A proteome-wide phospho-mass spectrometry screen identifies downstream mitotic effectors of CDKN3. (A) Strategy to identify the CDKN3-CDC2 targets. (B) Mass spectrometry reveals hyperphosphorylation of CKβpSer-209 upon CDKN3 knockdown. Two samples per siRNA were analyzed in duplicate runs (with a total of four LC-MS/MS experiments); representative images are shown. (C) Western blot verification of proteomic screen. Note the increased phosphorylation of endogenous CKβ upon CDKN3 knockdown, whereas the total endogenous CKβ protein level remains the same. (D) Endogenous CKβpSer-209 localizes to centrosomes. (E) Endogenous CKβpSer-209 localizes to centrosomes throughout mitosis and disappears from centrosomes in telophase. (F) Western blot verification of CKβ knockdown. (G) CKβ is essential for mitotic spindle checkpoint. HeLa cells were transfected with CK2β siRNAs and treated with 100 nm taxol (24 h) 2 d after transfection. (H) Quantification of SAC failure resulting from CKβ knockdown. P < 0.0001 in one-way ANOVA; n = at least 5 counts per siRNA. Error bars represent mean values ± SEM.
