FKBP51, FKBP52, and Cyp40 bind competitively to Hsp90 through their respective tetratricopeptide repeat (TPR) domains, and any one of the three immunophilins can be isolated in mature steroid receptor complexes. During cell-free assembly reactions, FKBP51 associates preferentially with progesterone and glucocorticoid receptors, but less preference is observed in FKBP51 association with estrogen receptor. A number of mutant FKBP forms were generated to map sequences responsible for FKBP51's preferred association with progesterone receptor. A double-point mutation in the peptidyl prolyl isomerase domain of FKBP51 that reduces enzymatic activity by greater than 90% had no observed effect on FKBP51 interactions with progesterone receptor or Hsp90. Coprecipitation of FKBP51 and FKBP52 truncation mutants with Hsp90 indicated that sequences both upstream and downstream of the TPR domain are necessary for Hsp90 binding. FKBP chimeric constructs were also generated and tested for Hsp90 binding and receptor association. The TPR domain of FKBP51 required appropriate downstream sequences for Hsp90 binding, but FKBP52's TPR domain did not. The C-terminal region of FKBP51 that functionally interacts with the TPR domain to permit Hsp90 binding also conferred preferential association with PR. In conclusion, despite the overall similarity of FKBP51 and FKBP52, these two immunophilins associate differentially with steroid receptors, and the difference relates to both the Hsp90-binding TPR domain and to poorly conserved C-terminal sequences.

Val-12 but had no effect on the ability of Ras Val-12 to induce membrane ruffling. These data indicate that KSR is a potent modulator of a signaling pathway essential to normal and oncogenic cell growth and development.

The kinase suppressor of Ras (KSR) is a loss-of-function allele that suppresses the rough eye phenotype of activated Ras in Drosophila and the multivulval phenotype of activated Ras in Caenorhabditis elegans. The physiological role of mammalian KSR is not known. We examined the mechanisms regulating the phosphorylation of this putative kinase in mammalian cells. Wild-type mouse KSR and a mutated KSR protein predicted to create a kinase-dead protein are phosphorylated identically in intact cells and in the immune complex. Phosphopeptide sequencing identified 10 in vivo phosphorylation sites in KSR, all of which reside in the 539 noncatalytic amino terminal amino acids. Expression of the amino terminal portion of KSR alone demonstrated that it was phosphorylated in the intact cell and in an immune complex in a manner indistinguishable from that of intact KSR. These data demonstrate that the kinase domain of KSR is irrelevant to its phosphorylation state and suggest that the phosphorylation of KSR and its association with a distinct set of kinases may affect intracellular signaling.