Related Experiment Video

Updated: May 22, 2026

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Published on: May 3, 2018

Successive phosphorylation of p27(KIP1) protein at serine-10 and C terminus crucially controls its potency to

Atish R Mohanty¹, Qiuming Kan, Saumya Srivastava

¹Department of Biochemistry and Molecular Biology, Graduate School and Faculty of Medicine, The University of Tokyo, Tokyo 113-0033, Japan.

The Journal of Biological Chemistry

|May 16, 2012

Abstract:

During the G(1)-S transition, the activity of Cdk2 is regulated by its association with p27(KIP1), which in rodent fibroblasts undergoes phosphorylation mainly at serine 10, threonine 187, and C-terminal threonine 197 by KIS, Cdk2, and Pim or ROCK, respectively. Recently Cdc6 the AAA+ ATPase, identified initially to assemble pre-replicative complexes on origins of replication and later to activate p21(CIP1)-inactivated Cdk2, was found also to activate p27-bound Cdk2 but only after the bound p27 is C-terminally phosphorylated. On the other hand, the biological significance of the serine 10 phosphorylation remains elusive aside from its involvement in the stability of p27 itself. We report here that serine 10 phosphorylation is required for efficient C-terminal phosphorylation of its own by PIM and ROCK kinases and critically controls the potency of p27 as a Cdk2 inhibitor. In vitro, PIM1 and active ROCK1 efficiently phosphorylated free as well as Cdk2-bound p27 but only when the p27 was phosphorylated at Ser-10 in advance. Consistently, a Ser-10 nonphosphorylatable mutant p27 protein was not phosphorylated at the C terminus in vivo. Furthermore, when double-phosphorylated, free p27 was no longer a potent inhibitor of Cdk2, and Cdk2-bound p27 could be removed by Cdc6 to reactivate the Cdk2. Thus, phosphorylation at these two sites crucially controls the potency of this CDK inhibitor in two distinct modes.

Insights

Phosphorylation of p27(KIP1) at serine 10 is crucial for its C-terminal modification and its ability to inhibit Cyclin-dependent kinase 2 (Cdk2). This dual phosphorylation controls p27

Area of Science:

Molecular Biology
Cell Cycle Regulation
Protein Phosphorylation

Background:

Cyclin-dependent kinase 2 (Cdk2) activity is tightly regulated during the G1-S transition, a critical phase for cell cycle progression.
p27(KIP1) is a key inhibitor of Cdk2, and its activity is modulated by phosphorylation at specific sites.
Previous studies identified phosphorylation sites on p27(KIP1) and the involvement of Cdc6 in Cdk2 activation, but the role of serine 10 phosphorylation was unclear.

Purpose of the Study:

To investigate the functional significance of serine 10 phosphorylation of p27(KIP1) in regulating its inhibitory activity against Cdk2.
To determine how serine 10 phosphorylation influences C-terminal phosphorylation and the interaction with Cdk2 and Cdc6.

Main Methods:

In vitro kinase assays using PIM1 and ROCK1 on wild-type and mutant p27(KIP1) proteins.
Analysis of p27(KIP1) phosphorylation status in vivo using a serine 10 nonphosphorylatable mutant.
Assessment of Cdk2 inhibition and Cdc6-mediated Cdk2 reactivation.

Main Results:

Serine 10 phosphorylation of p27(KIP1) is essential for efficient C-terminal phosphorylation by PIM and ROCK kinases.
A serine 10 nonphosphorylatable mutant of p27(KIP1) showed impaired C-terminal phosphorylation in vivo.
Double-phosphorylated p27(KIP1) exhibited reduced potency as a Cdk2 inhibitor, and Cdc6 could remove Cdk2-bound p27(KIP1) to reactivate Cdk2.

Conclusions:

Phosphorylation of p27(KIP1) at serine 10 is a prerequisite for its C-terminal modification, critically controlling its inhibitory function.
Dual phosphorylation of p27(KIP1) modulates its potency as a Cdk2 inhibitor through distinct mechanisms, impacting cell cycle control.
These findings elucidate a novel regulatory pathway for Cdk2 activity involving sequential phosphorylation of p27(KIP1).

