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Cell Growth & Differentiation Vol. 12, 409-417, August 2001
© 2001 American Association for Cancer Research

The Molecular Chaperone Hsp90 Is Required for Signal Transduction by Wild-Type Hck and Maintenance of Its Constitutively Active Counterpart1

Glen M. Scholz2, Steven D. Hartson, Kellie Cartledge, Lenora Volk, Robert L. Matts and Ashley R. Dunn

Molecular Biology Laboratory, Ludwig Institute for Cancer Research, Royal Melbourne Hospital, Victoria 3050, Australia [G. M. S., K. C., A. R. D.], and Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, Oklahoma 74078 [S. D. H., L. V., R. L. M.]


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
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.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Macrophages are a critical component of the innate immune response to microbial pathogens. In response to microbial infection, macrophages migrate to the site of invasion, whereupon they unleash their bactericidal activity, including the secretion of reactive nitrogen intermediates and inflammatory cytokines (e.g., TNF{alpha},3 IL-1, and IL-6), and the phagocytosis of bacteria and host cell debris (1 , 2) . The subsequent presentation of microbial antigens to T cells results in the induction of the adaptive immune response (1 , 2) . LPS, a major constituent of the outer wall of Gram-negative bacteria, is a potent activator of macrophages both in vivo and in vitro. The first step in the activation process is the binding of LPS and LPS-binding protein to CD14 on the cell surface of macrophages (3) . Functional interaction of this ternary complex with Toll-like receptor 4 then leads to the activation of networks of intracellular signal transduction pathways that mediate the biological response of macrophages (e.g., TNF{alpha} secretion) to LPS (4, 5, 6) .

Members of the Src-family of tyrosine kinases (e.g., Hck, Fgr, and Lyn) have been implicated previously in mediating some of the biological responses of macrophages to LPS. For example, stimulation of monocytes/macrophages with LPS has been reported to induce a rapid increase in the specific kinase activity of Hck, Fgr, and Lyn (7) . Moreover, Lowell and Berton (8) have shown that mice doubly deficient for Hck and Fgr are more resistant to LPS-induced toxic shock than their wild-type littermates. Additionally, we have shown that Hck enhances the cell adhesion properties of LPS-stimulated macrophages via Cbl and PI 3-kinase (9) .

Intriguingly, Hsp90 function has recently been demonstrated to be necessary for the activation of macrophages by LPS (10) . Specifically, pretreatment of macrophages with the Hsp90 inhibitor GA suppressed the activation of NF-{kappa}B and induction of TNF{alpha} expression by LPS (10) . Notably, the Src-family kinases Src and Lck have previously been shown to be dependent on Hsp90 for their catalytic competence (11, 12, 13, 14, 15) . Thus the requirement of Hsp90 function for the activation of macrophages by LPS might relate to its role in the folding of Src-family kinases into conformations competent to transduce intracellular signals in response to LPS stimulation.

Hsp90 is an abundant and highly conserved molecular chaperone that is essential for eukaryotic cell survival (16, 17, 18, 19) . It is unclear how Hsp90 mediates the folding of client proteins; nonetheless, the identification of an ATP-binding site within the NH2-terminal domain of Hsp90 and the demonstration that Hsp90 possesses an inherent ATPase activity that is necessary for its in vivo function suggests that its chaperoning activity is regulated by cycles of ATP binding and hydrolysis (20, 21, 22, 23) . The recruitment of Hsp90 to nascent and/or unstable protein kinases seems to be primarily facilitated by p50Cdc37. Coexpression of p50Cdc37 with cyclin-dependent kinase 4 (24) , Raf-1 (25) , or a temperature-sensitive Hck mutant (26) is sufficient to facilitate their association with endogenous Hsp90. Significantly, p50Cdc37 can bind both Hsp90 and protein kinases (24, 25, 26, 27) . On the basis of these findings, p50Cdc37 has been proposed to act as a protein kinase targeting cochaperone of Hsp90 that recruits Hsp90 to its client protein kinases (24, 25, 26) . Recent studies have elaborated on this model by demonstrating that the nucleotide-mediated conformational switching of Hsp90 might regulate, at least in part, the protein kinase-binding activity of p50Cdc37, thus establishing high-affinity interactions within the chaperone-kinase heterocomplex (26 , 28 , 29) . Here we have investigated the relationship between Hsp90 and the signal-transducing capacity of the Src-family kinase Hck.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
LPS-enhanced Macrophage Cell Adhesion Is an Hsp90-dependent Process.
To establish whether Hsp90 function is required for LPS to enhance the cell-adhesion properties of macrophages, murine Bac1.2F5 macrophage cells were pretreated for 16 h with the Hsp90 inhibitor GA or the drug vehicle DMSO and then stimulated with LPS. Differences in the adhesion properties of the cells were then determined by performing cell adhesion assays. LPS stimulation promoted an ~4–5-fold increase in the cell adhesion properties of Bac1.2F5 cells that had been pretreated with DMSO (Fig. 1)Citation . Notably, though, prior treatment of the Bac1.2F5 cells with GA almost completely abolished the ability of LPS to enhance the adhesion properties (1.5-fold versus 5-fold) of the cells (Fig. 1)Citation . GA treatment also modestly reduced (~1.5-fold) the basal cell adhesion properties of the Bac1.2F5 cells (Fig. 1)Citation .



