Tumor suppressor Tsc1 is a new Hsp90 co‐chaperone that facilitates folding of kinase and non‐kinase clients

Abstract

The tumor suppressors Tsc1 and Tsc2 form the tuberous sclerosis complex (TSC), a regulator of mTOR activity. Tsc1 stabilizes Tsc2; however, the precise mechanism involved remains elusive. The molecular chaperone heat‐shock protein 90 (Hsp90) is an essential component of the cellular homeostatic machinery in eukaryotes. Here, we show that Tsc1 is a new co‐chaperone for Hsp90 that inhibits its ATPase activity. The C‐terminal domain of Tsc1 (998–1,164 aa) forms a homodimer and binds to both protomers of the Hsp90 middle domain. This ensures inhibition of both subunits of the Hsp90 dimer and prevents the activating co‐chaperone Aha1 from binding the middle domain of Hsp90. Conversely, phosphorylation of Aha1‐Y223 increases its affinity for Hsp90 and displaces Tsc1, thereby providing a mechanism for equilibrium between binding of these two co‐chaperones to Hsp90. Our findings establish an active role for Tsc1 as a facilitator of Hsp90‐mediated folding of kinase and non‐kinase clients—including Tsc2—thereby preventing their ubiquitination and proteasomal degradation.

A methylated lysine is a switch point for conformational communication in the chaperone Hsp90

“…The human variants were omitted from this experiment as the intrinsic ATP turnover of the human variants and the inhibitory effect of p23 and Sti1 is quite low, which makes the detection of a further decrease difficult. Recent studies however showed that some newly discovered co-chaperones are impacting Hsp90's ATPase activity to a higher degree and might therefore be a viable sensor for future studies 21,22 . Sti1 binds to Hsp90 in its open conformation and prevents further rearrangements towards the closed state, thereby inhibiting the ATP turnover of the protein 19,53 .…”

“…This ATPase cycle can be modulated by a cohort of Hsp90 co-chaperones, which are only found in eukaryotes. Some co-chaperones accelerate the Hsp90 cycle, whereas others have a decelerating effect [19][20][21] . A further important regulatory element of the Hsp90 cycle are post-translational modifications (PTMs) 22 .…”

Co-chaperones TIMP2 and AHA1 Competitively Regulate Extracellular HSP90:Client MMP2 Activity and Matrix Proteolysis

“…The question remains whether this mechanism also applies in a similar fashion to other eHSP90 client proteins. In House (Dunn et al, 2015) HSP90His 6 In House (Woodford et al, 2017) oligonucleotide primers: GAPDH, TIMP1, TIMP2, TIMP3, TIMP4 and HSP70 control. mRNA expression levels were analyzed over GAPDH control.…”

“…CM were concentrated 10x using Amicon Ultra 3K or 10K centrifugal filters (Millipore) according to the manufacturer's protocol. Proteins were separated by SDS-PAGE, and either stained with Coomassie Blue Stain for loading control, or transferred to nitrocellulose membrane, and detected by immunoblotting with antibodies listed in Key Resources Table. Ni-NTA Pulldown and Immunoprecipitation IPs and pulldowns of both in vitro purified proteins, and in vivo cell extracts and conditioned media were performed as described in (Sá nchez-Pozo et al, 2018;Woodford et al, 2017). For immunoprecipitation, cell extracts and CM were incubated with anti-FLAG conjugate beads or anti-HA conjugate beads for 2 hours at 4 C or with HSP90a or TIMP2 antibodies for 1hr at 4 C followed by protein G agarose for 2 hours at 4 C. Pulldowns were prepared following incubation with Ni-NTA agarose or ATP conjugated agarose for 2 hours at 4 C. Pellets were washed 4 times with fresh lysis buffer (20mM HEPES pH7.0, 100mM NaCl, 1mM MgCl 2 , 0.1% NP40, protease inhibitor cocktail (Roche), and PhosSTOP (Roche)) and eluted in 5x Laemmli buffer.…”

“…Interestingly, the M-domain of HSP90 is a binding site for the N-terminal domain of AHA1 (Meyer et al, 2004). To test whether TIMP2 binds to a similar site as AHA1 on HSP90, we utilized the human HSP90a-V411E mutant (yeast HSP82p V391E ) that was previously shown to abolish HSP90 and AHA1 complex formation (Meyer et al, 2004;Retzlaff et al, 2010;Woodford et al, 2017). Following IP of WT and mutant HSP90a-V411E-FLAG from HEK293 CM, the data confirm that while both TIMP2 and AHA1 bind to WT HSP90a, their binding to HSP90a-V411E is abolished ( Figures 5D and S5C).…”