Recruitment of phosphorylated small heat shock protein Hsp27 to nuclear speckles without stress.

During stress, the mammalian small heat shock protein Hsp27 enters cell nuclei. The present study examines the requirements for entry of Hsp27 into nuclei of normal rat kidney (NRK) renal epithelial cells, and for its interactions with specific nuclear structures. We find that phosphorylation of Hsp27 is necessary for the efficient entry into nuclei during heat shock but not sufficient for efficient nuclear entry under control conditions. We further report that Hsp27 is recruited to an RNAse sensitive fraction of SC35 positive nuclear speckles, but not other intranuclear structures, in response to heat shock. Intriguingly, Hsp27 phosphorylation, in the absence of stress, is sufficient for recruitment to speckles found in post-anaphase stage mitotic cells. Additionally, pseudophosphorylated Hsp27 fused to a nuclear localization peptide (NLS) is recruited to nuclear speckles in unstressed interphase cells, but wildtype and nonphosphorylatable Hsp27 NLS fusion proteins are not. The expression of NLS-Hsp27 mutants does not enhance colony forming abilities of cells subjected to severe heat shock, but does regulate nuclear speckle morphology. These data demonstrate that phosphorylation, but not stress, mediates Hsp27 recruitment to an RNAse soluble fraction of nuclear speckles and support a site-specific role for Hsp27 within the nucleus.

Proliferative activity and expression of cyclin-dependent kinase inhibitor p21WAF1 and p53 protein in endothelial cells of human aorta during replicative aging in vitro.

Experiments with bromodeoxyuridine showed that the count of nonproliferating cells in a monolayer culture of aortic endothelial cells from adult humans rapidly increased during long-term subculturing. Cytochemical assay showed that these cells contain neutral b-galactosidase, the marker of aging cells. Immunocytochemical assay demonstrated that most cells express p53 protein and inhibitor of the cell cycle p21WAF1.