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Fig. 1. Hsp90 function is required for LPS-enhanced macrophage cell adhesion. Bac1.2F5 macrophage cells were treated for 16 h with either DMSO or 2.5 µM GA, then either left unstimulated (-) or stimulated (+) with 1 µg/ml LPS for 60 min. Differences in the cell adhesion properties of the Bac1.2F5 macrophage cells were then assessed. The data presented are the averages of three separate experiments performed in duplicate.

 
Hsp90 Is Required for the Stable Expression and Catalytic Competence of Src-Family Kinases in Macrophages.
Because we had previously observed that Hck plays a role in enhancing the cell adhesion properties of LPS-stimulated macrophages (9) , we sought to establish whether a loss of Hck function, or possibly other Src-family kinases (e.g., Fgr and Lyn), might underlie the cell-adhesion defect in GA-treated macrophages. Thus, murine Bac1.2F5 macrophage cells were treated for various periods of time (e.g., 0, 1, 4, and 16 h) with either DMSO or GA, and then the expression levels of Hck, Fgr, and Lyn were assessed by Western blotting. Only a modest decrease (~1.5-fold decrease) in the expression level of Hck occurred within 4 h of exposing the Bac1.2F5 cells to GA, however, a dramatic decrease (~5–10-fold) in the expression level of Hck was apparent by 16 h (Fig. 2A)Citation . An ~4–5-fold reduction in the expression level of Fgr was apparent after 16 h of GA treatment, whereas only a 2–3-fold decrease in the expression level of Lyn occurred within this same period of time (Fig. 2A)Citation . The 50 kDa anti-Lyn immunoreactive species detected in Bac1.2F5 cells that had been treated with GA for 16 h most likely represents a proteolytic degradation product of Lyn (Fig. 2A)Citation . The expression levels of Erk1 and Erk2 did not appear to be compromised by the same treatment with GA (Fig. 2A)Citation ; confirming that GA does not exert detrimental effects on the global population of cellular protein kinases. The effect of GA on the catalytic activity of Hck was also assessed. The relative specific activity of Hck was calculated by comparing its catalytic activity with its level of expression. An ~2-fold decrease in the specific activity of Hck was observed within 4 h of treating the cells with GA, whereas by 16 h, the little remaining Hck was catalytically inactive (Fig. 2B)Citation . Although treatment of the Bac1.2F5 cells with DMSO for 16 h had a modest effect (~2-fold reduction) on the activity of Hck in this experiment, the effect was not reproducible (Fig. 2BCitation and data not shown). A 2-fold decrease in the specific kinase activity of Lyn was found upon treating the Bac1.2F5 cells with GA for 16 h (data not shown). Concomitant with the decrease in the expression level and catalytic activity of these three Src-family kinases in GA-treated Bac1.2F5 cells was a decrease in the basal tyrosine phosphorylation of several cellular proteins (Fig. 2C)Citation .



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Fig. 2. Hsp90 function is required for the stable expression of Src-family kinases in macrophages. A, Bac1.2F5 macrophage cells were treated with either DMSO or 2.5 µM GA for 0, 1, 4, or 16 h and then lysed. Aliquots of the whole cell lysates were Western blotted with anti-Hck, anti-Fgr, anti-Lyn, and anti-Erk1/2 antibodies. B, Hck was immunoprecipitated from the whole cell lysates shown in A and subjected to an in vitro autophosphorylation reaction or Western blotting with an anti-Hck antibody. The relative specific activity (Hck Sp. Act.) of Hck is shown at the bottom. C, aliquots of the whole cell lysates shown in A were Western blotted with an anti-phosphotyrosine ({alpha}-pY) antibody. The positions of molecular mass markers (in kDa) are shown on the right.

