Summary Targeted intracellular protein degradation in eukaryotes is largely mediated by the proteasome. Here we report formation of an alternative proteasome isoform in human cells, previously found only in budding yeast, which bears an altered subunit arrangement in the outer ring of the proteasome core particle. These proteasomes result from incorporation of an additional α4 (PSMA7) subunit in the position normally occupied by α3 (PSMA4). Assembly of ‘α4-α4’ proteasomes depends on the relative cellular levels of α4 and α3, and on the proteasome assembly chaperone PAC3. The oncogenic tyrosine kinases ABL and ARG and the tumor suppressor BRCA1 regulate cellular α4 levels and formation of α4-α4 proteasomes. Cells primed to assemble α4-α4 proteasomes exhibit enhanced resistance to toxic metal ions. Taken together, our results establish the existence of a novel mammalian proteasome isoform and suggest a potential role in enabling cells to adapt to environmental stresses.

Gain-of-function p53 mutants such as p53-R175H form stable aggregates that accumulate in cells and play important roles in cancer progression. Selective degradation of gain-of-function p53 mutants has emerged as a highly attractive therapeutic strategy to target cancer cells harboring specific p53 mutations. We identified a small molecule called MCB-613 to cause rapid ubiquitination, nuclear export, and degradation of p53-R175H through a lysosome-mediated pathway, leading to catastrophic cancer cell death. In contrast to its effect on the p53-R175H mutant, MCB-613 causes slight stabilization of p53-WT and has weaker effects on other p53 gain-of-function mutants. Using state-of-the-art genetic and chemical approaches, we identified the deubiquitinase USP15 as the mediator of MCB-613’s effect on p53-R175H, and established USP15 as a selective upstream regulator of p53-R175H in ovarian cancer cells. These results confirm that distinct pathways regulate the turnover of p53-WT and the different p53 mutants and open new opportunities to selectively target them.

Background: MYC is rapidly degraded in cells, and its accumulation is associated with many human malignancies. Results: Sequential phosphorylation of MYC by protein kinase A (PKA) and polo-like kinase 1 (PLK1) protects MYC from proteasome-mediated degradation. Conclusion: A MYC-PKA-PLK1 signaling loop exists in cells. Significance: We highlight the importance of considering possible regulatory feedback loops while targeting molecules occupying hub positions in signaling pathways.

Elucidating kinase-substrate relationships is critical for understanding how phosphorylation affects signal transduction and regulatory cascades. Using the alpha catalytic subunit of protein kinase CK2 (CK2alpha) as a paradigm, we developed an in-gel method to facilitate identification of physiologic kinase substrates. In this approach, the roles of kinase and substrate in a classic in-gel kinase assay are reversed. In the reverse in-gel kinase assay (RIKA), a kinase is copolymerized in a denaturing polyacrylamide gel used to resolve a tissue or cell protein extract. Restoration of kinase activity and substrate structure followed by an in situ kinase reaction and mass spectrometric analyses results in identification of potential kinase substrates. We demonstrate that this method can be used to profile both known and novel human and mouse substrates of CK2alpha and cAMP-dependent protein kinase (PKA). Using widely available straightforward technology, the RIKA has the potential to facilitate discovery of physiologic kinase substrates in any biological system.

No abstract

Loss of NKX3.1 is an early and consistent event in prostate cancer and is associated with increased proliferation of prostate epithelial cells and poor prognosis. NKX3.1 stability is regulated post-translationally through phosphorylation at multiple sites by several protein kinases. Here, we report the paradoxical stabilization of the prostate-specific tumor suppressor NKX3.1 by the oncogenic protein kinase Pim-1 in prostate cancer cells. Pharmacologic Pim-1 inhibition using the small molecule inhibitor CX-6258 decreased steady state levels and half-life of NKX3.1 protein but mRNA was not affected. This effect was reversed by inhibition of the 26S-proteasome, demonstrating that Pim-1 protects NKX3.1 from proteasome-mediated degradation. Mass spectrometric analyses revealed Thr89, Ser185, Ser186, Ser195, and Ser196 as Pim-1 phospho-acceptor sites on NKX3.1. Through mutational analysis, we determined that NKX3.1 phosphorylation at Ser185, Ser186, and within the N-terminal PEST domain is essential for Pim-1-mediated stabilization. Further, we also identified Lys182 as a critical residue for NKX3.1 stabilization by Pim-1. Pim-1-mediated NKX3.1 stabilization may be important in maintaining normal cellular homeostasis in normal prostate epithelial cells, and may maintain basal NKX3.1 protein levels in prostate cancer cells.

No abstract

In this review, we provide a comprehensive overview of the known post-translational modifications and structural features that impact NKX3.1. Defining factors that regulate NKX3.1 in prostate epithelial cells will extend our understanding of molecular changes that may contribute to prostate cancer initiation and progression.