Read full analysis

More Related Videos

Studying Cell Cycle-regulated Gene Expression by Two Complementary Cell Synchronization Protocols

Studying Cell Cycle-regulated Gene Expression by Two Complementary Cell Synchronization Protocols

Published on: June 6, 2017

Development of Inhibitors of Protein-protein Interactions through REPLACE: Application to the Design and Development Non-ATP Competitive CDK Inhibitors

Development of Inhibitors of Protein-protein Interactions through REPLACE: Application to the Design and Development Non-ATP Competitive CDK Inhibitors

Published on: October 26, 2015

Related Concept Videos

Inhibition of Cdk Activity

Inhibition of Cdk Activity

The orderly progression of the cell cycle depends on the activation of Cdk protein by binding to its cyclin partner. However, the cell cycle must be restricted when undergoing abnormal changes. Most cancers correlate to the deregulated cell cycle, and since Cdks are a central component of the cell cycle, Cdk inhibitors are extensively studied to develop anticancer agents. For instance, cyclin D associates with several Cdks, such as Cdk 4/6, to form an active complex. The cyclin D-Cdk4/6 complex...

Inhibition of CDK Activity

Inhibition of CDK Activity

The orderly progression of the cell cycle depends on the activation of Cdk protein by binding to its cyclin partner. However, the cell cycle must be restricted when undergoing abnormal changes. Most cancers correlate to the deregulated cell cycle, and since Cdks are a central component of the cell cycle, Cdk inhibitors are extensively studied to develop anticancer agents. For instance, cyclin D associates with several Cdks, such as Cdk 4/6, to form an active complex. The cyclin D-Cdk4/6 complex...

Positive Regulator Molecules

Positive Regulator Molecules

Mitotic cell division results in daughter cells that exactly resemble the parent cell. However, errors in the DNA replication or distribution of genetic material may lead to genetic mutations that may be passed down to every new cell formed from the resulting abnormal cell. Propagation of such mutant cells is restricted through checkpoint mechanisms present at different stages of the cell cycle. These checkpoints involve regulator molecules that either promote or demote cell cycle events.

Positive Regulator Molecules

Positive Regulator Molecules

To consistently produce healthy cells, the cell cycle—the process that generates daughter cells—must be precisely regulated.

Anaphase Promoting Complex

Anaphase Promoting Complex

The stepwise destruction of specific proteins is necessary for the progression and completion of the cell cycle. Such proteins are ubiquitinated by ubiquitin ligases and then subsequently destroyed by the proteasome. The SCF (Skp1/Cullin/F-box) and the anaphase-promoting complex (APC) are two important ubiquitin ligases involved in cell cycle progression. While SCF is active throughout the cell cycle, APC gets activated during metaphase to anaphase transition. Cdc20 or Cdh1 binds to APC and...

M-Cdk Drives Transition Into Mitosis

M-Cdk Drives Transition Into Mitosis

Checkpoints throughout the cell cycle serve as safeguards and gatekeepers, allowing the cell cycle to progress in favorable conditions and slow or halt it in problematic ones. This regulation is known as the cell cycle control system.
Cyclin-dependent kinases, or Cdks, work in concert with cyclins to control cell cycle transitions. M-Cdk, a complex of Cdk1 bound to M cyclin, is a well-known example of this coordinated control that drives the transition from the G2 to the M phase.
M cyclin...

Articles linked to this work by shared authors, journal, and citation graph.

Application of blood rheology to develop hemolysis-free serum extraction sampling method for small-volume blood.

Practical laboratory medicine·2026

Severe pancreatic exocrine insufficiency presenting with hypoalbuminemia, anasarca, and pulmonary edema in a patient with prior gastrectomy and suspected immunoglobulin G4-related pancreatobiliary disease.

Clinical journal of gastroenterology·2026

Hybrid ZnCo2S4/Polyindole Electrode for Advanced Supercapacitor: DFT and Raman Mapping-Based Mechanistic Study of Improved Performance.

Small methods·2026

Efficacy and Safety of Evinacumab in Japanese Patients with Homozygous Familial Hypercholesterolemia: Long-term Results from an Open-label, Single-arm, Phase 3 Trial.

Journal of atherosclerosis and thrombosis·2026

Efficacy of an exercise oncology program on sleep quality and circadian rhythm in head and neck cancer patients undergoing chemo-radiation therapy: a randomized controlled trial.

PeerJ·2026

Recombinant kinesin protein of Leishmania donovani and Bacille Calmette-Guerin as an immunomodulatory agent: Insights into their role against visceral leishmaniasis.

Tropical parasitology·2026

See all related articles