Biomedical subjects

Sara J Felts

Publications and source records attributed to Sara J Felts.

5 recordsLinked to original sources

PubMed2006

GCUNC-45 is a novel regulator for the progesterone receptor/hsp90 chaperoning pathway.

The hsp90 chaperoning pathway is a multiprotein system that is required for the production or activation of many cell regulatory proteins, including the progesterone receptor (PR). We report here the identity of GCUNC-45 as a novel modulator of PR chaperoning by hsp90. GCUNC-45, previously implicated in the activities of myosins, can interact in vivo and in vitro with both PR-A and PR-B and with hsp90. Overexpression and knockdown experiments show GCUNC-45 to be a positive factor in promoting PR function in the cell. GCUNC-45 binds to the ATP-binding domain of hsp90 to prevent the activation of its ATPase activity by the cochaperone Aha1. This effect limits PR chaperoning by hsp90, but this can be reversed by FKBP52, a cochaperone that is thought to act later in the pathway. These findings reveal a new cochaperone binding site near the N terminus of hsp90, add insight on the role of FKBP52, and identify GCUNC-45 as a novel regulator of the PR signaling pathway.

Adaptor Proteins, Vesicular Transport↗

PubMed2005

Chaperoning checkpoint kinase 1 (Chk1), an Hsp90 client, with purified chaperones.

Checkpoint kinase 1 (Chk1), a serine/threonine kinase that regulates DNA damage checkpoints, is destabilized when heat shock protein 90 (Hsp90) is inhibited, suggesting that Chk1 is an Hsp90 client. In the present work we examined the interplay between Chk1 and Hsp90 in intact cells, identified a source of unchaperoned Chk1, and report the in vitro chaperoning of Chk1 in reticulocyte lysates and with purified chaperones and co-chaperones. We find that bacterially expressed Chk1 is post-translationally chaperoned to an active kinase. This reaction minimally requires Hsp90, Hsp70, Hsp40, Cdc37, and the protein kinase CK2. The co-chaperone Hop, although not essential for the activation of Chk1 in vitro, enhanced the chaperoning process, whereas the co-chaperone p23 did not stimulate the chaperoning reaction. Additionally, we found that the C-terminal regulatory domain of Chk1 affects the association of Chk1 with Hsp90. Collectively these results provide new insights into Hsp90-dependent chaperoning of a client kinase and identify a novel, biochemically tractable model system that will be useful to further dissect the Hsp90-dependent chaperoning of this important and ubiquitous class of Hsp90 clients.

Casein Kinase II↗

PubMed2003

Synthesis of novel fluorescent probes for the molecular chaperone Hsp90.

Heat shock protein 90 (Hsp90) is a molecular chaperone necessary for maintaining oncogenic transformation. There is substantial interest in developing novel agents that bind to the N-terminal of the chaperone. Here we report the synthesis and characterization of two fluorescent Hsp90 inhibitors and probe their use in an Hsp90 fluorescent polarization assay.

Binding, Competitive↗

PubMed2003

p23, a simple protein with complex activities.

p23 is a small but important cochaperone for the Hsp90 chaperoning pathway. It appears to facilitate the adenosine triphosphate-driven cycle of Hsp90 binding to client proteins. It enters at a late stage of the cycle and enhances the maturation of client proteins. Although this role of p23 is fairly well established, recent studies suggest that it may have additional functions in the cell that merit further exploration.

Animals↗

PubMed2001

Regulation of heat shock protein 90 ATPase activity by sequences in the carboxyl terminus.

Hsp90, in addition to being an abundant and pivotal cytoplasmic chaperone protein, has been shown to be a weak ATPase. In an effort to characterize the ATPase activity of hsp90, we have observed marked differences in activities among various species of hsp90. Chicken or human hsp90 hydrolyzed ATP with a k(cat) of 0.02 min(-1) and a K(m) greater than 300 microm. In contrast, yeast hsp90 and TRAP1, an hsp90 homologue found in mitochondria, were 10-100-fold more active as ATPases. Sedimentation studies confirmed that all are dimeric proteins. Chicken hsp90 mutants were then analyzed to identify regions within the protein that influence ATPase activity. A truncation mutant of chicken hsp90, N1-573, was found to be monomeric, and yet the catalytic efficiency (k(cat)/K(m)) was greater than 100 times that of the full-length protein (k(cat) of 0.24 min(-1) and K(m) of 60 microm). In contrast, an internal deletion mutant, Delta661-677, was also monomeric but failed to hydrolyze ATP. Finally, deletion of the last 30 amino acids resulted in a dimeric protein with an ATPase activity very similar to full-length hsp90. These data indicate that sequences within the last one-fourth of hsp90 regulate ATP hydrolysis.

Adenosine Triphosphatases↗