 
LPS-induced Tyrosine Phosphorylation of Cbl and Pyk2 Are Hsp90-dependent Events.
We have recently demonstrated that Hck enhances the cell-adhesion properties of LPS-stimulated Bac1.2F5 macrophage cells, at least in part, via its phosphorylation of Cbl, which in turn facilitates the physical association of PI 3-kinase with Cbl (9) . Additionally, Williams and Ridley (30) have reported that Pyk2 is tyrosine phosphorylated in a Src-family kinase-dependent manner in LPS-stimulated monocytes. Because the stable expression of catalytically active Src-family kinases in Bac1.2F5 macrophage cells was found to be dependent on Hsp90 function (Figs. 2ACitation and 3BCitation ), we sought to establish whether LPS-induced tyrosine phosphorylation of Cbl and Pyk2 might be compromised in cells pretreated with GA. Significantly, treating Bac1.2F5 cells with GA before stimulation with LPS perturbed the induction in tyrosine phosphorylation of several cellular proteins, including Cbl and Pyk2 (Fig. 3)Citation . These experiments revealed an ~2-fold decrease in basal tyrosine phosphorylation of Cbl and almost complete inhibition of LPS-induced tyrosine phosphorylation of Cbl in Bac1.2F5 cells pretreated with GA (Fig. 3C)Citation . Concomitant with the decrease in basal and LPS-induced tyrosine phosphorylation of Cbl was a decrease in its association with the p85 subunit of PI 3-kinase (data not shown). LPS-induced tyrosine phosphorylation of Pyk2 was also found to be significantly impaired by prior treatment of the Bac1.2F5 cells with GA, whereas basal tyrosine phosphorylation of Pyk2 was unaffected (Fig. 3D)Citation . Although we have not established the identities of other proteins whose basal and/or LPS-induced tyrosine phosphorylation is compromised after GA treatment (Fig. 3A)Citation , likely candidates include p190RhoGAP (31) and p145SHIP (32) .



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Fig. 3. Hsp90 function is required for LPS-induced tyrosine phosphorylation in macrophages. A, Bac1.2F5 macrophage cells were treated for 16 h with DMSO (-) or 2.5 µM GA (+) and then either left unstimulated (-) or stimulated (+) with 1 µg/ml LPS for 60 min. The cells were lysed, and aliquots of the whole cell lysates were Western blotted with an anti-phosphotyrosine ({alpha}-pY) antibody. The positions of molecular mass markers (in kDa) are shown on the right. B, whole cell lysates shown in A were Western blotted with anti-Hck, anti-Fgr, anti-Lyn, and anti-Erk1/2 antibodies. C, Cbl was immunoprecipitated from the whole cell lysates shown in A and then Western blotted with anti-phosphotyrosine and anti-Cbl antibodies. D, Pyk2 was immunoprecipitated from the whole cell lysates shown in A and then Western blotted with anti-phosphotyrosine and anti-Pyk2 antibodies.

 
Hsp90 Is Required for the Folding of Hck into a Stable and Active Conformation.
To more directly define the biochemical contribution of Hsp90 to signal transduction by Src-family kinases, we examined the role of Hsp90 in the de novo folding and maintenance of Src-family kinases. In particular, we focused on the functional relationship between Hsp90 and Hck. The effects of GA on both wild-type Hck and Hck499F, a constitutively active mutant of Hck, were studied. Experiments were performed in both in vivo (e.g., transfected 293T cells) and in vitro (e.g., rabbit reticulocyte lysates) systems to overcome potential complications associated with either system alone. The addition of GA, but not DMSO, to 293T cells immediately after their transfection almost completely inhibited the subsequent tyrosine phosphorylation of endogenous cellular proteins by transfected Hck499F (Fig. 4A)Citation . Consistent with these in vivo observations, in vitro kinase assays revealed that Hck499F synthesized in the presence of GA was catalytically inactive (Fig. 4B)Citation . Similar results were obtained when the cells were transfected with an expression vector encoding wild-type Hck (data not shown). In addition to its effects on kinase activity, GA also profoundly perturbed the accumulation of Hck499F when compared with cells that had been treated with DMSO (Fig. 4A)Citation . The detrimental effect of GA on Hck499F is unlikely to be a consequence of possible changes in the cell cycle parameters and/or transcriptional repertoire of the 293T cells, inasmuch as inhibition of Hsp90 function in rabbit reticulocyte lysates also resulted in the synthesis of Hck499F and wild-type Hck that was catalytically inactive (Fig. 4CCitation and data not shown). In contrast to the situation in 293T cells, GA did not have a detrimental effect on the accumulation of Hck499F in rabbit reticulocyte lysates (Fig. 4C)Citation . However, proteolytic nicking assays revealed that [35S]-methionine-labeled Hck499F translated in the presence of GA was highly susceptible to proteolysis, whereas kinase synthesized in the absence of the Hsp90 inhibitor was relatively resistant to the same treatment with chymotrypsin (Fig. 4D)Citation . The failure of [35S]-methionine-labeled Hck499F to fold into a compact conformation in the absence of Hsp90 function most likely explains its hypersensitivity to in vitro digestion with chymotrypsin. Notably, GA completely inhibited the association of Hck499F with Hsp90 and p50Cdc37 in rabbit reticulocyte lysates (data not shown). Given the very low levels of Hck499F in 293T cells treated with GA for 16 h (see Fig. 4ACitation , bottom panel), we did not attempt to establish whether the association of Hsp90 and p50Cdc37 with the kinase was likewise perturbed. However, treating the cells with GA for a shorter period of time (e.g., 60 min) resulted in a significant decrease in the association of Hsp90 and p50Cdc37 with both wild-type Hck and Hck499F (see Fig. 6CCitation ).



