Mitosis is initiated by the activation of cyclin B1-Cdk1 and the translocation of the active complexes to the nucleus. The all-or-none, irreversible nature of mitosis arises from a bistable trigger circuit composed of interlinked positive and double-negative feedback loops where active cyclin B1-Cdk1 promotes cyclin B1-Cdk1 activation, and nuclear cyclin B1-Cdk1 promotes cyclin B1-Cdk1 translocation. This systems-level behavior depends upon the kinetic properties of the individual components of the feedback loops, including the shapes of their response functions. During the last funding period, we showed that the multisite phosphorylation of Wee1A and Cdc25C allows them to generate highly ultrasensitive response, which in turn makes the bistability of the mitotic trigger possible. Here we propose to build upon these discoveries to determine how multisite phosphorylation leads to the ultrasensitive regulation of the key Cdk1 substrate and regulator Wee1A, and to examine the temporal order of protein dephosphorylation during mitotic exit. There are three Specific Aims:
Aim 1. The role of Cks proteins in the regulation of Cdk phosphorylation. Mitotic Cdk1 complexes consist of three proteins: the Cdk1 protein kinase, the allosteric activator cyclin B, and a third small protein referred to as Suc1 in S. pombe, Cks1 in S. cerevisiae, and Cks1 or 2 in vertebrates, whose function is more enigmatic. We have recently found that Xenopus Cks2 acts as a bivalent adaptor, binds independently to both Cdk1 and phospho-Wee1A, and builds a substantial threshold into the switch-like response of Wee1A to Cdk1. Here we aim to explore the generality of this phenomenon and examine the regulation of Cks2 by phosphorylation.
Aim 2. The mechanism of Wee1A inactivation. We have shown that the phosphorylation of Wee1A at T53 primes the protein for subsequent phosphorylations at T104 and T150, and that these phosphorylations result in Wee1A inactivation. Here we propose a series of biophysical and structural studies aimed at understanding how T104/T150 phosphorylation affects the Wee1A kinase domain.
Aim 3. Mechanisms of temporal organization in mitotic exit. Mitotic exit is a highly organized cell biological process. Here we propose to describe how the dephosphorylation of mitotic phosphoproteins is organized temporally, and determine what mechanisms allow the dephosphorylation to proceed in an orderly fashion.

Public Health Relevance

Mitosis is triggered by the activation of the protein kinase cyclin B1-Cdk1 and the translocation of the active cyclin B1-Cdk1 complexes to the nucleus. Here we propose to study the inactivation of the role of the Cks2 protein in targeting Cdk1 to particular substrates, the mechanism of inactivation of the critical Cdk1 regulator Wee1A, and complex process of protein dephosphorylation during mitotic exit. The overall aim is to understand how the systems-level organization of this critical regulatory system.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM046383-26
Application #
9054845
Study Section
Cellular Signaling and Regulatory Systems Study Section (CSRS)
Program Officer
Melillo, Amanda A
Project Start
1992-09-01
Project End
2019-02-28
Budget Start
2016-03-01
Budget End
2017-02-28
Support Year
26
Fiscal Year
2016
Total Cost
Indirect Cost
Name
Stanford University
Department
Pharmacology
Type
Schools of Medicine
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94304
Kamenz, Julia; Ferrell Jr, James E (2017) The Temporal Ordering of Cell-Cycle Phosphorylation. Mol Cell 65:371-373
Ha, Sang Hoon; Kim, Sun Young; Ferrell Jr, James E (2016) The Prozone Effect Accounts for the Paradoxical Function of the Cdk-Binding Protein Suc1/Cks. Cell Rep 16:2047
Ferrell Jr, James E (2016) Perfect and Near-Perfect Adaptation in Cell Signaling. Cell Syst 2:62-7
Ha, Sang Hoon; Kim, Sun Young; Ferrell Jr, James E (2016) The Prozone Effect Accounts for the Paradoxical Function of the Cdk-Binding Protein Suc1/Cks. Cell Rep 14:1408-1421
Ha, S H; Ferrell Jr, J E (2016) Thresholds and ultrasensitivity from negative cooperativity. Science 352:990-3
Gelens, Lendert; Huang, Kerwyn Casey; Ferrell Jr, James E (2015) How Does the Xenopus laevis Embryonic Cell Cycle Avoid Spatial Chaos? Cell Rep 12:892-900
Ferrell Jr, James E; Ha, Sang Hoon (2014) Ultrasensitivity part I: Michaelian responses and zero-order ultrasensitivity. Trends Biochem Sci 39:496-503
Gelens, Lendert; Anderson, Graham A; Ferrell Jr, James E (2014) Spatial trigger waves: positive feedback gets you a long way. Mol Biol Cell 25:3486-93
Tsai, Tony Y-C; Theriot, Julie A; Ferrell Jr, James E (2014) Changes in oscillatory dynamics in the cell cycle of early Xenopus laevis embryos. PLoS Biol 12:e1001788
Ferrell Jr, James E; Ha, Sang Hoon (2014) Ultrasensitivity part III: cascades, bistable switches, and oscillators. Trends Biochem Sci 39:612-8

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