How Hedgehog Signaling Pathway Induces Breast Cancer Metastasis

Intracellular accumulation of the abnormally modified tau is a pathology marker of Alzheimer’s disease. In AD brains, tau is hyperphosphorylated. Ubiquitin is an important component of the cellular defense system that tags abnormal proteins for their degradation by non-lysosomal proteases. Here, we report the crosstalk between SUMOylation and phosphorylation modifications of tau.

On one hand, to figure out tau SUMOylation affects its phosphorylation, eGFP-labeled SUMO-1 or the vector in HEK293/ tau cells that stably express human tau and analyzed the phosphorylation level of tau. The result shows tau SUMOylation induces tau hyperphosphorylation at multiple AD-associated sites, whereas site-specific mutagenesis of tau at K340R (the SUMOylation site) or simultaneous inhibition of tau SUMOylation by ginkgolic acid abolishes the effect of SUMO-1.

On the other hand, to figure out tau phosphorylation affects its SUMOylation, we treated the cells with okadaic acid to inhibit Pp2A, which leads to increased phosphorylation level of tau. The results suggest that tau hyperphosphorylation stimulates its SUMOylation at K340. Besides, the SUMOylation also regulate solubility of tau.

To summary, tau phosphorylation promotes its SUMOylation and thus inhibiting ubiquitination. A gradual impairment of ubiqutin-proteasomal system at later stages of AD is at least partially responsible for the reduced degradation of tau proteins.

Mutations of SPOP (E3 ubiquitin ligase substrate-binding protein) are associated with prostate cancer. SPOP mutants reduced ubiquitylation of a subset of proteins in a dominant-negative way. Here, we analyzed changes in the ubiquitin landscape by measuring glycine-glycine remnants of ubiquitylated lysines (K-e-GG) after trypsin digestion and stable isotope labeling of amino acids in cell culture (SILAC)–based mass spectrometry.

To account for ubiquitylation-related changes in protein expression, all K-e-GG values were normalized to protein abundance. There are only 12 of 7181 K-e-GG peptides detected exhibited more than twofold changes in peptide abundance in the SPOP-mutant contexts. Among them, we found DEK and TRIM24 is significant and reproducible.

We next explore whether prostate cancer SPOP mutants affected DEK protein stability. Treatment with the proteasome inhibitor Mg132 also increased DEK, consistent with a possible role for the proteasome in DEK regulation. As forced expression of SPOP reduced DEK protein level, we sought to determine whether SPOP interacts with DEK directly to promote ubiquitylation. The result shows direct interaction between SPOP and DEK.

In conclusion, DEK and TRIM24 emerged as effector substrates up-regulated by SPOP mutants. These results provide a way to unfold tumorigenic mechanisms linked to dysregulated ubiquitylation. Large-scale profiling of the ubiquitin landscape linked to tumor genomic alterations in protein homeostasis genes may speed up the identification of downstream targets.