Conformational processing of oncogenic v-Src kinase PNAS PLUS by the molecular

Edgar E. Boczeka, Lasse G. Reefschlägera, Marco Dehlinga,b, Tobias J. Strullera, Elisabeth Häuslera, Andreas Seidlb, Ville R. I. Kailaa, and Johannes Buchnera,1

aCenter for Integrated Science, Department Chemie, Technische Universität München, 85748 Garching, Germany; and bSandoz Biopharmaceuticals, Hexal AG, 82041 Oberhaching, Germany

Edited by F. Ulrich Hartl, Max Planck Institute of Chemistry, Martinsried, Germany, and approved May 14, 2015 (received for review January 5, 2015) Hsp90 is a molecular chaperone involved in the activation of its C terminus that includes a tyrosine at the position 527, whose numerous client , including many kinases. The most phosphorylation status regulates kinase activity (25, 26). In ad- stringent kinase client is the oncogenic kinase v-Src. To elucidate dition, v-Src differs from c-Src by several point mutations (27– how Hsp90 chaperones kinases, we reconstituted v-Src kinase 30). Some of these were shown to increase c-Src activity in vivo chaperoning in vitro and show that its activation is ATP-depen- and have been linked to cancer progression and metastasis in dent, with the cochaperone Cdc37 increasing the efficiency. Consis- humans (31–33). Due to these differences, v-Src cannot be down- tent with in vivo results, we find that Hsp90 does not influence the regulated and is permanently active, even in the absence of ac- almost identical c-Src kinase. To explain these findings, we designed tivating stimuli (26, 30). The analysis of proteins nearly identical Src kinase chimeras that gradually transform c-Src into v-Src and in sequence but highly different in chaperone dependence offers show that their Hsp90 dependence correlates with compactness and an excellent model system for understanding the features that folding cooperativity. Molecular dynamics simulations and hydro- render a protein Hsp90-dependent. We used these kinases to gen/deuterium exchange of Hsp90-dependent Src kinase variants reconstitute and dissect the chaperoning effect of Hsp90 on v-Src further reveal increased transitions between inactive and active kinase in vitro. The analysis of chimeras comprising elements of states and exposure of specific kinase regions. Thus, Hsp90 shifts an c-Src and v-Src allowed us to determine the molecular basis of ensemble of conformations of v-Src toward high activity states that the stringent Hsp90 dependence of v-Src. would otherwise be metastable and poorly populated. Results Cdc37 | kinase activation | metastable states | conformational ensembles | Hsp90-Dependent Chaperoning of v-Src in Vitro. It is well established chaperone mechanism that v-Src kinase activity is strictly Hsp90-dependent in vivo (5, 34). Furthermore, v-Src requires the Hsp90 cochaperone Cdc37 he 90-kDa heat shock protein (Hsp90) is an abundant to reach full activity in the cell (21, 35). Strikingly however, Tchaperone in the cytosol of eukaryotes (1). Together with its Hsp90 does not affect the highly homologous cellular form of

cochaperones, it functions in the conformational control of many v-Src, c-Src (21). We aimed to reconstitute the effects of Hsp90 on BIOCHEMISTRY – regulatory proteins (2 4). Kinases constitute the largest group of v-Src activity in vitro. To this end, we purified the v-Src and c-Src Hsp90 client proteins with more than 60% of the human kinases kinases from insect cells and determined the influence of Hsp90 that depend on Hsp90 in terms of their activity (5, 6). on their activity (Fig. 1). When we tested the effect of Hsp90 on Hsp90 forms V-shaped homodimers connected via a C-ter- c-Src kinase, we could not observe a significant change in its ac- minal domain. The middle domain (M-domain) is involved in tivity (Fig. 1B). In contrast, for v-Src, the presence of Hsp90 client binding (7, 8), and the N-terminal domain binds ATP. Upon ATP binding, the N-terminal domains dimerize, leading to Significance the closed state (9–13), whereas the open state is regained upon ATP-hydrolysis (14). Both conformation and ATPase activity are affected by interaction with a cohort of cochaperones (15). Given Hsp90 is a molecular chaperone involved in the activation of numerous client proteins, including 60% of the human kinases. the large number and diversity of client proteins, cochaperones – are believed to deliver specificity in this context. Previous studies on the Hsp90 kinase interaction were limited The Hsp90-mediated maturation of kinases is strictly de- due to the particular instability of client kinases. Here, we pendent on the cochaperone Cdc37 (cell division control protein reconstituted v-Src kinase chaperoning in vitro and used this to mechanistically elucidate how Hsp90 supports kinases. We 37) (16, 17) and phosphorylation of this cofactor is important for show that its activation is ATP-dependent and requires the its function (18, 19). Binding of Cdc37 to Hsp90 causes inhibition phosphorylated form of the cochaperone Cdc37. Hsp90 does of the ATPase activity of Hsp90 and has therefore been pro- not influence the almost identical c-Src kinase. The structural posed to facilitate client kinase loading onto the Hsp90 ma- analysis of Src kinase chimeras that gradually transformed c-Src chinery (20). into v-Src unveiled that Hsp90 dependence correlates with cli- The viral Src kinase (v-Src) is one of the most stringent known ent compactness, folding cooperativity, and lowered energy Hsp90 clients (5, 21). v-Src belongs to the family of nonreceptor barriers between different states. These findings establish a tyrosine kinases, which play important roles in many cellular new concept for the client specificity of Hsp90. pathways. v-Src kinase is constitutively active and leads to the

formation of sarcomas in chicken (22). It shows 98% sequence Author contributions: E.E.B., M.D., A.S., V.R.I.K., and J.B. designed research; E.E.B., L.G.R., identity with its cellular counterpart c-Src (cellular Src kinase), M.D., T.J.S., E.H., and V.R.I.K. performed research; E.E.B., L.G.R., M.D., T.J.S., E.H., A.S., the first identified protooncogene (23). Hsp90 binds to and V.R.I.K., and J.B. analyzed data; and E.E.B., V.R.I.K., and J.B. wrote the paper. stabilizes c-Src in its nascent state, but it dissociates after the The authors declare no conflict of interest. kinase folding is achieved (24). Due to this complete loss of in- This article is a PNAS Direct Submission. teraction, c-Src has been defined as a nonclient (5). Src consists Freely available online through the PNAS open access option. of a unique domain followed by the SH3 and SH2 domains and a 1To whom correspondence should be addressed. Email: [email protected]. flexible linker, which connects the SH2 domain with the highly This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. conserved kinase domain. c-Src contains an additional stretch at 1073/pnas.1424342112/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1424342112 PNAS | Published online June 8, 2015 | E3189–E3198 Downloaded by guest on September 23, 2021 A B

4,0 Hsp90β + Cdc37E KA = 0.23 μM 3,5 thermo stress activity Hsp90β K = 3.00 μM kinase rct. 3,0 A 30°C 2,5 on ice 30 °C cool c-Src + Hsp90β + Cdc37 2,0 E Cdc37E 1,5 +/- ATP / Hsp90 + *ATP Lysozyme / Cdc37 / substrate 1,0 normalized kinase 0 2 4 6 8 101214 (co-)chaperone concentration (μM) C D 4,0 5 10 min 3,5 3,0 30 min 4 activity 3,0 μ M) 2,5 2,5 3 2,0 2,0 1,5 2 – value (

