Fig. 6: The heat-shock protein DNAJA3 is recruited to hyperacetylated tubulin and induces MYC degradation.

A Interaction of acetylated tubulin (ac-tub) and DNAJA3. 293T cells were transfected with plasmids encoding DNAJA3-Flag and treated with 1 µM M-100 for 24 h. Cells were lysed in stringent lysis buffer containing M-100. Lysates were used for IP with α-Flag antibodies to precipitate DNAJA3-Flag. Overexpression of DNAJA3 generates unprocessed (up) precursor proteins. B Endogenous interaction of acetylated tubulin and DNAJA3 in Ramos cells. Cells were treated for 24 h with either 0.5 µM, 4 µM M-100, 5 µM MS-275, or left untreated. Lysates were used for IP with α-DNAJA3 antibodies and tested for interaction with ac-tub. IPs with unspecific IgG were used as control. C PLA was performed to detect endogenous co-localization of MYC and DNAJA3 in Ramos cells. Cells were treated with 4 µM M-100 for the indicated time points. Staining with unspecific IgG was used as a control. DAPI was used to stain nuclei. Scale bars indicate 20 µm, magnification 60x. PLA foci were counted and compared. Boxplots depict medium and min to max. One-Way ANOVA (Tukey’s posthoc). D Western Blot analysis of 293T cells overexpressing MYC and increasing amounts of small (S) or large (L) isoforms of DNAJA3. Vinculin was used as a loading control. Quantification of MYC was performed based on Vinculin. One-Way ANOVA (Dunnett’s posthoc). E Western Blot analysis of bone marrow lysates from Eµ-Myc mice after one i.p. injection with M-100 (30 mg/kg) or vehicle. Small and large isoforms of Dnaja3 can be noticed. Vinculin was used as a loading control. Each lane represents one individual mouse. Quantification of Dnaja3 protein levels is shown based on Vinculin. FC - fold change. F Scheme summarizing our findings. Hyperacetylation of tubulin by HDAC6 inhibition results in the recruitment of chaperone complexes including the HSP DNAJA3. High levels of DNAJA3 induce degradation of MYC preventing cancer-specific gene regulation. Data in A, B, and D are representative of n = 3 independent experiments, data in C are representative of n = 2 independent experiments. Data represent mean + SEM, if applicable.