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Fig. 4. Hsp90 function is required for the synthesis of catalytically active Hck499F. A, 293T cells were transfected with empty vector (-) or pEF-Hck499F (+) for 4 h, then immediately treated with DMSO (-) or 2.5 µM GA (+) for 16 h. The cells were lysed, and the whole cell lysates (WCL) were Western blotted with anti-phosphotyrosine ({alpha}-pY) and anti-Hck antibodies. The positions of molecular mass markers (in kDa) are shown on the right. B, Hck499F was immunoprecipitated from aliquots of the whole cell lysates shown in A and subjected to an in vitro autophosphorylation reaction or Western blotting with an anti-Hck antibody. C, Hck499F was synthesized for 35 min at 30°C in rabbit reticulocyte lysates containing [35S]-methionine and supplemented with either DMSO (-) or GA (+). Hck499F was then immunoprecipitated from aliquots of the rabbit reticulocyte lysates and subjected to an in vitro autophosphorylation reaction or directly subjected to autoradiography after SDS-PAGE. D, aliquots of the rabbit reticulocyte lysates from C were incubated with the indicated concentration of chymotrypsin, then subjected to SDS-PAGE and then autoradiography. The position of full-length [35S]-methionine-labeled Hck499F is indicated on the left.

 


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Fig. 6. Mature wild-type Hck and Hck499F have different requirements for on-going support by Hsp90. A, 293T cells expressing wild-type Hck or Hck499F were treated 48 h posttransfection with 100 µg/ml cycloheximide for 2 h and then with either DMSO (-) or 2.5 µM GA (+) for 60 min. The cells were lysed and the whole cell lysates Western blotted with anti-phosphotyrosine ({alpha}-pY) and anti-Hck antibodies. The positions of molecular mass markers (in kDa) are shown on the Xright. B, Hck499F was immunoprecipitated from aliquots of the whole cell lysates shown in A and subjected to an in vitro autophosphorylation reaction or Western blotting with an anti-Hck antibody. C, 293T cells expressing either wild-type Hck or Hck499F were treated 48 h posttransfection with 100 µg/ml cycloheximide for 2 h and then with either DMSO (-) or 2.5 µM GA (+) for 60 min. Wild-type Hck and Hck499F were immunoprecipitated from lysates of the cells and Western blotted with anti-Hsp90, anti-p50Cdc37, and anti-Hck antibodies.

 
Hsp90 Is Required to Maintain Mature Hck499F in a Catalytically Competent Conformation.
Although the preceding findings clearly indicate that Hsp90 was essential for the catalytic activity of wild-type Hck and Hck499F, they did not allow us to discriminate between a role for Hsp90 in the de novo folding and maturation of nascent Hck499F versus a role in the maintenance of mature kinase. To test whether Hsp90 is necessary for the maintenance of mature Hck499F, the effect of GA on the kinase postsynthesis was assessed. Specifically, 293T cells transiently expressing Hck499F were treated 48 h posttransfection with cycloheximide to inhibit additional protein synthesis. Then the cells were treated with either GA or DMSO for various periods of time, and the catalytic activity of mature Hck499F subsequently were assessed. Anti-phosphotyrosine Western blotting of whole cell lysates derived from the transfected cells indicated that the catalytic activity of mature Hck499F was severely compromised (~4–5-fold reduction in specific activity) within 1 h of exposing the cells to GA (Fig. 5A)Citation . The ability of Hck499F to autophosphorylate in vitro was similarly reduced upon treating the cells with GA (Fig. 5B)Citation . Notably, exposing the cells to GA for 4 h had only a modest effect (~1.5-fold reduction) on the expression level of Hck499F (Fig. 5ACitation , bottom panel). However, treating the cells with GA for longer periods of time (e.g., 8 h) in the absence of cycloheximide resulted in a more pronounced reduction (~10-fold) in the expression level of Hck499F (data not shown). The effect of GA on the catalytic activity of mature Hck499F in rabbit reticulocyte lysates was also examined. Specifically, Hck499F was synthesized at 30°C, and then reinitiation of protein synthesis was arrested. Then Hck499F molecules were allowed to fold into a mature conformation for 60 min at 30°C before being treated with GA. In vitro kinase assays revealed that the catalytic activity of mature Hck499F that had been exposed to GA for 60 min at 37°C was ~5-fold lower than that of kinase exposed to DMSO for the same length of time (Fig. 5C)Citation . To assess whether this loss in catalytic activity was accompanied by a reduction in the gross structural integrity of Hck499F, [35S]-methionine-labeled kinase was subjected to limited proteolysis. As shown in Fig. 5DCitation , although the catalytic activity of mature Hck499F was adversely affected by incubation at 37°C in the presence of GA, its gross structure did not seem to have been similarly affected.