1,0 A K 1 1,5 0,5 normalized kinase activity normalized kinase 0,0 0 * * * 1,0 w/o Hsp90 + Hsp90 + Hsp90 Hsp90 β α β β α β CM & Cdc37 & Cdc37 & Cdc37 Cdc37 WT E WT E E ERR E E E activation by Hsp90/Cdc37 + Rad E F

+ Hsp90 + Cdc37E 3,5 12 10 activity 3,0 + CM 2,5 + Hsp90-E42A 8 (°C) 6

2,0 1/2 4 1,5 + Hsp90 2

1,0 Δ T of I 0,5 v-Src only 0 + Hsp90-D88A -2 normalized kinase 0,0 c-Src v-Src -ATP +Rad Hsp90α 30 35 40 45 T (°C)

Fig. 1. In vitro influence of Hsp90 on Src kinases. (A) Assay scheme of in vitro stabilization and activation of Src kinases by chaperones. Before trans- phosphorylation, kinases were preincubated for 10 min on ice in the presence or absence of Hsp90 (and/or Cdc37 variants) and 20 μM ATP followed by 10 min at increasing temperatures (stabilization) or 30 °C (activation) and 10-min incubation on ice. Following, 10-fold excess (3.2 μM) of denatured enolase substrate and 20 μM of radioactively labeled ATP were added, and the reaction was incubated for 30 min at 30 °C to measure kinase activity. (B) v-Src kinase activation by chaperones in vitro. The normalized kinase activities were calculated by dividing the detected activities by the activity of the respective kinase alone.

Hsp90β (blue) and Cdc37-S13E (Cdc37E) (red) were titrated either alone or in combination (orange) to v-Src kinase and compared with a lysozyme control (green). Hsp90β together with Cdc37E had no significant effect on c-Src (gray). (C) Effect of Radicicol on the ability of 1.3 μM Hsp90 to activate 320 nM v-Src. Ten minutes after starting the kinase reaction, 250 μM Radicol was added and kinase activity was analyzed after 30 min. Here, all detected activities were normalized to the value of v-Src alone after 10 min. The error bars represent the SD of three independent experiments. (D) Activation of v-Src by different

combinations of Hsp90β (β) or Hsp90α (α) and Cdc37E (E), Cdc37WT (WT), a non–Hsp90-binding Cdc37 mutant [Cdc37-S13E-M164R-L205R, Cdc37ERR (ERR)], and a chaperone mix including Hsp90β, Cdc37E, Hdj-1 (Hsp40), Hsp70, and Hop (CM). Depicted are the activation constants KA (black bars) and the normalized kinase activity at a (co)chaperone concentration of 1.3 μM (blue bars). A black asterisk indicates activation curves for which no activation constant was calculable. (E) Influence of chaperones on v-Src during exposure to elevated temperatures. v-Src was incubated at different temperatures with 20 μM ATP in the absence (gray) or presence of 1.3 μM Hsp90 (blue), Hsp90-E42A (olive), Hsp90-D88A (cyan), 1.3 μM Hsp90 and 1.3 μM Cdc37 (orange), or the chaperone mix (red). (F) Stabilization of c-Src and v-Src by Hsp90 and ATPase dependence of the process. Plotted are the changes in the temperature (ΔT) of half-maximal in-

activation (I1/2). Leaving out ATP or addition of the Hsp90 inhibitor Radicicol during the preincubation steps compromised the ability of Hsp90 to rescue v-Src from inactivation. Unlike Hsp90β, Hsp90α was not able to stabilize v-Src.

resulted in a twofold increase in activity (Fig. 1B). This effect is used a Cdc37 variant in which the phosphorylation was mimicked ATP-dependent as shown by the addition of the Hsp90 in- by a glutamate substitution (Cdc37E), also this variant alone had hibitor Radicicol during the reaction (Fig. 1C), in which the no significant influence on v-Src (Fig. 1B), but together with Hsp90, activity of v-Src decreased to its basal, Hsp90-independent level. we observed a threefold increase in its activity at a chaperone This shows that activation is constantly dependent on Hsp90. In- concentration of 1.3 μM(Fig.1B and Fig. S1A). Importantly, the terestingly, only the human Hsp90 isoform, Hsp90β, but not the combination of Hsp90 and Cdc37E increased the apparent affinity Hsp90-α isoform, was active in chaperoning v-Src kinase (Fig. 1D). toward the kinase by roughly 15-fold, reflected by the activation The kinase-specific cochaperone Cdc37 alone or in combina- constants, KA, obtained by a Michaelis–Menten fit. For Hsp90β,we D tion with Hsp90 had no effect on v-Src activity (Fig. 1 ). How- obtain a KA value of 3.0 μM, whereas for the combination of ever, in vivo experiments had suggested that phosphorylation of Hsp90β and Cdc37E,theKA value decreased to 0.23 μM(Fig.1B). Cdc37 is involved in regulating its function (19, 36). When we Phosphorylated wild-type Cdc37 had the same effect. This increase

