then mixed with cell lysates and precipitated with glutathione-Sepharose, followed by immunoblotting. In vitro phosphatase assays were performed in 25 mM Hepes, pH 7.3, 5 mM EDTA, and 10 mM DTT (40 μl final volume), at 37°C, during the indicated times, as described in Zúñiga et al. 1999.