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G2/M transition requires multisite phosphorylation of oncoprotein 18 by two distinct protein kinase systems
N Larsson1, H Melander, U Marklund
1Department of Cell and Molecular Biology, University of Umeå, Sweden.
Abstract:
Oncoprotein 18 (Op18) is a conserved cytosolic protein that is a target for both cell cycle and cell surface receptor-regulated phosphorylation events. The four residues Ser16, Ser25, Ser38, and Ser63 are all subject to cell cycle-regulated phosphorylation. Ser25 and Ser38 are targets for cyclin dependent kinases (CDKs), while Ser16 and Ser63 are phosphorylated by an unidentified protein kinase. We have recently shown that induced expression of a CDK target site-deficient mutant, Op18-S25A,S38A, blocks human cell lines during G2/M transition. In the present report we show that mitosis is associated with complete phosphorylation of the two Op18 CDK target sites Ser25 and Ser38 and that Ser16 and Ser63 are also phosphorylated to a high stoichiometry. To evaluate the function of multisite phosphorylation of Op18, we expressed and analyzed the cell cycle phenotype of different kinase target site-deficient mutants. The data showed that induced expression of the S16A,S63A, S25A,S38A, and S16A,S25A,S38A,S63A mutants all resulted in an indistinguishable phenotype, i.e. immediate G2/M block and subsequent endoreduplication, a given fraction of G2 versus M-phase blocked cells, and a characteristic nuclear morphology of M-blocked cells. This result was unexpected; however, a likely explanation was provided by analysis of Op18 phosphoisomers, which revealed that mutations of the CDK sites interfere with phosphorylation of Ser16 and Ser63. The simplest interpretation of our results is that phosphorylation of Ser16 and Ser63 is essential during G2/M transition and that the phenotype of the S25A,S38A mutant is mediated by the observed block of Ser16/Ser63 phosphorylation.
Insights
Oncoprotein 18 (Op18) phosphorylation at Ser16 and Ser63 is crucial for G2/M cell cycle transition. Disrupting cyclin-dependent kinase (CDK) sites (Ser25, Ser38) unexpectedly impacts these other sites, leading to cell cycle arrest.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Oncoprotein 18 (Op18) is a key cytosolic protein involved in cell cycle regulation.
- Op18 phosphorylation occurs at four sites: Ser16, Ser25, Ser38, and Ser63.
- Ser25 and Ser38 are phosphorylated by cyclin-dependent kinases (CDKs), while Ser16 and Ser63 are targeted by an unknown kinase.
Purpose of the Study:
- To investigate the functional significance of Op18 multisite phosphorylation during the cell cycle.
- To determine the impact of specific phosphorylation site mutations on Op18 function and cell cycle progression.
Main Methods:
- Generation and expression of Op18 kinase target site-deficient mutants (S16A, S63A, S25A, S38A, and quadruple mutant).
- Analysis of cell cycle phenotypes, including G2/M transition, endoreduplication, and nuclear morphology.
- Examination of Op18 phosphoisomers to understand interdependencies between phosphorylation sites.
Main Results:
- Mutations at CDK sites (Ser25, Ser38) interfered with the phosphorylation of Ser16 and Ser63.
- Expression of S16A,S63A, S25A,S38A, and quadruple mutants induced an immediate G2/M block and subsequent endoreduplication.
- Cells expressing these mutants exhibited characteristic M-phase-blocked nuclear morphology.
Conclusions:
- Phosphorylation of Ser16 and Ser63 on Op18 is essential for proper G2/M transition.
- The observed G2/M block phenotype in Op18-S25A,S38A mutants is likely mediated by the disruption of Ser16/Ser63 phosphorylation.
- Multisite phosphorylation of Op18 involves complex interdependencies between different kinase target sites.