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The MAPK pathway triggers activation of Nek2 during chromosome condensation in mouse spermatocytes

Dipartimento di Sanità Pubblica e Biologia Cellulare, Facoltà di Medicina e Chirurgia, Università di Roma ‘Tor Vergata’, Via O. Raimondo 8, 00173 Rome, Italy

View larger version (45K): [in a new window]   Fig. 1. Chromosome condensation requires MAPK activation in mouse spermatocytes. The specific inhibitor U0126, which blocks activation of MAPK signaling, inhibits chromosome condensation during meiotic progression induced by okadaic acid. (A) Control cells; (B) cells treated for 6 hours with 0.5 µM OA, where condensation of chromatin into metaphase is observed; (C) cells preincubated with U0126 for 12 hours are no different from control cells; (D) cells preincubated for 12 hours with 10 µM U0126 before treatment with OA as described above, and in which chromatin condensation was strongly inhibited; cell nuclei were isolated and stained using 5% Giemsa. (E) Immunoblot analysis using anti-phospho-Erks monoclonal antibody (top) or anti-Erks polyclonal antibody (bottom) of protein extracts (30 µg/lane) from cells treated as described in A-D.

View larger version (50K): [in a new window]   Fig. 2. Specific expression of p90Rsk isoforms during mouse spermatogenesis. (A) Northern blot analysis of total RNA (20 µg) isolated from total testis or purified cell populations (see Materials and Methods) with specific cDNA probes for p90Rsk1, p90Rsk2 and p90Rsk3. (B) Western blot analysis of p90Rsk isoforms as in A confirms the results obtained with mRNAs analysis at the protein level. Immunoprecipitation of soluble protein extracts (500 µg) obtained from same cell populations as in A was performed using antibodies specific to p90Rsk1, p90Rsk2 or p90Rsk3, and immunoprecipitated proteins were analyzed by western blot using the same specific antibodies. As positive control, immunoprecipitated proteins obtained from COS cells transfected with recombinant p90Rsk1, p90Rsk2 or p90Rsk3 were used as control for antibody specificity. Molecular weights were calculated using the Gibco Benchmark protein ladder as standard.

View larger version (101K): [in a new window]   Fig. 3. Differential expression of p90Rsk2 and p90Rsk3 in mouse spermatocytes and spermatids. Histological sections obtained from adult testis were stained with antibodies directed against the three p90Rsk isoforms. Immunostaining was carried out using the anti-p90Rsk1, anti-p90Rsk2 or anti-p90Rsk3 antibody and a rhodamine-conjugated secondary antibody as described in the Materials and Methods. Sections were also stained with Hoechst 3332 to detect chromatin (right panels). In all panels, L stands for lumen of the tubule and B for basal lamina. In the anti-p90Rsk2 panels, the arrowheads indicate a representative spermatocyte, whereas in the anti-p90Rsk3 panels, the arrowheads point to a representative elongated spermatid (central tubule) and a representative round spermatid (lower tubule). The layer of spermatogonia just above the basal lamina is stained by both the anti-p90Rsk2 and the anti-p90Rsk3 antibody.

View larger version (11K): [in a new window]   Fig. 4. p90Rsk2 is activated during meiotic G2/M progression induced by okadaic acid. Soluble extracts (500 µg) obtained from either control pachytene spermatocytes or spermatocytes treated for 6 hours with 0.5 µM OA were immunoprecipitated using antibodies specific for the three p90Rsk isoforms. The immunoprecipitated kinase activity was assayed using 10 µM S6 peptide and 10 µM 32P--ATP as substrates. p90Rsk2 activity was increased 10-fold by incubation of spermatocytes with okadaic acid. Data represent the mean±s.d. of triplicate determinations from three separate experiments.

View larger version (19K): [in a new window]   Fig. 5. Activation of p90Rsk2 in mouse spermatocytes is mediated by the MAPK pathway. (A) Extracts from control or OA-treated cells preincubated in the absence or presence of 10 µM U0126 to inhibit signaling through the MAPK pathway were immunoprecipitated using the anti-p90Rsk2 antibody. Kinase activity present in the immunoprecipitates was assayed as described in the legend to Fig. 3. Data represent the mean±s.d. of triplicate determinations from three separate experiments. (B) Western blot analysis of p90Rsk2 in representative samples of the immunoprecipitates. The upward shift in electrophoretic mobility in SDS-PAGE of the sample obtained from cells treated with OA indicate that p90Rsk2 was hyperphosphorylated and activated during G2/M progression of mouse spermatocytes.

