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The nucleus, a site for signal termination by sequestration and inactivation of p42/p44 MAP kinases

¹ Institute of Signaling, Developmental Biology and Cancer Research, CNRS UMR-6543, Centre Antoine Lacassagne, 06189 Nice, France
² Serono Pharmaceutical Research Institute, Ares-Serono International SA, 1228 Plan-les-Ouates, Geneva, Switzerland

View larger version (38K): [in a new window]   Fig. 1. Both mitogenic and non-mitogenic agonists trigger the rapid entry of activated p42/p44 MAPKs into the nucleus. Quiescent CCL39 fibroblast cells were stimulated for the indicated times with either 100 µM thrombin receptor peptide agonist (TRP, A) or 10% serum (B). 0 minutes corresponds to non-stimulated cells. Indirect immunofluorescence detection was performed with the monoclonal antibody anti-activated p42/p44 MAPKs from Sigma. Identical results were obtained with the polyclonal antibody anti-activated p42/p44 MAPKs V6671 from Promega (data not shown). The specificity of the antibody from Sigma has been published (Yung et al., 1997) and was verified in Raf-1:ER cells in which the p42/p44 MAPKs can selectively be activated (see Fig. 2B for immunofluorescence and Fig. 5, middle blot, for western blot).

View larger version (60K): [in a new window]   Fig. 2. Long-term activation of p42/p44 MAPKs induces their nuclear accumulation in the dephosphorylated form. Quiescent CCL39 fibroblast cells (A) or quiescent Raf-1:ER cells (B), were stimulated for the indicated times with 10% serum or 1 µM estradiol, respectively. 0 minutes corresponds to non-stimulated cells. Indirect immunofluorescence detection was performed with the monoclonal antibody anti-activated p42/p44 MAPKs from Sigma. (C) Quiescent CCL39 fibroblast cells were left non-stimulated (NS) (a,c) or stimulated with 10% serum (FCS) for 3 hours (b,d,e,f). Indirect immunofluorescence detection was performed either with the polyclonal anti-p42/p44 MAPKs antibody from UBI (green; a,b,e) or with the monoclonal anti-activated p42/p44 MAPKs antibody (red; c,d,f). For e and f, confocal microscopy was performed as described in Materials and Methods.

View larger version (91K): [in a new window]   Fig. 3. Tyrosine phosphatase inhibitors prevent p42/p44 MAPKs inactivation in the nucleus. Quiescent CCL39 fibroblast cells were stimulated for 3 hours with 10% serum. Cells were then treated for the indicated times with bpV(phen) 1 mM (a,b,c,d). 0 minutes corresponds to non-treated cells (a). In e and f, cells were treated for 15 minutes with 1 mM estradiol (15 min est) or 30 µM cycloheximide (15 min CHX), respectively, following a 3 hour 10% serum stimulation. Indirect immunofluorescence detection was performed with the monoclonal antibody anti-activated p42/p44 MAPKs.

View larger version (53K): [in a new window]   Fig. 4. The phosphatases that dephosphorylate p42/p44 MAPKs in the nucleus are neo-synthesized. Quiescent CCL39 fibroblast cells were stimulated for 3 hours with 10% serum (a,d). 30 µM cycloheximide (b,e) or 5 µg/ml actinomycine D (c,f) were added throughout serum stimulation. Indirect immunofluorescence detection was performed either with the polyclonal antibody anti-p42/p44 MAPKs from UBI (green; a,b,c) or with the monoclonal antibody anti-activated p42/p44 MAPKs (red; d,e,f).

View larger version (65K): [in a new window]   Fig. 5. The time course of nuclear p42/p44 MAPKs activation correlates with the phosphorylation of a nuclear p42/p44 MAPKs substrate: HIF-1. Quiescent Raf-1:ER cells were either left untreated (normoxia, lane 1) or placed in hypoxic conditions for 5 hours (lanes 2-8). Before the end of the hypoxic period, Raf-1:ER cells were stimulated with 100 nM estradiol for the indicated times. After 180 minutes of estradiol treatment the cells were treated for 30 minutes with the tyrosine phosphatase inhibitor bpV(phen) 1 mM (lane 8). Immunoblotting was performed using an anti-HIF-1 antiserum (upper blot), the monoclonal antibody anti-activated p42/p44 MAPKs (middle blot) or an anti-MKP1 antiserum (lower blot).

View larger version (28K): [in a new window]   Fig. 6. The C-terminal peptide of p90rsk can disrupt the interaction of p42/p44 MAPKs with their nuclear phosphatases, thus preventing nuclear p42/p44 MAPKs inactivation. (A) Alignment of the amino acid sequences of the peptide corresponding to the docking site for p42/p44 MAPKs on p90rsk used in microinjection, the control peptide with the mutated residues shown in green and the docking sites on MKP1, MKP2 and MKP3 with the critical positively charged residues shown in red. (B) In vitro test of the inhibition of the MKP3 activity by the BSA-coupled peptides, the free peptides or BSA alone. (C) Nuclear microinjection of BSA-p90rskwt peptide (a,b,c,d) or BSA-p90rskmut peptide (e,f). Indirect immunofluorescence detection was performed either with the polyclonal antibody anti-BSA (green; a,b,e) or with the monoclonal antibody anti-activated p42/p44 MAPKs (red; c,d,f). (D) Cytoplasmic microinjection of BSA-p90rskwt peptide (a,b) or BSA-p90rskmut peptide (c,d). Indirect immunofluorescence detection was performed as described for C.

View larger version (76K): [in a new window]   Fig. 7. p42/p44 MAPKs constantly shuttle between the nucleus and the cytoplasm and their localization is dependent on the balance between nuclear and cytoplasmic anchors throughout stimulation. (A) Quiescent CCL39 cells (a,b) or 5 minutes 10% serum stimulated CCL39 cells (c,d) were either left untreated (a,c) or treated for 5 minutes with 5 ng/ml of the active nuclear export inhibitor Leptomycine B (LMB) (b,d). (B) Quiescent CCL39 cells were stimulated for 3 hours with 10% serum and either left untreated (a), treated for 10 minutes with 5 ng/ml LMB (b), treated for 10 minutes with 10 µM of the MEK inhibitor UO126 (c), or treated for 10 minutes with both LMB and UO126 (d). Indirect immunofluorescence detection was performed with the polyclonal antibody anti-p42/p44 MAPKs from UBI.

View larger version (33K): [in a new window]   Fig. 8. Proposed model for the nucleo-cytoplasmic shuttling of p42/p44 MAPKs. During stimulation, p42/p44 MAPKs localization and activity are regulated in three phases: (1) in quiescent cells, p42/p44 MAPKs are fully inactive and mainly located in the cytoplasm, sequestered by their interaction with MEK; (2) after 5 minutes of stimulation (mitogenic or not) there is a massive entry of activated p42/p44 MAPKs into the nucleus; (3) after 3 hours of mitogenic stimulation, p42/p44 MAPKs accumulate massively in the nucleus bound in an inactive form to nuclear anchors. Active forms of p42/p44 MAPKs are represented in red and inactive forms are shown in green.