SYSTEMATIC IDENTIFICATION OF MITOTIC PHOSPHOPROTEINS

Background: Cyclin-dependent kinases (CDKs) are thought to initiate an d coordinate cell division processes by sequentially phosphorylating k ey targets; in most cases these substrates remain unidentified. Result s: Using a screen that scores for phosphorylation of proteins, which w ere translated from pools of cDNA plasmids in vitro, by either phospho epitope antibody recognition or electrophoretic mobility shifts, we ha ve identified 20 mitotically phosphorylated proteins from Xenopus embr yos, 15 of which have sequence similarity to other proteins. Of these proteins, five have previously been shown to be phosphorylated during mitosis (epithelial-microtubule associated protein-115, Oct91, Elongat ion factor 1 gamma, BRG1 and Ribosomal protein L18A), five are related to proteins postulated to have roles in mitosis (epithelial-microtubu le associated protein-115, Schizosaccharomyces pombe Cdc5, inner-centr osome protein, BRG1 and the RNA helicase WM6), and nine are related to transcription factors (BRG1, negative co-factor 2 alpha, Oct91, S. po mbe Cdc5, HoxD1, Sox3, Vent2, and two isoforms of Xbr1b). Of 16 substr ates tested, 14 can be directly phosphorylated in vitro by the mitotic CDK, cyclin B-Cdc2, although three of these may be physiological subs trates of other kinases activated during mitosis. Conclusions: Examina tion of this broad set of mitotic phosphoproteins has allowed us to dr aw three conclusions about how the activation of CDKs regulates cell-c ycle events. First, Cdc2 itself appears to directly phosphorylate most of the mitotic phosphoproteins. Second, during mitosis most of the su bstrates are phosphorylated more than once and a number may be targets of multiple kinases, suggesting combinatorial regulation. Third, the large fraction of mitotic phosphoproteins that are presumptive transcr iption factors, two of which have been previously shown to dissociate from DNA during mitosis, suggests that an important function of mitoti c phosphorylation is to strip the chromatin of proteins associated wit h gene expression.