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-Nek2_1-272 and GST-Nek2_273-444 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-Nek2_1-272. (D) In vitro assay for phosphorylation of GST-Nek2_1-272 and GST-Nek2_273-444 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-Nek2_1-272 degradation products.

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