E3190 | www.pnas.org/cgi/doi/10.1073/pnas.1424342112 Boczek et al. Downloaded by guest on September 23, 2021 in apparent affinity could result from the independent action of (c-SrcΔC) and finally combined both elements in the variant PNAS PLUS Hsp90 and Cdc37E. To test whether the interaction between the c-Src3MΔC. To test the activity of the generated c-Src mutants in two proteins is required, we made use of a Cdc37E mutant de- vivo, we expressed these variants in . v-Src fective in Hsp90 binding (37) (Cdc37ERR). We found that this is known to be highly toxic for yeast, whereas c-Src does not affect variant was incapable of kinase activation in the presence of Hsp90, yeast growth (21). We were able to reproduce these findings and suggesting that the formation of a complex between Cdc37 and show that the expression of c-Src3M and c-Src3MΔCledtoa Hsp90 is important for activating v-Src (Fig. 1D and Fig. S1B). reduced growth of yeast, although to a lesser extent compared We could not observe a significant increase in activation by the with v-Src (Fig. 2C). c-SrcΔC did not affect yeast viability. Ap- addition of the Hsp70 chaperone system (Hsp70, Hsp40, and parently, the three introduced point mutations are responsible for Hop) to v-Src kinase (Fig. 1 D and E). These findings suggest the compromised growth observed in yeast. To test for a possible that, in this setup, Hsp90β together with phosphorylated Cdc37 Hsp90 dependence of the Src variants, cells were analyzed in the are the key players responsible for activating the client kinase. presence of the Hsp90 inhibitor Radicicol (Fig. 2C). Upon Hsp90 It is established that v-Src is less stable than c-Src against inhibition, yeast cells exhibited less pronounced lethality in the thermal unfolding (38). We thus asked whether Hsp90 could case of c-Src3M and normal growth upon expression of v-Src and stabilize v-Src kinase at elevated temperatures. To this end, we c-Src3MΔC. Thus, the toxic effect of c-Src3MΔC and v-Src was incubated v-Src at different temperatures with Hsp90 or with much more Hsp90-dependent than that of c-Src3M, and Hsp90 both Hsp90 and Cdc37E (Fig. 1A). With increasing temperatures, dependence is largely caused by the combination of the three point v-Src alone rapidly lost its activity compared with c-Src (Fig. 1E mutations and the missing C-terminal tail in v-Src. To further in- and Fig. S1C). The presence of ATP had no influence on kinase vestigate Src kinase activity in vivo, we analyzed the presence of stability under these conditions. However, in the presence of phosphotyrosines in yeast proteins (Fig. 2D). For c-Src and c-SrcΔC, Hsp90β (but not Hsp90α), the loss of kinase activity was pre- no phosphotyrosines were detectable. However, for c-Src3M, vented. For that, a concentration of 1.3 μM Hsp90 was sufficient c-Src3MΔC, and v-Src, a distinct phosphorylation pattern ap- (Fig. S1D). peared. The intensity increased from c-Src3M to c-Src3MΔC with Remarkably, this stabilizing effect became Cdc37-independent v-Src exhibiting the most pronounced effects. Interestingly, in at higher temperatures (Fig. 1E) as the two curves converge at vivo activity did not seem to correlate with protein abundance around 40 °C. The presence of the Hsp70 chaperone system had (Fig. 2D). The expression of the toxic c-Src3M mutant was in- no influence on the action of Hsp90 (Fig. 1E). creased, although this kinase showed less activity relative to the To analyze how Hsp90 couples ATP binding and hydrolysis to c-Src3MΔC and v-Src variants. Furthermore, the mutants with a v-Src activation and stabilization, we used two well-characterized deleted C-terminal tail were less abundantly expressed, indicating ATPase-deficient variants of human Hsp90β and tested them for a lower stability of these constructs (Fig. 2D). To further inves- activation of v-Src kinase (Fig. 1E). Hsp90-E42A binds ATP but tigate the in vivo stability of the kinases, we performed a pulse does not hydrolyze it. Hsp90-D88A on the other hand neither chase experiment and analyzed the protein levels. We found that binds nor hydrolyzes ATP (39, 40). Both variants showed a v-Src showed a strongly and c-Src3MΔC showed a slightly reduced similar stability compared with wild-type Hsp90 (Fig. S1E). half-life during the time course compared with c-Src (Fig. S2). Analysis of these Hsp90 mutants showed that Hsp90-D88A in Interestingly, analyzing the stability of the constructs in a Cdc37- BIOCHEMISTRY combination with Cdc37E did not affect v-Src activity, whereas knockdown background revealed that the variants c-Src3M, Hsp90-E42A was able to activate v-Src approximately twofold at c-Src3MΔC, and v-Src exhibited a significant dependence on Cdc37 30 °C (Fig. 1E). Furthermore, we found that Hsp90-D88A could with c-Src3M showing less Cdc37 dependence than c-Src3MΔCand not stabilize v-Src (Fig. 1E), which is in agreement with the necessity v-Src. The Hsp90-independent variants c-Src and c-SrcΔCwerenot of ATP for this reaction (Fig. 1F). Intriguingly, Hsp90-E42A was affected by the Cdc37 knockdown (Fig. S2). able to stabilize v-Src similar to wild-type Hsp90 (Fig. 1E). Next, we tested the different Src variants for their potential to In the presence of Hsp90, the temperature of half-maximal be activated by Hsp90 and Cdc37E in vitro. We found that v-Src inactivation I1/2 was shifted by 9 °C (Fig. 1F), and the apparent and c-Src3MΔC were readily activated, whereas, in accordance stability corresponded to that of c-Src (Fig. S1C). For c-Src, the with the in vivo results, c-Src, c-SrcΔC, and c-Src3M showed only presence of Hsp90 did not affect the inactivation process (Fig. low activation potential (Fig. 2E). This was consistent with the 1F). The stabilizing effect of Hsp90β on v-Src was dependent on observation of a lower in vivo activity of c-Src3M regardless of its the presence of ATP and could be suppressed by the addition of robust expression. A similar picture emerges at elevated tem- Radicicol (Fig. 1F). In summary, we observed that, for stabili- peratures: c-Src3M and c-SrcΔC were not significantly stabilized, zation of v-Src during heat exposure, ATP binding by Hsp90 is and, in contrast, the inactivation of c-Src3MΔC and v-Src was sufficient. However, for activation of v-Src under physiological strongly affected by Hsp90 (Fig. 2F and Fig. S1C). To further conditions, both the presence of the cochaperone Cdc37E and evaluate the effect of Hsp90 and Cdc37 in vivo and in vitro, Fig. ATP hydrolysis by Hsp90 is needed. 2G shows the in vivo activities of the different variants normal- ized to their respective expression level in comparison with their Src Chimeras Reveal Client Features of v-Src. Having reconstituted in vitro activities in the presence and in the absence of Hsp90 the Hsp90-dependent chaperoning of v-Src kinase in vitro, we and Cdc37E. Notably, the in vitro analysis faithfully recapitulates probed factors that render v-Src a chaperone client but leave in vivo effects and the ratios of the activities for the different c-Src unaffected. variants in the presence of Hsp90 and Cdc37E reflect the ratios v-Src differs from its cellular isoform c-Src in only a few amino of the in vivo activities. acids and in the absence of the C-terminal tail (Fig. 2 A and B). Specifically, three mutations have been shown to be responsible What Renders a Kinase Hsp90-Dependent? Our set of mutants for the transforming potential of v-Src (31–33). These point allowed us to define characteristics that correlate with differ- mutations are located in the SH3–linker–kinase domain in- ences in Hsp90 dependence. When comparing the activity of the terface. To determine how these changes affect the function of different Src variants, we found that isolated v-Src has a fourfold the kinase as well as its structure and Hsp90 dependence, we higher activity than c-Src (Fig. 3A). In agreement with results created chimeras of c-Src. In the c-Src variant c-Src3M, three from the in vivo activity (Fig. 2D), deletion of the C-terminal tail residues were exchanged for their respective residues in v-Src in c-Src did not significantly alter the activity of the kinase. In- (R95W, D117N, and R318Q). Furthermore, we created a c-Src troduction of the three point mutations (c-Src3M), however, led variant, in which the C-terminal tail of c-Src was deleted to a nearly 2.5-fold activation of c-Src, whereas the combination

Boczek et al. PNAS | Published online June 8, 2015 | E3191 Downloaded by guest on September 23, 2021 A D117N R95W R318Q C-terminus B N unique

SH3 N-term. lobe

C SH2 C- term. lobe

C D phospho-tyrosine signal + DMSO + Rad c-Src ΔC3v-Src 3M M c-Src ΔC 240 140 c-SrcΔC 100 70 c-Src3M

c-Src3MΔC 50

v-Src expression levels

E F G 1,2 c-Src in vitro activity 3,0 in vitro activity 3M 10 1,0 C + Hsp90-Cdc37E 2,5 3M C 8 0,8 in vivo activity (norm.) v-Src (°C) 6 0,6 2,0 1/2 4 0,4 T of I

1,5 Δ 2 0,2 0 normalized kinase activity normalized kinase1,0 activity 0,0 activation by Hsp90/Cdc37 -2 c-Src c-Src c-Src c-Src v-Src c-Srcc-Src c-Src c-Src v-Src 3M ΔC 3MΔC ΔC 3M 3MΔC

