Evidence for Cytosolic p27(Kip1) Ubiquitylation and Degradation during Adipocyte Hyperplasia

Authors


Department of Nutrition, 318 Stone Building, UNC Greensboro, Greensboro, NC 27402. E-mail: ron_morrison@uncg.edu

Abstract

Objective: Subcellular localization has been shown to play an important role in determining activity and accumulation of p27 protein during cell cycle progression. The purpose of this study was to examine p27 localization and ubiquitylation in relation to E3 ligase expression during adipocyte hyperplasia.

Research Methods and Procedures: This study used the murine 3T3-L1 preadipocyte model to examine p27 regulation during synchronous cell cycle progression. Cell lysates were isolated over time after hormonal stimulation, fractionated to cytosolic and nuclear compartments, and immunoblotted for relative protein determinations.

Results: Data presented in this study show that p27 was present in the cytosol and nucleus in density-arrested preadipocytes and that abundance in both compartments decreased in a phase-specific manner as preadipocytes synchronously re-entered the cell cycle during early phases of adipocyte differentiation. Blocking CRM1-mediated nuclear export did not prevent degradation, nor did it cause nuclear accumulation of p27, suggesting that distinct mechanisms mediating cytosolic and nuclear p27 degradation were involved. Treating preadipocytes with a potent and specific proteasome inhibitor during hormonal stimulation prevented Skp2 accumulation and p27187 phosphorylation, which are essential events for SCFSkp2 E3 ligase activity and nuclear p27 ubiquitylation during S/G2 phase progression. Proteasome blockade also resulted in the first evidence of cytosolic p27 ubiquitylation during late G1 phase as preadipocytes undergo the transition from quiescence to proliferation.

Discussion: These data are consistent with the postulate that p27 is ubiquitylated and targeted for degradation by the 26S proteasome in a phase-specific manner by distinct ubiquitin E3 ligases localized to the cytosol and nucleus during adipocyte hyperplasia.

Ancillary