View larger version (43K): [in a new window]   Fig. 6. Subcellular localization of p90Rsk2 in mouse spermatocytes. The panels on the left show the immunofluorescence analysis of control (top) or OA-treated (6 hours with 0.5 µM, bottom) cells. Immunostaining was carried out using the anti-p90Rsk2 antibody and a rhodamine-conjugated secondary antibody as described in the Materials and Methods. Cells were also stained with Hoechst 3332 to detect chromatin (right panels).

View larger version (64K): [in a new window]   Fig. 7. Activated Erk1/2 and p90Rsk2 associate with meiotic chromosomes. Chromatin and/or chromosomes were obtained by hypotonic treatment of spermatocytes and were attached directly on a microscope slide for immunofluorescence analysis as described in the legend to Fig. 5. (A) Chromatin was stained with anti-p90Rsk2 antibody (right panels) or Hoechst dye (left panels). (B) Chromatin was stained with anti-phosphoErk1/2 antibody (right panels) or Hoechst (left panels). Both p90Rsk2 and activated Erks colocalize with meiotic chromosomes.

View larger version (40K): [in a new window]   Fig. 8. Histone H3 phosphorylation is not mediated by the MAPK pathway in mouse spermatocytes. (A) Immunokinase assay of p90Rsk2 activity using H3 as substrate. p90Rsk2 was immunoprecipitated as described in the legend to Fig. 4 from cells treated as indicated in the figure and in the text, and activity was assayed using 0.1 mg/ml H3 and 10 mM ATP as substrates. At the end of the incubation, proteins were separated on a 10% SDS-PAGE and analyzed by western blot using either anti-phospho-H3 to detect phosphorylation of Ser10 in H3, or anti-p90Rsk2 to verify that equal amounts of enzyme were used in the assay. (B) Western blot analysis of extracts (20 µg) of cells treated as indicated in the figure and in the text using either the anti-phosphoH3 antibody to detect phosphorylation of Ser10 in H3 in intact cells, or the anti-H3 antibody to quantify the amount of H3 in the samples.

View larger version (34K): [in a new window]   Fig. 9. Activation of the Ser/Thr protein kinase Nek2 in mouse spermatocytes is MAPK dependent. (A) Immunokinase assay of Nek2 activity. Nek2 was immunoprecipitated from control or OA-treated cells preincubated or not with U0126 (10 µM for 12 hours) and incubated for 20 minutes at 30°C in the presence of 1 µg full-length MBP and 10 µM 32P--ATP as substrates. Reactions were terminated by adding SDS-sample buffer, samples were boiled for 5 minutes and protein were separated on a 13% SDS-PAGE. The dried gel was then autoradiographed. (B) Reconstitution of Nek2 activation in vitro. Active and inactive p90Rsk2 were immunopurified as described in the Materials and Methods, and incubated with cytosolic extracts from control spermatocytes for 30 minutes at 30°C in the presence of 10 µM ATP. At the end of the incubation, cytosolic extracts were separated by centrifugation and Nek2 was immunopurified using 1 mg anti-Nek2 antibody and proteinA-sepharose beads. The activity of Nek2 was assayed as described in A and the dried gel was autoradiographed. (C) Coomassie Blue staining of GST-Nek21-272 and GST-Nek2273-444 purified from E. coli that were used in D as substrates for p90Rsk2. Arrowheads on the left side point to degradation products routinely observed in purified GST-Nek21-272. (D) In vitro assay for phosphorylation of GST-Nek21-272 and GST-Nek2273-444 by p90Rsk2. Active or inactive immunopurified p90Rsk2 was incubated with GST-Nek2 proteins for 30 minutes at 30°C in the presence of 10 µM ATP. The reaction was terminated adding SDS-PAGE sample buffer and proteins were separated on a 10% SDS-PAGE. The gel was dried and autoradiographed. Arrowhead on the left shows the position of GST-Nek21-272 degradation products.

View larger version (20K): [in a new window]   Fig. 10. Hypothetical model of the activation of Nek2 and induction of chromatin condensation by Erk1 and p90Rsk2. Erk1 may be activated by local extracellular signals when spermatocytes are mature to enter the meiotic divisions. Activation of Erk1 allows its translocation into the nucleus where it phosphorylates and activates p90Rsk2. Activated Erk1 and p90Rsk2 associates with chromatin, and p90Rsk2 phosphorylates and activates the chromatin-bound Nek2. Activation of Nek2 triggers chromatin condensation into metaphase chromosomes.