Maturation of the tyrosine kinase c-src as a kinase and as a substrate depends on the molecular chaperone Hsp90

Abstract

Although Hsp90 displays general chaperone activity in vitro, few substrates of the chaperone have been identified in vivo, and the characteristics that render these substrates dependent on Hsp90 remain elusive. To investigate this issue, we exploited a paradoxical observation: several unrelated oncogenic viral tyrosine kinases, including v-src, attain their native conformation after association with Hsp90, yet their nearly identical cellular homologs interact only weakly with the chaperone. It has been controversial whether Hsp90 is vital for normal maturation of the cellular kinases or is simply binding a misfolded subfraction of the proteins. By modulating Hsp90 levels in Saccharomyces cerevisiae, we determined that Hsp90 is indeed necessary for the maturation of c-src (the normal homolog of v-src). c-src maturation is, however, less sensitive to Hsp90 perturbations than is v-src maturation. Dependence of the two proteins on Hsp90 does not correspond to their relative efficiency in reaching their final destination (the plasma membrane); we observed that in yeast, unlike in vertebrate cells, neither c-src nor v-src concentrate in the membrane. Expression of different v/c-src chimeras in cells carrying wild-type or temperature-sensitive Hsp90 alleles revealed that the difference between the proteins instead arises from multiple, naturally occurring mutations in the C-terminal region of v-src.

Figure 1

Effects of Hsp90 mutant strains on c-src and v-abl. (A) Accumulation of c-src protein in wild type (WT) and G170D mutant (TS) cells at 34°C. (B) c-src-mediated tyrosine phosphorylation in the two strains. Cells were grown to early logarithmic phase in SRaf at 25°C then transferred to SGal at 34°C for 5 hr. (C) From Left to Right, accumulation of c-src in wild-type (WT) and ILEP cells; c-src-mediated tyrosine phosphorylation in wild-type and ILEP strains; and v-abl-mediated tyrosine phosphorylation in wild-type and ILEP1 cells.

Figure 2

src proteins used in this work. (A) Diagram of c-src, indicating the boundaries of the variable (Var), SH3, SH2, and kinase domains. Also shown are the location of the negative regulatory phosphorylation site (Y527), the major autophosphorylation site (Y416), and the conserved lysine in the ATP binding site (K295). M, B, and S, MluI, BglI, and SphI restriction sites, respectively, used to make v-src/c-src chimeras. (B) Diagram of v-src, indicating the amino acid changes (marked by X) and the C-terminal sequence (dark shading) that differ from c-src (see text). (C–F) v-src/c-src chimeras. Shaded portions represent sequences derived from v-src. The c-src/v-src chimeras are named according to the v-src restriction fragments they contain (e. g., MS is a chimera in which a MluI-SphI fragment of v-src is flanked by c-src sequences) (10).

Figure 3

Effect of the temperature-sensitive mutation hsp90^G17OD on the activity of the c-src regulatory kinase Csk and on the maturation of c-src^K295M at the semirestrictive temperature. (A) Csk produced at 32°C: accumulation of c-src^K295M and Csk and Csk-mediated tyrosine phosphorylation of c-src^K295M in wild-type (WT) and G170D (TS) cells. (B) Csk and c-src^K295M produced at 32°C: accumulation of c-src^K295M and Csk and Csk-mediated tyrosine phosphorylation of c-src^K295M in wild-type (WT) and G170D (TS) cells.

Figure 4

Immunofluorescece analysis of c-src and v-src distribution in wild-type yeast cells. Intracellular distribution of c-src (A), v-src (B), and a GST-ras fusion protein (C). Cells were grown in raffinose medium to early logarithmic phase then transferred to galactose-containing medium for 5 hr to induce expression of the indicated protein. As expected from its lower level of accumulation, the fluorescence signal from v-src was lower than that of c-src but well above background (staining with secondary antibody in the absence of primary antibody), which was not detectable at this level of exposure (not shown).

Figure 5

Regions of v-src responsible for increased dependence on Hsp90. Tyrosine phosphorylation mediated by src kinases (A) and accumulation of src proteins in wild-type (WT) and G170D (TS) strains at the semirestrictive temperature (B). See Fig. 2 and text for description of the src chimeras.