ABSTRACT
We have investigated the relationship between the molecular chaperone heat shock protein-90 (Hsp90) and the signal transducing capacity of the Src-family kinase Hck. Inhibition of Hsp90 with geldanamycin suppressed the ability of bacterial lipopolysaccharide to enhance the cell adhesion properties of macrophages, a phenomenon most likely explained by the reduced expression and activity of Hck in macrophages lacking Hsp90 function. The contribution of Hsp90 to signal transduction by Hck was biochemically dissected further by examining its role in the de novo folding and maintenance of wild-type Hck and its constitutively active counterpart, Hck499F. The folding of nascent wild-type Hck and Hck499F into catalytically active conformations, and their accumulation in cells was found to be dependent on Hsp90 function. Notably, mature Hck499F had a greater requirement for on-going support from Hsp90 than did mature wild-type Hck. This particular finding might have important implications for our understanding of the evolution of oncogenic protein kinases.
Subject(s)
Cell Adhesion/physiology , HSP90 Heat-Shock Proteins/metabolism , Lipopolysaccharide Receptors/metabolism , Macrophage Activation/physiology , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Signal Transduction/physiology , Animals , Benzoquinones , Catalytic Domain/drug effects , Catalytic Domain/physiology , Cells, Cultured/cytology , Cells, Cultured/drug effects , Cells, Cultured/metabolism , Enzyme Inhibitors/pharmacology , Focal Adhesion Kinase 2 , Lactams, Macrocyclic , Lipopolysaccharides/pharmacology , Macrophages/cytology , Macrophages/drug effects , Macrophages/metabolism , Mice , Oncogene Protein v-cbl , Phosphorylation/drug effects , Protein Folding , Protein Structure, Tertiary/drug effects , Protein Structure, Tertiary/physiology , Protein-Tyrosine Kinases/drug effects , Proto-Oncogene Proteins/drug effects , Proto-Oncogene Proteins c-hck , Quinones/pharmacology , Retroviridae Proteins, Oncogenic/drug effects , Retroviridae Proteins, Oncogenic/metabolism , Tyrosine/drug effects , Tyrosine/metabolism , src-Family Kinases/drug effects , src-Family Kinases/metabolismABSTRACT
Although little is known about the precise mechanisms by which the molecular chaperone Hsp90 recognizes its client proteins, Cdc37 has been shown to play a critical role in the targeting of Hsp90 to client protein kinases. Described here is the identification and characterization of a novel 35-kDa human protein that is 31% identical to Cdc37. We have named this novel protein Harc (Hsp90-associating relative of Cdc37). Northern blot analysis revealed the presence of Harc mRNA in several human tissues, including liver, skeletal muscle, and kidney. Biochemical fractionation and immunofluorescent localization of epitope-tagged Harc (i.e. FLAG-Harc) indicated that it is present in the cytoplasm of cells. FLAG-Harc binds Hsp90 but unlike Cdc37 does not bind Src family kinases or Raf-1. Mapping experiments indicate that the central 120 amino acids of both Harc and Cdc37 constitute a Hsp90-binding domain not described previously. FLAG-Harc is basally serine-phosphorylated and hyperphosphorylated when co-expressed with an activated mutant of the Src family kinase Hck. Notably, FLAG-Harc forms complexes with Hsp90, Hsp70, p60Hop, immunophilins, and an unidentified p22 protein but not with the Hsp90 co-chaperone p23. Thus Harc likely represents a novel participant in Hsp90-mediated protein folding, potentially targeting Hsp90 to Hsp70-client protein heterocomplexes.
