Hsp90 is essential for the synthesis and subsequent membrane association, but not the maintenance, of the Src-kinase p56(lck)

Abstract

Tyrosine kinases of the Src family are synthesized as cytosolic proteins that subsequently translocate to membranes. Little is known of the mechanisms responsible for targeting these proteins to membranes, although a role for the cytosolic chaperone Hsp90 has been proposed. Here, we have studied the involvement of Hsp90 in the synthesis, membrane binding, and maintenance of the Src-kinase Lck. Using specific inhibitors of Hsp90, geldanamycin and radicicol, we found that functional Hsp90 is essential for the stability of newly synthesized, but not mature, Lck. Similar results were obtained for two other Src-kinases, c-Src and Lyn. In contrast, LckY505F and LckDeltaSH2, constitutively active Lck mutants lacking the C-terminal regulatory tyrosine or the entire Src homology 2 domain, respectively, required Hsp90 activity to stabilize the mature proteins. Lck synthesized in the absence of Hsp90 activity was degraded within 30-45 min. This unstable Lck was myristoylated normally but did not associate with membranes or CD4, interactions that normally start within minutes of the completion of Lck synthesis. A construct composed of the N-terminal unique domain of Lck fused to green fluorescent protein did not require Hsp90 activity during synthesis. In addition, this protein associated with membranes efficiently in the absence of Hsp90 activity. Together these data suggest that interaction with Hsp90 is necessary for the correct synthesis and subsequent membrane binding of Lck. However, Hsp90 does not appear to play a direct role in Lck membrane, or CD4, association.

Figure 1

Hsp90 inhibition affects newly synthesized, but not mature, Src-kinases. (A—C) Cells were labeled with [³⁵S]methionine/cysteine for 3.5 h in the absence (−) or presence of 5 μM geldanamycin (GA) or of 10 μM radicicol (Rad). Cell lysates were analyzed by immunoprecipitation (ip; A), immunoblotting (B), or autoradiography (C). SupT1 cells were used for the detection of Lck, Daudi cells were used for Lyn, and transfected NIH-3T3 fibroblasts were used for Src. (A) Eighty percent of the volume of each lysate was used for immunoprecipitations. The anti-Lck serum recognizes in addition to Lck a nonrelated, slower-migrating protein (★) (Bijlmakers and Marsh, 1999). Lyn exists as two splice variants of 53 and 56 kDa (Yi et al., 1991). (B) Ten percent of the volume of each lysate was used for immunoblotting. (C) The blot shown in B for Daudi lysates was exposed to film to detect total labeled proteins. Molecular weight markers are indicated in kilodaltons on the right. (D) Lck was immunoprecipitated from unlabeled SupT1 cells treated with or without Hsp90 inhibitors as described above. Immunoprecipitates were analyzed for kinase activity. Autophosphorylated Lck is shown.

Figure 2

Stable expression of LckY505F and LckΔSH2 requires Hsp90. NIH-3T3 cells transfected with wt Lck (A), LckY505F (B), or LckΔSH2 (C) were treated without (−) or with 5 μM geldanamycin (GA), 10 μM radicicol (Rad), or 100 μg/ml cycloheximide (Chx) for 3.5 h. Cell lysates, containing equal amounts of protein, were immunoblotted with antibodies against Lck (A) or against both Lck and Src (B and C).

Figure 3

Lck synthesized in the absence of Hsp90 activity is rapidly degraded. SupT1 cells were labeled with [³⁵S]methionine/cysteine for 5 min and chased in an excess of unlabeled amino acids for the indicated times. Cells were either not treated (−; A) or treated as described in MATERIALS AND METHODS with 5 μM geldanamycin only (GA; B), with both 5 μM geldanamycin and 50 μM LLnL (GA + LLnL; C), with 10 μM radicicol only (Rad; D), or with both 10 μM radicicol and 10 μM lactacystin (Rad + Lac; E). Lck was immunoprecipitated with anti-Lck serum. In addition to Lck, coimmunoprecipitated CD4 and a background band (★) were detected.

Figure 4

Hsp90 activity is required only during the synthesis of Lck. SupT1 cells were labeled for 5 min with [³⁵S]methionine/cysteine and chased as indicated. Radicicol (Rad) was either absent (A), added at 10 μM together with [³⁵S]methionine/cysteine at the beginning of the pulse and present during the chase (B), or present during the chase only (C). Lck was immunoprecipitated as indicated in MATERIALS AND METHODS. Lck, coimmunoprecipitated CD4, and the background band (★) are indicated.

Figure 5

In the absence of Hsp90 activity, newly synthesized Lck does not associate with membranes. (A) SupT1 cells were labeled for 5 min with [³⁵S]methionine/cysteine and chased as indicated, either in the absence (−) or presence (+) of 5 μM geldanamycin (GA) as described in MATERIALS AND METHODS. Cells were homogenized, nuclei were removed, and cellular membranes were recovered by centrifugation at 100,000 × g. Lck was immunoprecipitated from both membrane (M) and soluble (S) fractions. Lck, coimmunoprecipitated CD4, and the background band (★) are indicated. (B) SupT1 cells were pulse labeled with [³⁵S]methionine/cysteine for 5 min and chased as indicated, in the presence of either 5 μM geldanamycin only (GA) or both 5 μM geldanamycin and 50 μM LLnL (GA + LLnL) as described in MATERIALS AND METHODS. Lck was immunoprecipitated from membrane (M) and soluble (S) fractions, prepared as described above.

Figure 6

In the absence of Hsp90 activity, newly synthesized Lck is myristoylated. (A) LSTRA cells were labeled for 15 min in the absence (−) or presence (+) of 10 μM radicicol (Rad) with [³⁵S]methionine/cysteine and chased as indicated. Lck was immunoprecipitated. (B) LSTRA cells were labeled for 15 min in the absence (−) or presence (+) of 10 μM radicicol with [³H]myristic acid and chased as indicated. Lck was immunoprecipitated.

Figure 7

In the absence of Hsp90 activity, newly synthesized Lck does not associate with CD4. SupT1 cells were labeled for 5 min with [³⁵S]methionine/cysteine and chased as indicated in the absence (−; A) or presence (+; B) of 10 μM radicicol (Rad) as described in MATERIALS AND METHODS. For each time point, cell lysates were split into two equal parts. One part was subjected to immunoprecipitation with anti-CD4 antibodies (CD4); the other was subjected to immunoprecipitation with anti-Lck antibodies (Lck). (★) indicates the background band.

Figure 8

UD-GFP does not require Hsp90 for synthesis. HeLa cells transfected with UD-GFP (A) or Lck (B) were labeled for 5 min with [³⁵S]methionine/cysteine and chased as indicated in either the absence (−) or presence (+) of 5 μM geldanamycin (GA) as described in MATERIALS AND METHODS. UD-GFP and Lck were immunoprecipitated with anti-Lck serum from detergent lysates.

Figure 9

UD-GFP does not require Hsp90 for membrane binding. (A) HeLa cells transfected with UD-GFP were homogenized, and total cellular membranes were recovered by centrifugation at 100,000 × g. Membrane (M) and soluble (S) fractions were analyzed for UD-GFP by immunoblotting with anti-Lck serum. Molecular weight markers are indicated in kilodaltons on the right. (B) HeLa cells transfected with UD-GFP were labeled for 5 min with [³⁵S]methionine/cysteine and chased as indicated in the absence (−) or presence (+) of 10 μM radicicol (Rad) as described in MATERIALS AND METHODS. UD-GFP was immunoprecipitated from both membrane (M) and soluble (S) fractions.