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Fig. 5. Hsp90 function is required to maintain mature Hck499F in a catalytically active conformation. A, 293T cells expressing Hck499F were treated 48 h posttransfection with 100 µg/ml cycloheximide for 2 h and then with either DMSO or 2.5 µM GA for 0, 1, or 4 h. The cells were lysed and the whole cell lysates Western blotted with anti-phosphotyrosine ({alpha}-pY) and anti-Hck antibodies. The positions of molecular mass markers (in kDa) are shown on the right. B, Hck499F was immunoprecipitated from aliquots of the whole cell lysates shown in A and subjected to an in vitro autophosphorylation reaction or Western blotting with an anti-Hck antibody. C, Hck499F was synthesized for 35 min at 30°C in rabbit reticulocyte lysates containing [35S]-methionine. Reinitiation of protein synthesis was inhibited, and reactions were incubated at 30°C for 60 min. Subsequently, DMSO (-) or GA (+) was added, and the reactions were incubated for an additional 60 min at 37°C. Then Hck499F was immunoprecipitated from aliquots of the rabbit reticulocyte lysates and subjected to an in vitro autophosphorylation reaction or directly subjected to autoradiography after SDS-PAGE. D, aliquots of the rabbit reticulocyte lysates from C were incubated with the indicated concentration of chymotrypsin, then subjected to SDS-PAGE and then autoradiography. The position of full-length [35S]-methionine-labeled Hck499F is indicated on the left.

 
Mature Hck499F Has a Greater Requirement for On-going Support from Hsp90 than Does Mature Wild-Type Hck.
Because the conformation of Src-family kinases in their activated state differs from that in their repressed state (33) , we wanted to determine whether the activation status of mature Hck might influence its requirement for on-going support from Hsp90. Thus, the effect of GA on the catalytic activity of mature wild-type Hck and Hck499F in 293T cells was compared. Although an ~4-fold decrease in the catalytic activity of mature Hck499F occurred upon treating the cells for 60 min with GA, no comparable decrease in the catalytic activity of mature wild-type Hck was apparent over the same time frame (Fig. 6A)Citation . Results obtained from in vitro kinase assays performed on wild-type Hck and Hck499F immunoprecipitated from the whole cell lysates likewise revealed that GA had a more detrimental effect on the catalytic activity of mature Hck499F than on that of mature wild-type Hck (Fig. 6B)Citation . Similarly in rabbit reticulocyte lysates, mature Hck499F was found to be more dependent on Hsp90 function for maintenance of its catalytic activity than was mature wild-type Hck (data not shown). If mature Hck499F has a greater requirement for on-going support from Hsp90 than does wild-type Hck, it would be expected that higher levels of endogenous Hsp90 and p50Cdc37 would be associated with Hck499F than wild-type Hck. Indeed, ~5–10-fold higher levels of Hsp90 and p50Cdc37 were associated with Hck499F than with wild-type Hck (Fig. 6C)Citation . Notably, the association of Hsp90 and p50Cdc37 with both wild-type Hck and Hck499F was dramatically reduced upon treating the cells with GA (Fig. 6C)Citation . Thus the inhibitory effect of GA on the catalytic activity of Hck correlated with the ability of GA to disrupt Hsp90-p50Cdc37-Hck complexes.


    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The molecular chaperone Hsp90 has been reported previously to be required for the activation of macrophages by LPS (10) . Moreover, the ability of Hsp90 to bind LPS in vitro led to the proposal that Hsp90 might be directly involved in LPS trafficking and signaling in macrophages (10) . Although possible, an alternative role for Hsp90 in the activation of macrophages would be to fold and maintain in a catalytically active conformation protein kinases that transduce intracellular signals in response to LPS.

This alternative hypothesis predicts that GA should have a deleterious impact on the biological functions of Src-family kinases (e.g., Hck, Fgr, and Lyn) in LPS-stimulated macrophages. Indeed, the inhibition of Hsp90 with GA severely compromised the ability of LPS to enhance the cell adhesion properties of Bac1.2F5 macrophage cells, a biological response that has been shown previously to be mediated by Src-family kinases (9 , 30) . GA treatment of Bac1.2F5 macrophage cells perturbed the expression and catalytic activity of Hck, Fgr, and Lyn, although differences in the kinetics with which the kinases were affected by the inhibition of Hsp90 function were observed. Hck seemed most sensitive to GA treatment, and Lyn seemed least sensitive.

