The regulation of casein gene expression by both PRL and glucocorticoids has been a well studied paradigm for understanding how the signaling pathways regulated by these two hormones interact in the nucleus. Previous studies have demonstrated that the downstream effectors of these pathways, signal transducer and activator of transcription 5 (STAT5) and the glucocorticoid receptor (GR), are associated via protein-protein interactions and act synergistically to enhance beta-casein gene transcription. Indirect immunofluorescence microscopy was used to demonstrate that PRL-activated STAT5 can translocate GR into the nucleus, and that ligand-bound GR can translocate STAT5 into the nucleus. This provided further support of an interaction between the two proteins. To better understand the mechanism of transcriptional synergy between STAT5 and GR, experiments were performed in cells transiently transfected with STAT5 alone or with STAT5 and GR. GR cotransfection enhanced the DNA-binding activity of STAT5 without affecting STAT5 protein levels. The enhancement of STAT5 DNA binding by GR resulted in the formation of a complex that exhibited prolonged DNA binding after PRL treatment. This was correlated with increased STAT5 tyrosine phosphorylation, suggesting that GR enhances STAT5 DNA binding by modulating the rate of STAT5 dephosphorylation. In contrast, cotransfection of the estrogen receptor resulted in an overall decrease in STAT5 tyrosine phosphorylation, without changing the kinetics of dephosphorylation. Enhancement of STAT5 activity by GR is, therefore, one component of the transcriptional synergy exhibited by STAT5 and GR at the beta-casein promoter and is an example of how transcription factors at a composite response element may modulate each other's activity.

We have used a fluorometric assay to determine the relative amounts of polymerized actin (F-actin) in wild-type and profilin mutant yeast cells. Our results indicate that profilin plays a role in maintaining normal F-actin levels in response to shifts to high temperature. Cells lacking prof'din display a greater drop in F-actin levels upon such temperature shifts, and are slower to recover to initial F-actin levels than are wild-type cells. Interestingly, shifts to cold temperatures result in rapid increases of F-actin levels in wild-type and profilin null cells. We have further determined that shifting to high-osmolarity growth conditions causes a relatively slow decrease in F-actin levels in wild-type cells, and a small but rapid increase in the F-actin levels in profilin null cells. Profilin null cells contain normal concentrations of F-actin while growing exponentially at room temperature, indicating that profilin is not essential for maintaining F-actin concentrations during steady-state growth. Our data suggest that actin is inherently unstable in vivo at high temperatures, and that profilin helps to maintain actin in its filamentous state at these temperatures, perhaps by stimulating actin polymerization in a proper temporal and spatial fashion. direct interaction between yeast profilin and adenylate cyclase (S. Palmieri and B. Haarer, unpublished data).Despite these many interactions attributed to profilins from various species, it is still unclear which roles are central to profilin function, and how such properties relate to the in vivo control of the actin cytoskeleton. Convincing arguments can be made for an in vivo role as an inhibitor [13][14][15], or stimulator [8,16] of actin polymerization. Indeed, it is entirely likely that profilin could be playing both roles, depending on species, cell type, or temporal regulation within an individual cell. To further address profilin's potential in vivo functions, we have utilized a fluorometric assay to measure the relative filamentous actin content in cells before and after changing growth conditions. Our findings are consistent with profilin's playing a significant role in the stimulation of actin polymerization under conditions that are perturbing to the yeast actin cytoskeleton. Materials and methods

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