Distinct Pools of cdc25C Are Phosphorylated on Specific TP Sites and Differentially Localized in Human Mitotic Cells

Abstract

BackgroundThe dual specificity phosphatase cdc25C was the first human cdc25 family member found to be essential in the activation of cdk1/cyclin B1 that takes place at the entry into mitosis. Human cdc25C is phosphorylated on Proline-dependent SP and TP sites when it becomes active at mitosis and the prevalent model is that this phosphorylation/activation of cdc25C would be part of an amplification loop with cdk1/cyclin B1.Methodology/Principal FindingsUsing highly specific antibodies directed against cdc25C phospho-epitopes, pT67 and pT130, we show here that these two phospho-forms of cdc25C represent distinct pools with differential localization during human mitosis. Phosphorylation on T67 occurs from prophase and the cdc25C-pT67 phospho-isoform closely localizes with condensed chromosomes throughout mitosis. The phospho-T130 form of cdc25C arises in late G2 and associates predominantly with centrosomes from prophase to anaphase B where it colocalizes with Plk1. As shown by immunoprecipitation of each isoform, these two phospho-forms are not simultaneously phosphorylated on the other mitotic TP sites or associated with one another. Phospho-T67 cdc25C co-precipitates with MPM2-reactive proteins while pT130-cdc25C is associated with Plk1. Interaction and colocalization of phosphoT130-cdc25C with Plk1 demonstrate in living cells, that the sequence around pT130 acts as a true Polo Box Domain (PBD) binding site as previously identified from in vitro peptide screening studies. Overexpression of non-phosphorylatable alanine mutant forms for each isoform, but not wild type cdc25C, strongly impairs mitotic progression showing the functional requirement for each site-specific phosphorylation of cdc25C at mitosis.Conclusions/SignificanceThese results show for the first time that in human mitosis, distinct phospho-isoforms of cdc25C exist with different localizations and interacting partners, thus implying that the long-standing model of a cdc25C/cdk1 multi-site auto amplification loop is implausible.

Phosphorylation Mediated Regulation of Cdc25 Activity, Localization and Stability

“…A phospho-T130 specific antibody shows that Cdc25C phosphorylated on this site localizes to the centrosome. [145] A localization for the de-inhibition of Cdc25C fits well with the putative centrosomal localization of Cdc25C activation.…”

“…S99, S148, S178, S192, S204, S206, T226, S234, S359, T561, S567, T569 [60,156,157,162] Cdk1-cyclin B S18, S40, S88, S116, S261, S283, S321 [149,151,167] Chk1 S76, S124, S178, T507 [168][169][170][171][172][173]200] Chk2 S124, S278 [173,175] Casein Kinase 1ε S82 [199] Casein Kinase 1α S79, S82 [198] p38 S76, S124 [168,175] GSK-3β S76 [193,194] NEK11 S82, S88 [187] Plk3 S80 [193,194] Cdk1-cyclinB S50, S160, S321 [135,136,246] Chk1 S151, S230, S323, S563 [236,238,239] Aurora S353 [124] Casein Kinase 2 S186, S187 [235] JNK S101, S103 [280] MEK/ERK S249 [259] p38 S323 [208] MK2 S323 [210] Plk1 T167, S209, T404, S465 [136] Cdk1/cyclin B T48, T67, S122, T130, S168, S214 [42,143,145,246] Chk1 S216, S247, S263 [219-221, 225, 226] Chk2 S216 [209,242] Casein Kinase 2 T236 …”

“…[144] Use of phospho-specific antibodies recently showed that phosphorylation of T48, T67 and T130 occur on spatially separate pools of human Cdc25C. [145] T67-phosphorylated Cdc25C is chromatin associated from prophase until telophase while T130-phosphorylated Cdc25C localizes to the centrosomes. T130 phosphorylation creates a Plk1 binding site on Cdc25C.…”

Phosphatases and kinases regulating CDC25 activity in the cell cycle: clinical implications of CDC25 overexpression and potential treatment strategies

“…The non-phosphorylated mutant forms of CDC25C impair mitotic progression in human cells. This finding contradicts auto-amplification mechanism involving CDC25C and Cdk1 in mitotic activation [74] (Figure 3; Table 3). …”

The Centrosome and Its Duplication Cycle

“…This appears to require the activity of the Cdc25B isoform (Gabrielli et al 1996;De Souza et al 2000;Lindqvist et al 2005) whose association with centrosomes is promoted by Aurora A (Dutertre et al 2004). Specific phosphorylation events on centrosomal Cdc25C in late G 2 phase could also play some role (Franckhauser et al 2010). Although the relationship to the spindle pole is less well developed than in fission yeast, Plk1 activity levels do determine the timing of mitotic commitment in human cells (Gavet and Pines 2010).…”