The loss of Src-family kinase function in GA-treated Bac1.2F5 macrophage cells correlated with a decrease in both basal and LPS-induced tyrosine phosphorylation of several cellular proteins, including Cbl and Pyk2. Significantly, tyrosine phosphorylation of Cbl and Pyk2 by Src-family kinases has been postulated to mediate the enhancement in the cell adhesion properties of LPS-stimulated macrophages and monocytes (9 , 30) . The findings presented here are therefore consistent with the hypothesis that the dependence of macrophages on Hsp90 function for their activation by LPS relates to the critical role Hsp90 plays in the folding of Src-family kinases, and possibly other protein kinases, into signal transduction competent conformations.

There are at least three nonexclusive models that might describe the relationship between Hsp90 and Src-family kinases: (a) to facilitate de novo folding of immature Src-family kinases; (b) to provide obligatory full-time support to mature Src-family kinases; and (c) to provide conditional support to mature Src-family kinases. To define precisely the contribution of Hsp90 to the signal transducing capacity of Src-family kinases, we specifically examined the effect of GA on wild-type Hck and its constitutively active counterpart, Hck499F. As was the case for Bac1.2F5 macrophage cells, Hsp90 function was obligatory for the expression of catalytically active wild-type Hck and Hck499F in 293T cells and rabbit reticulocyte lysates. However, this finding did not allow us to discriminate between the three models proposed above. We directly tested the second and third models by assessing the impact of GA on kinase molecules that had folded into a mature conformation. The adverse effect of GA on the specific activity of mature Hck499F, but not mature wild-type Hck, and the higher levels of Hsp90 and p50Cdc37 associated with Hck499F relative to those associated with wild-type Hck, clearly support model 3 and not model 2 (i.e., conditional versus full-time support). Recent studies have similarly shown that constitutively active mutants of Lck and Src (e.g., Lck505F and v-Src) exhibit a greater dependence on Hsp90 function for their stable expression and activity than do their wild-type counterparts (15 , 34 , 35) . Thus, the activation status of mature Src-family kinases seems to determine their need for on-going support from Hsp90. In their repressed state, Src-family kinases are "locked" in a relatively inflexible and stable conformation by virtue of an intramolecular interaction between the phosphorylated COOH-terminal regulatory tyrosine residue (e.g., tyrosine-499 in Hck) and their own SH2 domain (33) . Activation of Src-family kinases, either as a consequence of dephosphorylation, mutation, or deletion of their regulatory tyrosine residues, releases them from such structural constraints. The associated increase in the flexibility of the kinases is likely to be accompanied by an increase in their propensity to unfold into inactive conformations; thereby explaining their greater requirement for support from Hsp90. This relationship is exaggerated in the case of temperature-sensitive mutants of Src-family kinases (e.g., ts v-Src and tsHck499F). Amino acid substitutions within the catalytic domains of these mutant kinases destabilize the structure of the domains, particularly at elevated temperatures, and in doing so, promote the enhanced association of the kinases with Hsp90 and p50Cdc37 (26 , 36 , 37) . A role for Hsp90 in the maintenance of mature protein kinases might not be restricted solely to Src-family kinases. For instance, Uma et al. (38) have provided evidence that Hsp90 function is required for the maintenance of the heme-regulated eIF2{alpha} kinase, whereas An et al. (35) have demonstrated that p210bcr-abl requires Hsp90 function for its stable expression in cells. Additionally, Xu et al. (39) have reported recently that mature ErbB2 but, interestingly, not ErbB1 is similarly dependent upon Hsp90 for its stable expression in cells.

Our data, however, are also consistent with a role for Hsp90 in the de novo folding of Hck into an active and stable conformation, because the magnitude and rate of onset of the effects of GA on Hck499F were dependent upon the maturation state of the kinase. The addition of GA to rabbit reticulocyte lysates before the synthesis of Hck499F resulted in the production of kinase molecules that were catalytically inactive and hypersensitive to proteolytic cleavage. By contrast, Hck499F that was allowed to fold into a mature conformation before the addition of GA was significantly less sensitive to proteolytic cleavage. Therefore the detrimental effect of adding GA before protein kinase synthesis cannot solely be explained by it subsequently causing inactivation of mature Hck499F. It can, nonetheless, be explained by Hsp90 function also being required for the folding and maturation of nascent Hck molecules into stable and active conformations. Indeed, early studies on v-Src by Brugge et al. (36) demonstrated that Hsp90 and p50Cdc37 preferentially associated with newly synthesized molecules of v-Src. More recently, Xu et al. (40) have shown genetically that Hsp90 is required for the maturation of nascent c-Src in yeast cells, whereas both Hartson and colleagues (14 , 41) and Bijlmakers and Marsh (15) have demonstrated a role for Hsp90 in folding nascent Lck into an active and stable conformation. Thus, Hsp90 plays a role in the de novo folding of Src-family kinases into catalytically active and stable conformations, and, dependent upon the phosphorylation status of the kinases’ regulatory tyrosine residues, may also provide on-going support. Interestingly, Hsp90 has also been postulated to play a role in the translocation of mature Src-family kinases to the plasma membrane (15 , 37 , 42) . However, a recent study has shown that in the case of Lck rather than Hsp90 being directly involved in the translocation of the kinase to the plasma membrane, Hsp90-mediated maturation of Lck is simply a prerequisite for its subsequent association with the plasma membrane (15) .

