Conformational processing of oncogenic v-Src kinase PNAS PLUS by the molecular chaperone Hsp90 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 Protein 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 proteins, 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 M) μ 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 T of I 2 1,0 Δ 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.