Figure 1
AKAP4 is an ERK1/2 substrate.
(A) AKAP4 is a potential MAPK substrate. Human spermatozoa were stimulated with 100 nM PMA for 15 min and subjected to western-blot with anti-MPM2, a phospho-specific anti-MAPK substrate antibody. A major PMA-stimulated band (~90 kDa) was subjected to MALDI-TOF mass spectrometry. The peptide coverage map of the specific identified protein indicated that the protein is AKAP4 (82 kDa). (B) proAKAP4 is phosphorylated in vitro by ERK2 on Thr265. HEK293T cells were transfected with proAKAP4 fused to turboGFP in its N-terminus using the calcium phosphate method. Cells were also transfected with turboGFP, turboGFP-proAKAP4130–266 deletion mutant and turboGFP-proAKAP4T265A mutant. Later cells were lysed, and the substrates were immunoprecipitated with an anti-turboGFP antibody. The precipitates were subjected to an in vitro phosphorylation assay with activated ERK2 as mentioned under experimental procedures. The upper panel is the autoradiogram, and the lower panel is a western blot of anti-tGFP. Lanes from left to right are as followes: tGFP, tGFP-proAKAP4, tGFP-proAKAP4 delta (130–266), and tGFP-proAKAP4-T265A. The experiment is a representative result from three experiments. (C) proAKAP4 is phosphorylated by ERK1/2 in spermatozoa in vivo. Human spermatozoa were washed and pretreated with U0126 (25 μM) or GDC-0994 (10 μM) for 20 min. Thereafter, the cells were treated with or without PMA (25 nM) for 15 min. Cells lysate were immunoprecipitated with anti-proAKAP4, followed by western blotting with anti-MPM2, or anti-proAKAP4 for loading control. The experiment is a representative result.