The depletion of Src-family kinases from cells treated with GA is not attributable to the inhibitor directly inducing their proteolysis. In vivo, the exposure of 293T cells to GA caused a rapid decline in the catalytic activity of mature Hck499F, and subsequently a slow decline in its expression level. In contrast, GA caused dramatic inhibition of kinase activity without any loss of Hck499F protein in rabbit reticulocyte lysates. These observations indicate that proteolysis is a derivative effect of Hck mis-/unfolding in the absence of Hsp90 function. Accordingly, studies that solely assess the effect of GA on the expression level rather than the catalytic activity of a protein kinase may underestimate its requirement for Hsp90 function. Although we have not investigated the proteolytic pathway by which Hck is degraded in response to inhibition of Hsp90 function, previous studies have implicated both the proteasome and lysosome in the proteolytic degradation of other protein kinases (e.g., Src, Lck, and Raf-1) in GA-treated cells (35 , 41 , 43) . It seems likely that the degradation of Hck in GA-treated cells is similarly mediated via proteasomal and/or lysosomal-dependent pathways.

A genetic study in Drosophila by Rutherford and Lindquist (44) has provided evidence that Hsp90 can often "buffer" the detrimental effect of genetic variations within organisms by stabilizing the structure of the mutant protein. However, when Hsp90 function becomes compromised (e.g., following heat or chemical-induced protein denaturation), these cryptic genetic differences can become the subject of natural selection, leading Rutherford and Lindquist to propose that Hsp90 can act as a capacitor for morphological evolution. The potential of Hsp90 to support protein kinase evolution may be important to the microevolution of tumor cell populations by allowing tumor cells to evolve hyperactive kinases and thus extend their malignant phenotypes. Examination of the effects of GA on the function of oncogenic protein tyrosine kinases (e.g., v-Src, Lck505F, Hck499F, and p210bcr-abl) supports this possibility (Refs. 11 , 13 , 15 , 34 , and 35 , and herein). Consequently, GA and other drugs that specifically inhibit Hsp90, or perhaps p50Cdc37, might represent potentially useful chemotherapeutic agents by targeting aberrant signal transduction kinases and tumor cells that rely upon them for growth and survival. Indeed, 17-allylamino 17-demethoxy GA is currently undergoing Phase I clinical trial in solid tumors at five institutions world-wide.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Reagents.
Cell culture medium and supplements were from Trace Biosciences, Ltd. (Melbourne, Australia) and Life Technologies, Inc., respectively. FCS was from CSL, Ltd. (Melbourne, Australia). The rat anti-Hck monoclonal antibodies H34 and H42 were developed in this laboratory (45) . The rabbit anti-Fgr, anti-Lyn, and anti-Erk1/2 antibodies were from Santa Cruz Biotechnology. The anti-phosphotyrosine monoclonal antibody (4G10) was from Upstate Biotechnology, whereas the anti-Pyk2 monoclonal antibody was from Transduction Laboratories. The polyclonal rabbit anti-Cbl antibody was from Dr. Wallace Langdon (Department of Pathology, University of Western Australia). Goat antirat IgG Sepharose beads were from Zymed Laboratories. Protein A-Sepharose, enhanced chemiluminescence reagents and [{gamma}-32P]ATP (3000 Ci/mmol) were from Amersham-Pharmacia-Biotech. Pfu DNA polymerase was obtained from Stratagene, whereas Complete protease inhibitors and FuGENE-6 were from Roche. Geldanamycin, cycloheximide, and lipopolysaccharide (Escherichia coli 0111:B4) were purchased from Sigma Chemical Co.-Aldrich. All other reagents were of the highest grade available.

Plasmid Construction.
The mammalian expression vectors pEF-Hck and pEF-Hck499F were as described previously (26) . The in vitro translation vector pSP64T-Hck499F was constructed as follows. The NH2-terminal end of Hck (amino acids 1–134 of the p59 isoform) was generated by PCR using Pfu DNA polymerase. The sense primer used in the PCR resulted in deletion of the 5' untranslated region from the Hck cDNA (46) and converted the CTG codon that initiates translation of the p59 isoform of Hck to ATG. The purified PCR product was ligated to a cDNA fragment encoding the COOH-terminal end (amino acids 135–503) of Hck499F and subcloned into pSP64T (a generous gift of Dr. Douglas Melton, Harvard University, Boston, MA; Ref. 47 ). It should be noted that only the p59 isoform of Hck499F is translated from the pSP64T-Hck499F vector in rabbit reticulocyte lysates (see Fig. 1CCitation ). The fidelity of all constructs was confirmed by restriction mapping and/or automated DNA sequencing.