Fig. 2. Design of a Hsp90 client kinase. (A) Structure of c-Src (PDB ID 1Y57) (66). Coloring from N to C terminus: SH3 domain in green, SH2 domain in red, linker connecting SH2 domain and N-lobe of the kinase domain in yellow, kinase domain N-lobe in violet, and C-lobe in blue. Tryptophan residues are depicted as gray spheres. The mutated or deleted residues in the c-Src variants are depicted as blue spheres. (B) Schematic representation of c-Src. Colors are the same as in the crystal structure. The unique domain, which is missing in the crystal structure, is shown in gray. Intramolecular contacts made by the mutated residues are represented by dotted light blue lines. (C) Serial dilutions of S. cerevisiae expressing different Src variants in the absence or presence of the Hsp90 inhibitor Radicicol in the medium. (D) In vivo activity of Src kinases were tested by detecting phosphotyrosines in yeast lysates. In addition, ex-

pression levels were determined by a Src-specific antibody. (E) Activation of Src variants by 1.3 μMHsp90β and Cdc37E.(F) Stabilization of Src variants by 1.3 μM Hsp90β is represented as the shift of the temperature of half-maximal inactivation. (G) Comparison of v-Src chaperoning in vivo and in vitro. Shown are the in

vitro activities of Src variants alone (orange) and their Hsp90/Cdc37E-dependent in vitro-activated activities (red) in comparison with their in vivo activities normalized to their respective expression levels.

of the amino acid substitutions and the C-terminal deletion The structural changes of the Src variants during thermal (c-Src3MΔC) gave rise to a threefold increase. It is noteworthy unfolding were monitored by circular dichroism (CD) spectros- that c-Src and c-SrcΔC did not show detectable activity in yeast copy (Fig. 3B and Table 1). We found that c-Src was most stable but were active as purified proteins in vitro. Overall, the activities with a melting temperature (TM) of 50 °C, whereas the TM for of the three toxic kinase variants were comparably increased in v-Src was 46 °C. The c-SrcΔC mutant showed a T of 44 °C, and K M vitro. Consistent with the literature, the M of c-Src for ATP was the c-Src3M mutant exhibited an unfolding behavior in-between ∼ μ K in the 13 M range (41) (Table 1). We found that the M of those observed for c-Src and v-Src. v-Src and the mutant Δ ∼ μ c-Src C was slightly increased to about 20 M. Notably, all Src c-Src3MΔC showed remarkably low cooperativities, as indicated variants carrying the three point mutations showed a threefold to K ∼ μ by the less sigmoidal melting curves, and had already begun to fivefold increase in the ATP affinity with M values of 4 M. unfold around 20 °C. Interestingly, the unfolding process was To determine the stability of the Src variants, we incubated completed only above 70 °C. Thus, deletion of the C-terminal tail each of them at elevated temperatures and measured their ac- in v-Src contributes mainly to the observed instability at lower tivities. In agreement with previous results (38), c-Src lost its temperatures, whereas the combination of the point mutations activity at higher temperatures compared with v-Src (Fig. S1A). and the C-terminal deletion strongly decrease the cooperativity The temperature of half-maximal kinase activity (I1/2) was 49.5 °C for c-Src and 40.2 °C for v-Src (Table 1). The C-terminal tail of unfolding. This revealed a correlation between the unfolding contributes significantly to the loss of activity of v-Src already at cooperativity of the kinases and their respective in vivo Hsp90 Δ moderate temperatures. The additional three single amino acid dependence: the two stringent client kinases v-Src and c-Src3M C substitutions (c-Src3MΔC) destabilized the protein even further unfolded over a broad temperature range, which is reflected in the dx Δ with an I1/2 of 33.3 °C. Hence, grafting of v-Src elements onto c-Src high values of 6.4 and 5.8 of the Boltzmann fits for c-Src3M C kinase generally decreases kinase activity at elevated tempera- and v-Src, respectively. For the less Hsp90-dependent constructs tures. In this context, the C-terminal tail deletion seems to play a c-Src, c-SrcΔC, and c-Src3M, this value was in the range of 2.0 (Fig. pivotal role, with a loss of stability of at least 10 °C observed for 3B, Inset, and Table 1). Consistent with previous findings (5), we all C-terminally deleted kinase variants. observed a regain of cooperativity by the addition of a kinase inhibitor.

E3192 | www.pnas.org/cgi/doi/10.1073/pnas.1424342112 Boczek et al. Downloaded by guest on September 23, 2021 Table 1. Characteristics of investigated Src variants PNAS PLUS 5 Src variant I1/2,°C TM,°C dx, value of unfolding Aggregation, % Tryptophan accessibility, fa ANS fluorescence, 10 a.u. KM for ATP, μM

c-Src 49.5 50 2.4 55 0.65 (± 0.041) 6.48 12.8 (± 1.4) c-Src3M 45.1 46.5 2.1 58 0.68 (± 0.017) 9.73 4.9 (± 0.8) c-SrcΔC 38.8 44 2.0 89 0.66 (± 0.042) 11.46 19.8 (± 3.6) c-Src3MΔC 33.3 43.5 6.4 92.5 0.83 (± 0.083) 13.91 4.0 (± 0.7) v-Src 40.2 46 5.8 95 0.92 (± 0.111) 17.17 4.0 (± 0.8) c-Src-K295R — 51 1.7 44 0.64 (± 0.037) 8.35 — v-Src-K295R — 32.6 9.2 99.8 0.79 (± 0.033) 19.34 —

Separating the Src variants incubated at 45 °C into soluble and Investigation of surface hydrophobicity revealed that the three insoluble fractions revealed that, although one-half of the c-Src point mutations only led to a moderate increase in 1-anilino-8- protein remained soluble, 95% of v-Src was found in the in- naphthalene sulfonate (ANS) binding (Fig. 3E). However, in ac- soluble fraction (Fig. 3C). The two Src variants missing the cordance with the aggregation behavior, the kinase variants lacking C-terminal tail behaved similar to v-Src. Analogous to c-Src, the C-terminal tail exhibited considerably stronger ANS binding, 58% of the c-Src3M protein was found in the insoluble fraction. arguing for an increased exposure of hydrophobic patches (Fig. 3E). Thus, deletion of the C-terminal tail led to increased aggregation Taken together, our results suggest that the point mutations propensity, suggesting that the point mutations alone did not together with the C-terminal tail deletion in v-Src destabilize the influence the aggregation of c-Src. protein and reduce its rigidity, thereby increasing hydrophobicity To gain further insight into conformational changes, we ana- and aggregation propensity. lyzed the accessibility of tryptophan residues in the Src variants. Particular Regions Are Exposed in Hsp90 Client Kinases. To in- In Src kinase, tryptophans are widely distributed across the A timately compare Hsp90 client and nonclient kinase conforma- protein (Fig. 2 , gray spheres). Higher molecular flexibility will tions, we used hydrogen/deuterium (H/D) exchange experiments. result in an increased exposure of these hydrophobic residues to For this, we used the catalytically inactive v-Src mutant v-SrcK295R – quenchers like acrylamide (42). Modified Stern Volmer plots (43), due to significantly increased protein yields. Its characterization f revealed the corresponding accessibility factors, a, of the ana- in comparison with v-Src, c-Src, and c-SrcK295R by our established D f lyzed Src variants (Fig. 3 ). The a value of v-Src was 0.92 and set of methods revealed that, although minor differences were the one corresponding to c-Src3MΔC was 0.83, indicating that in observed, this version sufficiently resembled wild-type v-Src prop- these variants most of the tryptophan residues were readily ac- erties (Fig. S3 A–D and Table 1). We determined the regions of cessible to the quenching molecules. c-Src and the c-Src3M and v-SrcK295R with increased H/D exchange relative to the non- c-SrcΔC mutants had an fa value of 0.6–0.7, suggesting that the Hsp90 client c-Src and the weak Hsp90 client c-Src3M (Fig. 4A,

tryptophan residues were more buried in these proteins. Figs. S4–S8,andTable S1). For c-Src3M, these regions are located BIOCHEMISTRY

