Two Populations of p27 Use Differential Kinetics to Phosphorylate Ser-10 and Thr-187 via Phosphatidylinositol 3-Kinase in Response to Fibroblast Growth Factor-2 Stimulation

Jeong Goo Lee,Eunduck P Kay

doi:10.1074/jbc.m607808200

Jeong Goo Lee, Eunduck P Kay

Open Access

https://doi.org/10.1074/jbc.m607808200

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Abstract

The cyclin-dependent kinase inhibitor p27 regulates cell cycle progression. We investigated whether FGF-2 uses PI 3-kinase to facilitate phosphorylation of p27 on serine 10 (Ser-10) and threonine 187 (Thr-187) and whether the two phosphorylation sites were differentially regulated. FGF-2 stimulation dramatically increased p27 phosphorylation at Ser-10 and Thr-187 using differential kinetics, and the FGF-2-induced p27 phosphorylation was completely blocked at both sites by LY294002. We determined the physical and biochemical interaction of p27 with the Cdk2-cyclin E complex in response to FGF-2 stimulation. Maximal p27 binding to Cdk2-cyclin E occurred at 12 h; the maximal level of p27 phosphorylation at Thr-187 in the ternary complex was observed at 16 h; ubiquitination of the Thr-187-phosphorylated p27 (pp27Thr-187) was observed starting at 12 h and continuing up to 24 h. However, maximum p27 phosphorylation at Ser-10 occurred in the nucleus 6 h after FGF-2 stimulation; maximal export of Ser-10-phosphorylated p27 (pp27Ser-10) occurred 8 h after FGF-2 treatment, and pp27Ser-10 was simultaneously ubiquitinated. We further investigated which of the two phosphorylated p27 was involved in G(1)/S progression. LY294002 blocked 64% of the cell proliferation stimulated by FGF-2. Use of leptomycin B to block nuclear export of pp27Ser-10 greatly decreased the FGF-2-stimulated cell proliferation (44%), suggesting that phosphorylation of p27 at Ser-10 is the major mechanism for G(1)/S transition. Our results suggest that differential kinetics are observed in p27 phosphorylation at Ser-10 and Thr-187 and that pp27Thr-187 and pp27Ser-10 may represent two populations of p27 observed in the G(1) phase of the cell cycle.

Highlights

The PI 3-kinase pathway is centrally involved in cell proliferation and survival, and the importance of p27 as a regulator of PI 3-kinase-mediated cell cycle progression is well established (10 –12)
Among the phenotypes altered during endothelial mesenchymal transformation (EMT), fibroblast growth factor-2 (FGF-2) directly regulates cell cycle progression through the action of phosphatidylinositol (PI) 3-kinase and that this phenotype alteration leads to a marked stimulation of cell proliferation [7], as opposed to the G1-arrested corneal endothelial cells (CECs)
Degradation of p27 at the G0–G1 transition is known to be independent of Skp2 and to occur in the cytoplasm [22]. This information, suggests differential regulation of the two populations of the phosphorylated p27 at different phases of the cell cycle. These data further indicate that at least the two populations of p27 respectively regulate the different stage of the cell cycle: pp27Ser-10 regulates via the nuclear export of p27 during reentry of quiescent cells into the cell cycle, while pp27Thr-187 regulates the cell cycle during late G1-S transition

Summary

EXPERIMENTAL PROCEDURES

Materials—LY294002 and monoclonal antibodies against ␤-actin and proliferating cell nuclear antigen (PCNA) were obtained from Sigma-Aldrich. Staining in CECs; Fig. 1A demonstrated that CEC progression to S phase in response to FGF-2 stimulation took 16 h Another control experiment was performed to determine the time period for CECs to re-enter G0 phase following serum starvation. When the cially purified histone H1 (Upstate Biotechnology Inc.) was kinetics of p27 phosphorylation at Thr-187 and Ser-10 were added to the immunoprecipitates, and the mixtures were determined using specific antibodies to either pp27Thr-187 or incubated for 30 min at 30 °C. The reaction mixtures were pp27Ser-10, differential kinetics were observed: the maximum resolved by SDS-PAGE, and phosphorylated histone H1 was phosphorylation of p27 at the Ser-10 residue was observed in detected by immunoblotting with anti-phosphohistone H1- cells stimulated for 4 h with FGF-2; this high level was mainspecific antibody. Pp27Thr-187 immune complex was assayed for Cdk2-associated kinase was barely detectable in cells treated for 8 h, after which the activity using histone H1 as a substrate as stated above

RESULTS

When cells were stimulated with

Findings

DISCUSSION