The molecular chaperone Hsp90 is required for signal transduction by wild-type Hck and maintenance of its constitutively active counterpart.

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.

Identification and characterization of Harc, a novel Hsp90-associating relative of Cdc37.

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.

Generation and characterization of monoclonal antibodies to the Src-family kinase Hck.

Hck, a member of the Src-family of protein tyrosine kinases, is expressed primarily in hematopoietic cells of the myeloid and B-lymphocyte lineages. Hybridoma cell lines were established that secrete monoclonal antibodies (MAbs) to Hck. Three of the MAbs were extensively characterized and designated H7, H34, and H42. The MAbs H7 and H34 recognized an epitope within the SH3 domain of Hck, while the epitope recognized by the H42 MAb resides within the Unique domain. All three MAbs specifically recognized the p59 and p56 isoforms of Hck in transiently transfected 293T cells and in a murine macrophage cell line. Notably, the antibodies did not cross-react with other Src-family kinases tested. Under native conditions, the MAbs H34 and H42 efficiently immunoprecipitated Hck from transfected cells. Both MAbs were also successfully used for the immunofluorescent staining of Hck in intact cells.Thus, the MAbs described herein should be useful in studies of Hck function and expression.

p50(Cdc37) can buffer the temperature-sensitive properties of a mutant of Hck.

Genetic studies have previously revealed that Cdc37p is required for the catalytic competence of v-Src in yeast. We have reasoned that temperature-sensitive mutants of Src family kinases might be more sensitive to the cellular level of p50(Cdc37), the mammalian homolog of Cdc37p, than their wild-type counterpart, thus potentially providing a unique opportunity to elucidate the involvement of p50(Cdc37) in the folding and stabilization of Src family kinases. A temperature-sensitive mutant of a constitutively active form of Hck (i.e., tsHck499F) was created by mutating two amino acids within the kinase domain of Hck499F. Significantly, overexpression of p50(Cdc37) rescues the catalytic activity of tsHck499F at 33 degrees C, while partially buffering it against inactivation at higher temperatures (e.g., 37 and 39 degrees C). Hsp90 function is required for tsHck499F activity and its stabilization by p50(Cdc37), but overexpression of Hsp90 is not sufficient to stabilize tsHck499F. Overexpression of p50(Cdc37) promotes the association of tsHck499F with Hsp90, suggesting that the cellular level of p50(Cdc37) might be the rate-limiting step in the association of tsHck499F with Hsp90. A truncation mutant of p50(Cdc37) that cannot bind Hsp90 still has a limited capacity to rescue the catalytic activity of tsHck499F and promote its association with Hsp90. This is a particularly important observation, since it argues that rather than solely acting as a passive adapter protein to tether tsHck499F to Hsp90, p50(Cdc37) may also act allosterically to enhance the association of tsHck499F with Hsp90. The findings presented here might also have implications for our understanding of the evolution of protein kinases and tumor development.

Hck enhances the adherence of lipopolysaccharide-stimulated macrophages via Cbl and phosphatidylinositol 3-kinase.

Src family tyrosine kinases have previously been proposed to mediate some of the biological effects of lipopolysaccharide on macrophages. Accordingly, we have sought to identify substrates of Src family kinases in lipopolysaccharide-stimulated macrophages. Stimulation of Bac1.2F5 macrophage cells with lipopolysaccharide was found to induce gradual and persistent tyrosine phosphorylation of Cbl in an Src family kinase-dependent manner. Immunoprecipitation experiments revealed that Cbl associates with Hck in Bac1.2F5 cells, while expression of an activated form of Hck in Bac1.2F5 cells induces tyrosine phosphorylation of Cbl in the absence of lipopolysaccharide stimulation. The Src homology 3 domain of Hck can directly bind Cbl, and this interaction is important for phosphorylation of Cbl. Association of the p85 subunit of phosphatidylinositol (PI) 3-kinase with Cbl is enhanced following lipopolysaccharide stimulation of Bac1.2F5 cells, and transient expression experiments indicate that phosphorylation of Cbl by Hck can facilitate the association of p85 with Cbl. Lipopolysaccharide treatment also stimulates the partial translocation of Hck to the cytoskeleton of Bac1.2F5 cells. Notably, lipopolysaccharide enhances the adherence of Bac1.2F5 cells, an effect that is dependent on the activity of Src family kinases and PI 3-kinase. Thus, we postulate that Hck enhances the adherence of lipopolysaccharide-stimulated macrophages, at least in part, via Cbl and PI 3-kinase.