A B in vitro Transphosphorylation Thermal Unfolding 1,0 1,0 c-Src 0,8 0,8 structure c-SrcΔC 0,6 0,6 6 dx c-Src3M 0,4 0,4 4 c-Src3MΔC

secondary 0,2 2 v-Src 0,2 0,0 0

normalized kinase0,0 activity c-Src ΔC3M3MΔC v-Src loss of 20 30 40 50 60 70 80 T (°C)

C Aggregation D Tryptophan Quenching E ANS Binding 6 100100 3,0 2,0x10 2,5 9090 1,5x106 2,0 8080 F Δ 6 /

0 1,5 1,0x10 F 7070 1,0 1,0 f 5 a 5,0x10 6060 0,5 0,8 insoluble fraction (%)

fluorescence signal (a.u.) 0,0 0,0 0,6 01234 400 450 500 550 600 c-Src 3M ΔC 3MΔCv-Src acrylamide-1 (M-1) wavelength (nm) S ISI S I S I S I

Fig. 3. Biophysical characterization of Src variants. (A) In vitro transphosphorylation signal of 320 nM Src variants and the corresponding SDS/PAGE auto- radiograph. (B) Stability and unfolding cooperativity of 1.6 μM Src variants measured by CD spectroscopy. Thermal transitions were recorded at 222 nm. The Inset shows the dx values of the Boltzmann plot. (C) Analysis of aggregation propensity. Src variants at a concentration of 1.6 μM were incubated for 10 min at 45 °C, and soluble and insoluble fraction separated by SDS/PAGE and densitometrically quantified. (D) Quenching of tryptophan fluorescence. Acrylamide was titrated to 500 nM Src variants and fluorescence emission after excitation at 295 nm was monitored at 340 nm. Shown are modified Stern–Volmer plots for the

different Src mutants. In the Inset,thefa values representing the accessibility of tryptophans are plotted for the different proteins. (E) Analysis of ANS binding to hydrophobic protein patches. The 20 μM ANS was incubated with 1.6 μM Src variants, and fluorescence spectra after excitation at 380 nm were recorded and buffer corrected.

Boczek et al. PNAS | Published online June 8, 2015 | E3193 Downloaded by guest on September 23, 2021 A

active center β1-5- strands rotate 90° N-lobe αC-helix A-loop

C-lobe αE-helix E310-K295

BC 8

) displacement c-Src 2

Å SASA 20 c-Src3MΔC

12 6 ) 2 Å )

Å 15 4 ( 8 2 10 4 0 distance 0 5 -2 ( relative SASA relative displacement ( 0 -4 -4 0 50 100 150 200 250 300 350 400 450 500 time (ns) log(p) residue D

c-Src c-Src3MΔC

Fig. 4. Surface-exposed regions and dynamics of client kinases. (A) Hydrogen/deuterium exchange of Src variants. Increased exchange in c-Src3M compared with c-Src in pale green and blue, increased exchange in v-SrcK295R compared with c-Src in red and regions overlapping with c-Src3M in pale green, increased exchange in v-SrcK295R compared with c-Src3M in magenta. The Glu310–Lys295 ion pair is depicted as stick model. The three point mutations in c-Src3M and v-Src are depicted as orange spheres. (B) The dynamics of Glu310–Lys295 distances in c-Src (red) and c-Src3MΔC (blue). The Glu310–Lys295 distances are 3.4 and 14.0 Å in the active [A(EK)] and inactive [I(EK)] states of c-Src, respectively. (C) Difference in c-Src3MΔC vs. c-Src displacement and solvent-accessible surface area (SASA) for the kinase domain. The SASA was calculated as the average over the 250-ns runs for the complete residues. The displacement was calculated from the displacement of the Cα position obtained from end point structures at 250 ns. (D) Structural features of the active-site region of c-Src3MΔC and c-Src.

in the SH2 domain, in the kinase linker, and around the active stability in v-Src. For the c-Src model, we find that the Glu310– center, including the active-site residues as well as the αC-helix, the Lys295 ion pair remains closed for most of the 250-ns simulation αC-β4–loop, the β1-, β2-, and β3-strands, and the β4- and β5- (Fig. 4B), suggesting that the structure is trapped in the active − strands (Fig. 4A). Moreover, we observed an increased exchange in state. This finding is consistent with the 4 kcal·mol 1 activation the A-loop region, including the site of autophosphorylation barrier recently estimated by Shukla et al. (46). In contrast, for Tyr416, and around the C-terminal part of the kinase domain. the c-Src3MΔC variant, we observe several transient dissociation Strikingly, the exposed regions in v-Src almost completely events of the Glu310–Lys295 ion pair (Fig. 4B), and a consistent overlap with those of c-Src3M (Fig. 4A). However, we observe shortening of the Glu310–Arg409 distance, indicating fluctuations additional exposed parts in v-Src and even higher solvent ac- between active and inactive states. Our simulations also suggest cessibility in the regions that are crucial for kinase activation, conformational changes in the A-loop region in c-Src3MΔCrel- which include the active center region containing the αC-helix, the ative to c-Src, indicating significant displacement and partial P-loop, and the β1- to β3-strands of the kinase domain N-lobe. unfolding (Fig. 4C). Moreover, we observe a subtle outward tilting The accessibility of the A-loop and of the C-terminal part of the of the αC-helix and an increase in the solvent accessibility and kinase domain is increased in v-Src over c-Src3M. In addition, in residue flexibility of the active-site and the A-loop regions (Fig. v-Src also the αE-helix is more accessible (Fig. 4A). 4C), which may affect the accessibility for substrate binding. To probe intrinsic dynamics of the proteins, we performed Similarly, the β1-, β2-, and β3-strands show increased displacement classical atomistic molecular dynamics (MD) simulations on the and surface exposure, and, consistent with our H/D exchange data, in silico mutated Src variant c-Src3MΔC and compared these to the αE-helix seems to be more flexible compared with c-Src (Fig. MD simulations of wild-type c-Src. The dynamics of the central 4C). The MD simulations can reveal possible reasons for the Glu310-Lys295 ion pair, and the activation-loop (A-loop) region distinct behavior of the c-Src3MΔC variant relative to c-Src. In the represent dynamical indicators of the kinase activation process c-Src simulation, Arg95 forms ion pairs with two glutamate resi- (44, 45), which we use here to address the underlying molecular dues (Glu305 and Glu93), which are involved in stabilizing the αC- mechanism for the remarkable increase in activity and loss of helix and Arg409 of the A-loop (Fig. 4D). The R95W mutation

E3194 | www.pnas.org/cgi/doi/10.1073/pnas.1424342112 Boczek et al. Downloaded by guest on September 23, 2021 leads to interaction between Arg409 and Trp95, and partial dis- ATP PNAS PLUS – sociation of the Arg95 Glu305/Glu93 ion pairs, thus destabilizing H sp9 0 p9 0 s H Hsp90β the αC-helix and A-loop regions. We also observe that the in- teraction between Arg318, at the end of the αC-helix, with Trp260 is weakened by replacement with Gln318, which is likely to con- unfolded aggregates tribute to the flexibility of the helix and further increase of the rate Hsp90- of H/D exchange. inhibitor Discussion The Conformational Activation of v-Src Kinase. In vivo, the Hsp90 heat shock machinery activates v-Src kinase. Reconstitution of the chaper- one effect in vitro shows that, although Hsp90 alone is able to increase v-Src activity, Cdc37 strongly enhances this effect. The v-Src physiological v-Src affinity of Hsp90 toward the kinase is increased in the presence inactive conditions active of Cdc37 about 15-fold, resulting in an apparent affinity of 0.23 μM.

