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A novel regulatory mechanism of MAP kinases activation and nuclear translocation mediated by PKA and the PTP-SL tyrosine phosphatase.

Blanco-Aparicio C, Torres J, Pulido R - J. Cell Biol. (1999)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Investigaciones Citológicas, 46010 Valencia, Spain.

ABSTRACT
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.

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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.
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Figure 3: 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.

Mentions: PTP-SL retains ERK2 in the cytoplasm in a KIM-dependent manner (Zúñiga et al. 1999). To study the effect of phosphorylation of PTP-SL by PKA on its ability to retain MAP kinases outside of the nucleus, immunofluorescence analysis was performed on COS-7 cells cotransfected with HA-ERK2 or HA-p38α, and PTP-SL. Overexpression of HA-ERK2 or HA-p38α alone resulted in their accumulation in the nucleus (see Fig. 4 A; and data not shown); however, in the presence of PTP-SL, the nuclear accumulation of these kinases was abolished, colocalizing with the phosphatase outside of the nucleus (Fig. 3 and Fig. 4 A). Interestingly, neither the PTP activity nor the PTP domain of PTP-SL itself was required to retain HA-ERK2 outside of the nucleus, as observed by coexpression with PTP-SL catalytically inactive mutants (C480S or R486M) or with truncated PTP-SL forms lacking the PTP domain (PTP-SL 1-288) (Fig. 4 A). On the other hand, upon coexpression with the PTP-SL S231E mutant, the cytoplasmic retention of HA-ERK2 or HA-p38α was significantly reduced, as compared with wild-type PTP-SL (Fig. 4B and Fig. C). Also, when cells coexpressing wild-type PTP-SL and HA-ERK2 or HA-p38α were treated with dibutyryl-cAMP, the nuclear localization of both MAP kinases was partially restored, and such an effect was prevented by cell preincubation with H89 (Fig. 4B and Fig. C). However, no effect was observed upon cell treatment with agents that activate other kinase pathways, such as EGF or PMA (data not shown). Furthermore, cotransfection with an inducible expression vector coding the Cα catalytic subunit of PKA (cPKAα), also favored the nuclear localization of these MAP kinases in the presence of PTP-SL (Fig. 4B and Fig. C). Remarkably, the effect of PKA activation on the colocalization of HA-ERK2 and HA-p38α with wild-type PTP-SL was not observed with the PTP-SL S231A mutant (Fig. 4B and Fig. C), demonstrating that phosphorylation of the Ser231 residue of PTP-SL by PKA inhibits the in vivo association of PTP-SL with HA-ERK2 and HA-p38α, and favors the nuclear translocation of these kinases.


A novel regulatory mechanism of MAP kinases activation and nuclear translocation mediated by PKA and the PTP-SL tyrosine phosphatase.

Blanco-Aparicio C, Torres J, Pulido R - J. Cell Biol. (1999)

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.
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Related In: Results  -  Collection

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Figure 3: 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.
Mentions: PTP-SL retains ERK2 in the cytoplasm in a KIM-dependent manner (Zúñiga et al. 1999). To study the effect of phosphorylation of PTP-SL by PKA on its ability to retain MAP kinases outside of the nucleus, immunofluorescence analysis was performed on COS-7 cells cotransfected with HA-ERK2 or HA-p38α, and PTP-SL. Overexpression of HA-ERK2 or HA-p38α alone resulted in their accumulation in the nucleus (see Fig. 4 A; and data not shown); however, in the presence of PTP-SL, the nuclear accumulation of these kinases was abolished, colocalizing with the phosphatase outside of the nucleus (Fig. 3 and Fig. 4 A). Interestingly, neither the PTP activity nor the PTP domain of PTP-SL itself was required to retain HA-ERK2 outside of the nucleus, as observed by coexpression with PTP-SL catalytically inactive mutants (C480S or R486M) or with truncated PTP-SL forms lacking the PTP domain (PTP-SL 1-288) (Fig. 4 A). On the other hand, upon coexpression with the PTP-SL S231E mutant, the cytoplasmic retention of HA-ERK2 or HA-p38α was significantly reduced, as compared with wild-type PTP-SL (Fig. 4B and Fig. C). Also, when cells coexpressing wild-type PTP-SL and HA-ERK2 or HA-p38α were treated with dibutyryl-cAMP, the nuclear localization of both MAP kinases was partially restored, and such an effect was prevented by cell preincubation with H89 (Fig. 4B and Fig. C). However, no effect was observed upon cell treatment with agents that activate other kinase pathways, such as EGF or PMA (data not shown). Furthermore, cotransfection with an inducible expression vector coding the Cα catalytic subunit of PKA (cPKAα), also favored the nuclear localization of these MAP kinases in the presence of PTP-SL (Fig. 4B and Fig. C). Remarkably, the effect of PKA activation on the colocalization of HA-ERK2 and HA-p38α with wild-type PTP-SL was not observed with the PTP-SL S231A mutant (Fig. 4B and Fig. C), demonstrating that phosphorylation of the Ser231 residue of PTP-SL by PKA inhibits the in vivo association of PTP-SL with HA-ERK2 and HA-p38α, and favors the nuclear translocation of these kinases.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Investigaciones Citológicas, 46010 Valencia, Spain.

ABSTRACT
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.

Show MeSH