Cell Culture, Transient Transfection, and Lysis.
Murine Bac1.2F5 macrophage cells were maintained in HEPES-buffered DMEM supplemented with 10% FCS and 25% L-cell conditioned medium (as a source of colony stimulating factor-1) and grown at 37°C in a humidified atmosphere of 5% CO2. Human 293T cells were maintained in RPMI 1640 supplemented with 10% FCS and grown at 37°C in a humidified atmosphere of 5% CO2. 293T cells were transfected for 4 h with polyethylenimine (48) or FuGENE-6. Cells were lysed directly in tissue culture dishes with 1% NP40 lysis buffer [20 mM HEPES (pH 7.4), 100 mM NaCl, 2 mM EGTA, 1 mM DTT, 1% NP40, 10% glycerol, 1 mM sodium orthovanadate, 0.1 mM sodium molybdate, and Complete protease inhibitors] for 30 min on ice. The lysates were clarified by centrifugation at 13,000 x g for 10 min at 4°C, then protein concentrations were measured with a Bio-Rad protein assay kit.

Macrophage Adhesion Assays.
Bac1.2F5 macrophage cells were seeded in 6-well tissue culture plates and grown until ~75% confluent. The cells were then treated for 16 h with either DMSO or 2.5 µM GA and then stimulated with 1 µg/ml LPS for 60 min. Differences in the adherence of the cells were then assessed by incubating the cells in 2 ml of PBS containing 10 mM EDTA (PBS/EDTA) for 30 min on a rotating platform. After this treatment, the nonadherent cells (i.e., the "nonadherent" fraction) were collected and counted with the aid of a hemocytometer. Cells that remained attached to the dishes (i.e., the "adherent" fraction) were collected by vigorous pipetting in PBS/EDTA and counted (9) .

In Vitro Translation of Hck.
Coupled in vitro transcription/translation reactions in rabbit reticulocyte lysate were programmed for the synthesis of Hck499F as described previously (13) . Proteolytic fingerprinting of in vitro translated Hck499F, using exogenous chymotrypsin, was performed as described previously (14 , 34 , 49) .

Western Blotting and Immunoprecipitation.
Western blotting of whole cell lysates was performed by standard techniques. Hck was immunoprecipitated from aliquots of cell lysates using a rat anti-Hck monoclonal (H34 or H42) antibody. The immunoprecipitates were washed four times with lysis buffer before fractionation on SDS-PAGE and Western blotting with the appropriate antibody. Immune complexes were visualized by using enhanced chemiluminescence and exposure to X-ray film. The results were digitized using a Computing Densitometer, quantified using the ImageQuaNT program, version 4.2, then converted to TIF files using the Convert 16 to 8 program, version 1.5a (all from Molecular Dynamics).

Kinase Assays.
Anti-Hck or anti-Lyn immunoprecipitates were incubated at room temperature for 10 min in 30 µl of kinase buffer [20 mM HEPES (pH 7.4), 10 mM MnCl2, 0.1% NP40, and 0.1 mM sodium orthovanadate] containing 10 µCi [{gamma}-32P] ATP. Reactions were terminated by the addition of an equal volume of 2x SDS-PAGE sample buffer and heating for 5 min at 95°C. Phosphorylated proteins were analyzed by SDS-PAGE and then exposed to X-ray film or to a PhosphorImager screen (Molecular Dynamics).


    Acknowledgments
 
We thank Drs. Wallace Langdon and Douglas Melton for gifts of reagents.


    Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by grants from the National Health and Medical Research Council (G. M. S. and A. R. D.), by Oklahoma Center for the Advancement of Science and Technology Grant HN6-018 (to S. D. H.), by NIH Grant GM51608 (to R. L. M.), and by the Oklahoma Agricultural Experiment Station (Project 1975; to R. L. M.). Back

2 To whom requests for reprints should be addressed, at Molecular Biology Laboratory, Ludwig Institute for Cancer Research, Post Office Box 2008, Royal Melbourne Hospital, Parkville, Victoria 3050, Australia. Phone: 61-3-9341-3155; Fax: 61-3-9341-3191; E-mail: Glen.Scholz{at}ludwig.edu.au Back

3 The abbreviations used are: TNF, tumor necrosis factor; IL, interleukin; LPS, lipopolysaccharide; PI 3-kinase, phosphatidylinositol 3-kinase; Hsp90, heat shock protein-90; GA, geldanamycin. Back

Received for publication 3/ 2/01. Revision received 5/30/01. Accepted for publication 5/31/01.


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