H s We assume the physiological concentration of Hsp90 to be in the p9 p90 0 s H

7 ATP

3 low micromolar range, and the concentration of Cdc37, mid- c

Cd nanomolar (47, 48). Cdc37 thus allows Hsp90 to interact with ki- Hsp90- Hsp90β nases in the cell under normal growth conditions. Consistent with inhibitor Cdc37 the literature (36), we find that the phosphorylated form of Cdc37 is required for efficient activation of v-Src, without going through phosphorylation/dephosphorylation cycles. Furthermore, we show that the formation of the Cdc37–Hsp90 complex is mandatory for v-Src activation. Although this finding is in agreement with the v-Src need of the Hsp90–Cdc37 interaction for kinase-dependent cell active proliferation (49), alternative pathways for certain kinases may Fig. 5. A model for kinase maturation by Hsp90. Client kinases exist in an exist (37). In this context, the affinity of a kinase for ATP does not equilibrium of metastable and stabilized states. Unfolding stress and in- seem to correlate strictly with its chaperone dependence as dif- trinsic instability shift the equilibrium toward the less active state. Hsp90 ferent effects were observed for B-Raf kinase (50) and the Src stabilizes and maturates the kinase toward its active state. Cdc37 is able to kinase pair studied here. The finding of an Hsp90-isoform spec- enhance Hsp90’s affinity toward the kinase. Inhibition of Hsp90 prevents the ificity of v-Src implies that the client spectrum of Hsp90β and process and leads to kinase inactivation, aggregation, or degradation. Hsp90α may differ, as Hsp90α has been shown to be able to ac- tivate other kinases (51). To figure out which properties of a protein render it a client for Hsp90, we made use of the few sequence differences between

Chaperoning of v-Src at Elevated Temperatures. Under thermal BIOCHEMISTRY the v-Src and c-Src kinase pair. By grafting of client elements stress, a different picture emerges for the effect of Hsp90 on v-Src kinase. Here, Hsp90 alone stabilizes v-Src in a reaction onto the scaffold of the nonclient c-Src kinase and comparing the where ATP binding, but not hydrolysis, is crucial. Notably, Cdc37 characteristics of the chimeras, we could identify structural prop- erties that cause chaperone dependence. Interestingly, the deletion does not seem to be required for Hsp90 to stabilize v-Src. At around Δ 38 °C, we observed a transition point for the shift from the Hsp90- of the regulatory C-terminal tail in c-Src C, which is the primary Cdc37–mediated activation toward an Hsp90-dependent stabilization. reason for the conversion of c-Src to a transforming protein in mammals (54), did not result in increased Hsp90 dependence in In summary, we found that for stabilization at elevated tem- Δ peratures ATP binding is sufficient. However, for activation of vivo and in vitro. Nevertheless, c-Src C showed a significantly de- v-Src under physiological conditions (30 °C), both the presence of creased stability compared with the wild-type c-Src kinase, as well as a high aggregation propensity. Thus, these properties per se are not the cochaperone Cdc37E and ATP hydrolysis by Hsp90 is needed. If only ATP binding is possible, kinase activation is inefficient. sufficient for defining a protein an Hsp90 client. The observed stabilizing effect might require different in- Another c-Src variant, carrying three point mutations teractions of v-Src with Hsp90 for which Cdc37 is dispensable (Fig. (c-Src3M) that are relevant for the development of transforming – 5). It is tempting to speculate that the thermal destabilization of properties in cancer (31 33), was toxic in yeast as well as the v-Src kinase leads to the accumulation of a kinase conformation combination of the C-terminal tail deletion and the three point Δ with increased affinity for Hsp90, and this may bypass the initial mutations (c-Src3M C). However, only the latter chimera was step of Cdc37 association. Of note, in yeast, only Hsp90 and the similarly Hsp90-dependent as v-Src. cochaperones Sti1 (stress-inducible protein) and Cpr6, but not To resolve this conundrum, we aimed to define the structural Cdc37 are up-regulated under heat shock (52). Also, a Cdc37- differences between the variants. We found that the introduction independent association of Hsp90 with kinases was previously of v-Src elements into c-Src leads to a general decrease in sta- Δ observed (50), and Cdc37 is not present in all organisms (53), bility. However, only the two strong Hsp90 clients c-Src3M C suggesting a distinct role of Hsp90 in client kinase maturation. and v-Src show remarkable low unfolding cooperativity, in- dicating that the protein is flexible and may exist in an ensemble Identifying the Characteristics of an Hsp90 Client. ATP binding and of folded structures. Furthermore, the Hsp90 dependence cor- hydrolysis by Hsp90 is a prerequisite for kinase activation under relates with an increased surface hydrophobicity. This finding is physiological conditions. We found that generating and maintaining in agreement with the notion of increased structural dynamics the activated state of v-Src is strictly dependent on the continued and molecular compactness of the Hsp90 client. Of note, the ATPase cycle of Hsp90 as its inhibition decreases kinase activity to three mutations and the deletion of the C-terminal tail did not its basal Hsp90-independent level. This implies that the kinase fully convert c-Src into v-Src with respect to Hsp90 dependence molecules are constantly activated by the chaperone. We propose and its biophysical qualities. In addition to the altered C-termi- that Hsp90/Cdc37 shift the conformational ensemble of different nal tail, v-Src in total carries 11 point mutations compared with activation states toward a more active distribution. c-Src that might well be involved in causing the differences.

