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Published on: February 22, 2014
Systematic identification of mitotic phosphoproteins
P T Stukenberg1, K D Lustig, T J McGarry
1Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Background:
Cyclin-dependent kinases (CDKs) are thought to initiate and coordinate cell division processes by sequentially phosphorylating key targets; in most cases these substrates remain unidentified.
Results:
Using a screen that scores for phosphorylation of proteins, which were translated from pools of cDNA plasmids in vitro, by either phosphoepitope antibody recognition or electrophoretic mobility shifts, we have identified 20 mitotically phosphorylated proteins from Xenopus embryos, 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, Elongation factor 1gamma, BRG1 and Ribosomal protein L18A), five are related to proteins postulated to have roles in mitosis (epithelial-microtubule associated protein-115, Schizosaccharomyces pombe Cdc5, innercentrosome protein, BRG1 and the RNA helicase WM6), and nine are related to transcription factors (BRG1, negative co-factor 2alpha, Oct91, S. pombe Cdc5, HoxD1, Sox3, Vent2, and two isoforms of Xbr1b). Of 16 substrates tested, 14 can be directly phosphorylated in vitro by the mitotic CDK, cyclin B-Cdc2, although three of these may be physiological substrates of other kinases activated during mitosis.
Conclusions:
Examination of this broad set of mitotic phosphoproteins has allowed us to draw three conclusions about how the activation of CDKs regulates cell-cycle events. First, Cdc2 itself appears to directly phosphorylate most of the mitotic phosphoproteins. Second, during mitosis most of the substrates 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 transcription factors, two of which have been previously shown to dissociate from DNA during mitosis, suggests that an important function of mitotic phosphorylation is to strip the chromatin of proteins associated with gene expression.
Insights
This study identified 20 mitotic phosphoproteins in Xenopus embryos, revealing that cyclin-dependent kinases (CDKs) directly phosphorylate many substrates, suggesting combinatorial regulation and chromatin stripping during cell division.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Cyclin-dependent kinases (CDKs) regulate cell division by phosphorylating key targets, but these substrates are often unknown.
- Understanding CDK substrates is crucial for deciphering cell cycle control mechanisms.
Purpose of the Study:
- To identify novel mitotic phosphoproteins in Xenopus embryos.
- To investigate the direct substrates of the mitotic CDK, cyclin B-Cdc2.
- To elucidate the regulatory roles of mitotic phosphorylation in cell cycle progression.
Main Methods:
- A screen was developed to detect protein phosphorylation in vitro using phosphoepitope antibody recognition or electrophoretic mobility shifts.
- Proteins were translated from cDNA plasmid pools.
- Xenopus embryos were used as the biological system.
Main Results:
- Twenty mitotically phosphorylated proteins were identified from Xenopus embryos.
- Fifteen of these proteins showed sequence similarity to known proteins.
- Fourteen of 16 tested substrates were directly phosphorylated by cyclin B-Cdc2 in vitro, indicating Cdc2 directly phosphorylates most mitotic phosphoproteins.
- A significant fraction of identified proteins were transcription factors, suggesting a role in chromatin regulation.
Conclusions:
- Cdc2 directly phosphorylates the majority of identified mitotic phosphoproteins.
- Mitotic phosphoprotein substrates are often multiply phosphorylated and may be targeted by multiple kinases, indicating combinatorial regulation.
- Mitotic phosphorylation of transcription factors suggests a mechanism for stripping gene expression-associated proteins from chromatin during cell division.