Boczek et al. PNAS | Published online June 8, 2015 | E3195 Downloaded by guest on September 23, 2021 In summary, a combination of qualities seems to cause Hsp90 Our results are in line with this suggestion as they show that the dependence: client kinases are destabilized, aggregation prone, degree of exposition of these sites correlates with the strength of with hydrophobic, less compact structures, and can hence be the dependence on Hsp90, increasing from c-Src, over c-Src3M, designated as metastable proteins. to v-Src. Furthermore, we find that the C-terminal tail deletion strongly increases the accessibility of the distant active center Exposure of Key Kinase Regions Determines Hsp90 Dependence. The and the A-loop. backbone hydrogen/deuterium exchange experiments showed a These results support the notion that v-Src may exist in mul- significant increase in the dynamics of regions that are located tiple native-like states. We suggest that the activated state of the within previously suggested chaperone binding sites. These in- kinase is instable and reverts to the less active state unless Hsp90 α clude the glycine-rich P-loop, the C-helix, the adjacent loop is present, which may induce or select this state. This, in effect, α β – β β ( C- 4 loop), as well as the 1- to 5-strands, all located in the leads to an apparent higher activity of v-Src in the presence – N-lobe of the kinase domain (55 58). In addition to the in- of Hsp90. creased exposure of these sites, the P-loop and the αE-helix are more accessible in v-Src compared with c-Src and c-Src3M, Materials and Methods which could account for the differences in response to Hsp90. Cloning and Expression of c-Src, v-Src, and Src Mutants in Sf9 Cells. Chicken Our molecular dynamics simulations confirm these notions c-Src and v-Src from Rous sarcoma virus (Schmidt-Ruppin strain A) were used and extend the picture. Specific regions show increased exposure in this study. Coding were obtained by PCR using 5′-CATCGCCCATG- and/or flexibility including the αC-helix, the proximal portion of GCCGGGAGCAGCAAGAGCAAGC-3′ (containing a NcoI restriction site) as the αC-β4–loop, and the αE-helix, consistent with the H/D ex- forward primer and 5′-CACATAAGCTTTCACTATAGGTTCTCTCCAGGCTG-3′ change data. Because the P-loop is rich in glycines, the overall and 5′-CACATAAGCTTCTACTCAGCGACCTCCAACACACAA-3′ (both contain- solvent-accessible surface area (SASA) is small for this region. ing a HindIII restriction site) as reverse primers for c-Src and v-Src, re- Nevertheless, there seems to be a structural difference, as in- spectively. For the C-terminal truncation mutants of c-Src, 5′-GATCAAGCTTC- ′ dicated by the peak in displacement. TACAGGAAGGCCTGCAGGTACTCAAAAGTC-3 (containing a HindIII restriction The increased “active-to-inactive” transitions in the c-Src3MΔC site) was used as reverse primer. Templates of Src were generated as described elsewhere (38). Point mutants of all genes were generated by site-directed variant suggest that the kinetic barrier for activation is effectively mutagenesis. After amplification, the Src variants were cloned into the lowered, which might explain the experimentally observed in- pFastBacHTA expression plasmid (Invitrogen) and subsequently transformed Δ creased activity and conformational flexibility in c-Src3M C. We into DH10Bac Escherichia coli cells (Invitrogen) for producing recombinant observe transient structures with partial structural features from Bacmid DNA. For generating recombinant baculovirus, Sf9 cells in monolayer both the active and inactive states in c-Src3MΔC. Together with culture were transfected with 1 μg of Bacmid-DNA at a density of 9 × 105 cells our results on their unfolding cooperativity, this indicates that per well and incubated a further 7–10 d. For expression, 20 μLofviruspermL there exist more metastable structures in v-Src, which could be were added to 2 × 106 cells per mL in suspension culture and incubated for stabilized by the interaction with Hsp90. Thermodynamically, an 48 h at 27 °C. increased population of different native-like structures would be expected to result in an entropic gain, which render the Protein Purification. For purification of Src kinases, Sf9 cells resuspended in immobilized metal ion affinity chromatography (IMAC) buffer [40 mM activation process more efficient. A general increase in con- · formational flexibility corroboratespreviousstudiessuggesting Tris HCl, 150 mM NaCl, 5% (vol/vol) glycerine, pH 7.5] containing a protease inhibitor mixture (Sigma) were sonicated on ice and centrifuged (20,000 × g, a correlation between chaperone binding and the openness of 40 min, 4 °C). The cleared lysate was loaded onto a Cobalt-Talon column (BD thekinasefold(59). β β Biosciences) preequilibrated in IMAC buffer. To remove unspecifically bound Although the 4- and 5-strands in the kinase domain have proteins, the column was washed with IMAC buffer and subsequently with previously been suggested to be important for chaperone–kinase IMAC buffer containing 15 mM imidazole. The kinase was eluted with IMAC interaction (55), the exposure of the β1-, β2-, and β3-strands in v-Src buffer containing 300 mM imidazole. Src kinase containing fractions were shown here has not yet been correlated with chaperone recognition. pooled, concentrated to 5 mL, loaded onto a Superdex 75 16/60-pg column For the ErbB2 kinase domain, it was found that introducing a (GE Healthcare) for size exclusion chromatography, and eluted with IMAC negative charge in the αC-β4–loop strongly decreased its Hsp90 buffer containing 5 mM DTT (Src buffer). dependence (60). Interestingly, in the case of v-Src, the increased Human Hsp90α and Hsp90β wild-type and mutants were expressed in the Hsp90 dependence cannot be explained by increased hydro- E. coli strain BL21 as a His6-tagged version using a pET28 vector. Human phobicity of this region as the R318Q mutation and the presence Cdc37 wild-type and Cdc37 mutants were expressed in the E. coli strain of other hydrophilic residues in v-Src leaves the αC-β4–loop HB101 as a His6-tagged version using a pQE30 vector containing the re- polar. This implies that Hsp90 does not generally interact with a spective . Hsp90, Cdc37, Hop, Hdj1, and Hsp70 were expressed and purified as described (62–64). hydrophobic αC-β4–loop, but rather with a kinase that shows more flexibility in this region. In addition, the finding that mu- Kinase Activity Assay. For activity measurements of Src variants, 320 nM of the tated ErbB2 remained Hsp90-dependent during its initial folding respective kinase variant was incubated at 30 °C for 30 min in Src buffer 32 (60) is in line with our suggestion that Hsp90 and Cdc37 may supplemented with 10 mM MgCl2, 1 mM MnCl2, and 40 μM[γ- P]ATP with recognize more exposed “epitopes” in client kinases. an activity of 0.5 μCi. A 10-fold molar excess (3.2 μM) of acid-denatured Previous screens found that determinants of the mature enolase (Sigma) was used as a substrate. For that enolase was incubated with kinase–chaperone interaction are distributed over both lobes of 25 μM acetic acid (pH 3.0) for 5 min at 30 °C and subsequently neutralized by thekinasedomainandcorrelatedkinasestabilitywithHsp90 adding 200 mM Tris·HCl buffer (pH 8.0). The kinase reaction was stopped by dependence (5), supporting an early study on the stability of adding Laemmli buffer and boiling the sample. The samples were separated c-Src and v-Src (38). Here, we extend the picture and show that by SDS/PAGE, and transphosphorylation was detected by applying a phos- Hsp90 dependence directly correlates with unfolding coopera- phor image screen onto the gel. The screen was analyzed using a Typhoon tivity, tryptophan accessibility, and hydrophobicity, and fur- 9200 PhosphorImager and the program ImageQuant (GE Healthcare). The kinase reaction was performed either in the absence or presence of different thermore with the extent of exposure of structural elements in amounts of chaperones. thekinasedomain. Given the high similarities between chaperone-dependent Temperature Dependence of Kinase Activation and Stabilization. To investigate and -independent kinases, the idea has been raised that client the influence of the temperature on the activity of Src variants and the in- kinases would frequently expose Hsp90 binding sites, whereas fluence of chaperones on this process, the kinases were first preincubated on

stable kinases only do so during initial folding and therefore only ice in the presence or absence of chaperones in Src buffer with 5 mM MgCl2 transiently interact with chaperones during maturation (38, 61). for 10 min, and subsequently incubated at increasing temperatures for

E3196 | www.pnas.org/cgi/doi/10.1073/pnas.1424342112 Boczek et al. Downloaded by guest on September 23, 2021 10 min and cooled down on ice for 10 min. Then enolase and [γ-32P]ATP upon excitation at 295 nm was recorded using a FluoroMax-3 fluorescence PNAS PLUS were added, and the activity was measured as described above. spectrometer (Spex). The slit widths were set to 3 and 5 nm for excitation and emission, respectively. The changes in fluorescence intensity were CD Spectroscopy: Thermal Transition. The thermal unfolding of the proteins plotted against the acrylamide concentration according to a modified Stern– was monitored at 222 nm in 30 mM sodium phosphate buffer, 30 mM NaCl, Volmer equation (42): 2 mM DTT, pH 7.5, using a 0.1-cm quartz cuvette and a protein concentration of 100 μg/mL by measuring changes of the CD signal between 20 and 80 °C, at F 1 1 0 = + , [1] a heating rate of 20 °C per h. The curves were fitted to a sigmoidal transition. ΔF faKa½Q fa Δ Aggregation Assay. The 1.6 μM kinase was incubated at 45 °C for 10 min. To where F0 is the total fluorescence in the absence of quencher, F is the remove insoluble aggregates, the proteins were subsequently centrifuged fluorescence intensity change at a given quencher concentration [Q], fa is – for 60 min (20,000 × g; 4 °C). Pellet and supernatant proteins were dena- the accessible tryptophan fraction, and Ka is the Stern Volmer quenching tured in Laemmli buffer, boiled at 95 °C, and subsequently analyzed by SDS/ constant of the accessible fraction (38). PAGE and densitometry. Hydrogen/Deuterium Exchange–Mass Spectrometry. Hydrogen/deuterium ex- Yeast Viability Assay. The Hsp90 double-knockout strain ECU82α containing change–mass spectrometry (HDX-MS) experiments were performed on a yeast Hsp82 on a plasmid (yHsp82 in pRS426) was transformed with plasmids fully automated system equipped with a Leap robot (HTS PAL; Leap Tech- containing the Src variants under a galactose promoter (Src in pY413). After nologies), a Waters ACQUITY UPLC, a HDX manager, and the Synapt G2-S growing in glucose-containing URA/LEU/HIS selection medium to stationary mass spectrometer as described elsewhere (Waters) (65). Src kinases were μ phase, cells were diluted in a 10-fold dilution series and 3 L of each dilution vacuum concentrated in the presence of 100 mM trehalose (Sigma). The were spotted on agar plates containing galactose as sole sugar source. Cells protein samples were diluted in a ratio of 1:4 with deuterium oxide-con- were grown at 30 °C for 3 d. In a second approach, the Hsp90 inhibitor taining Src buffer [40 mM Tris, pH 7.1, 150 mM NaCl, 5% (vol/vol) glycerine, Radicicol was added at a final concentration of 5 μM to the plates for de- 5 mM DTT] and incubated at 25 °C for 20, 60, 600, 3,600, and 7,200 s. After termining the Hsp90 dependence of the expressed Src variants. the labeling reaction, the protein was denatured and the exchange was stopped by diluting the labeled protein 1:1 in quenching buffer [50 mM Western Blotting. Src-plasmid–transformed yeast cells were grown at 30 °C in Na HPO × 2HO, 50 mM NaH PO × 2HO; 35 mM tris(2-carboxyethyl) raffinose-containing URA/LEU/HIS selection medium to stationary phase, 2 4 2 2 4 2 spun down, and resuspended in glucose-free URA/LEU/HIS medium con- phosphine, 4 M Gua-HCl, pH 2.6, 4 °C]. Digestion was performed on-line by taining galactose for induction of Src expression. The cells were grown for an immobilized pepsin column (Applied Biosystems, Poroszyme). Peptides 6 h at 30 °C and spun down, and the pellet resuspended in 250 μLofex- were trapped and subsequently separated on a Waters UPLC CSH C 18 col- μ × traction buffer [8 M urea, 5% (wt/vol) SDS, 40 mM Tris, 0.1 M EDTA, pH 7.5] umn (1.7 m, 1.0 100 mm) with a H2O plus 0.1% formic acid (vol/vol) and and additionally lysed with glass beads. After centrifugation at 20,000 × g for ACN plus 0.1% formic acid (vol/vol) gradient. Trapping and chromatographic 5 min, the protein concentration of the supernatant was measured using a separation were carried out at 0 °C to minimize back-exchange. Eluting Nanodrop (Peqlab) and normalized. After SDS buffer addition and incubation peptides were directly subjected to the time-of-flight mass spectrometer by at 95 °C for 5 min, a 10% SDS/PAGE was run for Western blotting. The blotted electrospray ionization. Before fragmentation by MSE and mass detection in membrane was blocked with 5% (wt/vol) milk in TBS-T (0.1% Tween 20) for resolution mode, the peptide ions were additionally separated by drift time 30 min and subsequently treated with mouse anti-phosphotyrosine antibody within the mobility cell. Data processing was performed using the Waters clone 4G10 (1:1,000 in TBS-T, 1% milk) (Merck Millipore). After three 10-min Protein Lynx Global Server PLGS (Version 2.5.3) and DynamX (Version 2.0). washing steps with TBS-T, the membrane was treated with the secondary BIOCHEMISTRY rabbit peroxidase (POD)-conjugated anti-mouse antibody (1:10,000 in TBS-T, Molecular Dynamics Simulations. The structure of wild-type c-Src [Protein Data 1% milk) (Sigma). The blot was washed with TBS-T, POD reaction was per- Bank (PDB) ID 1Y57] (66) was obtained from Brookhaven Databank and used formed using ECLplus Western Detection System (GE Healthcare), and the Δ chemiluminescence was visualized by exposing the blot to Kodak X-Omat. for constructing the c-Src3M C variant by introducing point mutations Δ For detection of Src kinase in the yeast lysates, after blocking with 3% (R95W, D117Q, R318Q) in silico and by removing the C-terminal tail ( C). The (wt/vol) milk in PBS-T (0.1% Tween 20) for 30 min the membrane was treated models were solvated in a water box using a 100 mM sodium chloride with mouse anti-avian Src antibody clone EC10 (1:1,000 in PBS-T, 1% milk) concentration. The systems were simulated for 250 ns with a 2-fs integration (Merck Millipore). After washing in PBS-T, the membrane was treated with time step at T = 310 K, and by using the CHARMM27 force field (67). The the secondary rabbit POD-conjugated anti-mouse antibody and de- simulations were performed using NAMD (68), and Visual Molecular Dy- veloped as described. namics (69) was used for analysis.

ANS Binding Assay. A concentration of 1.6 μM of the respective kinase variant ACKNOWLEDGMENTS. We thank L. Neckers for kindly providing Hsp90β- was incubated with 30 μM ANS (Sigma) in Src buffer for 20 min at room E42A and Hsp90β-D88A plasmids, A. Liebscher for excellent practical assis- temperature and subsequently analyzed in a FluoroMax-3 fluorescence tance, O. Lorenz for providing purified Hop, P. Sahasrabudhe for supporting spectrometer (Spex) with an excitation wavelength of 380 nm and an yeast experiments, and all members of the J.B. laboratory for discussions. We emission wavelength of 470 nm. The obtained emission spectra of the pro- acknowledge the high-performance Biowulf cluster (biowulf.nih.gov)atthe National Institutes of Health for providing computational resources. V.R.I.K. teins were normalized against the Src buffer blank. acknowledges the Jane and Aatos Erkko Foundation for financial support. The project was funded by a grant of the Deutsche Forschungsgemeinschaft Tryptophan Quenching. Five molar acrylamide was titrated to a 500 nM Src (SFB1035) (to J.B.). E.E.B. acknowledges a PhD scholarship from the Studien- kinase in Src buffer at 25 °C. Tryptophan fluorescence emission quenching stiftung des Deutschen Volkes.

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