Most human tumors have abnormal numbers of chromosomes, a condition known as aneuploidy. The mitotic checkpoint is an important mechanism that prevents aneuploidy by restraining the activity of the anaphase-promoting complex (APC). The deubiquitinase USP44 was identified as a key regulator of APC activation; however, the physiological importance of USP44 and its impact on cancer biology are unknown. To clarify the role of USP44 in mitosis, we engineered a mouse lacking Usp44. We found that USP44 regulated the mitotic checkpoint and prevented chromosome lagging. Mice lacking Usp44 were prone to the development of spontaneous tumors, particularly in the lungs. Additionally, USP44 was frequently downregulated in human lung cancer, and low expression correlated with a poor prognosis. USP44 inhibited chromosome segregation errors independent of its role in the mitotic checkpoint by regulating centrosome separation, positioning, and mitotic spindle geometry. These functions required direct binding to the centriole protein centrin. Our data reveal a new role for the ubiquitin system in mitotic spindle regulation and underscore the importance of USP44 in the pathogenesis of human cancer.

De-ubiquitinating enzymes (DUBs) can reverse the modifications catalyzed by ubiquitin ligases and as such are believed to be important regulators of a variety of cellular processes. Several members of this protein family have been associated with human cancers; however, there is little evidence for a direct link between deregulated de-ubiquitination and neoplastic transformation. Ubiquitin C-terminal hydrolase (UCH)-L1 is a DUB of unknown function that is overexpressed in several human cancers, but whether it has oncogenic properties has not been established. To address this issue, we generated mice that overexpress UCH-L1 under the control of a ubiquitous promoter. Here, we show that UCH-L1 transgenic mice are prone to malignancy, primarily lymphomas and lung tumors. Furthermore, UCH-L1 overexpression strongly accelerated lymphomagenesis in Eμ-myc transgenic mice. Aberrantly expressed UCH-L1 boosts signaling through the Akt pathway by downregulating the antagonistic phosphatase PHLPP1, an event that requires its de-ubiquitinase activity. These data provide the first in vivo evidence for DUB-driven oncogenesis and suggest that UCH-L1 hyperactivity deregulates normal Akt signaling.

The family of ubiquitin-like molecules (UbLs) comprises several members, each of which has sequence, structural, or functional similarity to ubiquitin. ISG15 is a homolog of ubiquitin in vertebrates and is strongly upregulated following induction by type I interferon. ISG15 can be covalently attached to proteins, analogous to ubiquitination and with actual support of ubiquitin conjugating factors. Specific proteases are able to reverse modification with ubiquitin or UbLs by hydrolyzing the covalent bond between their C-termini and substrate proteins. The tail regions of ubiquitin and ISG15 are identical and we therefore hypothesized that promiscuous deubiquitinating proteases (DUBs) might exist, capable of recognizing both ubiquitin and ISG15. We have cloned and expressed 22 human DUBs, representing the major clades of the USP protease family. Utilizing suicide inhibitors based on ubiquitin and ISG15, we have identified USP2, USP5 (IsoT1), USP13 (IsoT3), and USP14 as ISG15-reactive proteases, in addition to the bona fide ISG15-specific protease USP18 (UBP43). USP14 is a proteasome-associated DUB, and its ISG15 isopeptidase activity increases when complexed with the proteasome. By evolutionary standards, ISG15 is a newcomer among the UbLs and it apparently not only utilizes the conjugating but also the deconjugating machinery of its more established relative ubiquitin. Functional overlap between these two posttranslational modifiers might therefore be more extensive than previously appreciated and explain the rather innocuous phenotype of ISG15 null mice.

Mammalian target of rapamycin (mTOR) is a serine/threonine kinase that regulates processes including mRNA translation, proliferation, and survival. By assembling with different cofactors, mTOR forms two complexes with distinct biological functions. Raptor-bound mTOR (mTORC1) governs cap-dependent mRNA translation, whereas mTOR, rictor, and mSin1 (mTORC2) activate the survival and proliferative kinase Akt. How the balance between the competing needs for mTORC1 and -2 is controlled in normal cells and deregulated in disease is poorly understood. Here, we show that the ubiquitin hydrolase UCH-L1 regulates the balance of mTOR signaling by disrupting mTORC1. We find that UCH-L1 impairs mTORC1 activity toward S6 kinase and 4EBP1 while increasing mTORC2 activity toward Akt. These effects are directly attributable to a dramatic rearrangement in mTOR complex assembly. UCH-L1 disrupts a complex between the DDB1-CUL4 ubiquitin ligase complex and raptor and counteracts DDB1-CUL4-mediated raptor ubiquitination. These events lead to mTORC1 dissolution and a secondary increase in mTORC2. Experiments in Uchl1-deficient and transgenic mice suggest that the balance between these pathways is important for preventing neurodegeneration and the development of malignancy. These data establish UCH-L1 as a key regulator of the dichotomy between mTORC1 and mTORC2 signaling.

Cdc20-anaphase promoting complex/cyclosome (Cdc20-APC/C) E3 ubiquitin ligase activity is essential for orderly mitotic progression. The deubiqituinase USP44 was identified as a key regulator of APC/C and has been proposed to suppress Cdc20-APC/C activity by maintaining its association with the inhibitory protein Mad2 until all chromosomes are properly attached to the mitotic spindle. However, this notion has been challenged by data in which a lysine-less mutant of Cdc20 leads to premature anaphase, suggesting that it's ubiquitination is not required for APC/C activation. To further evaluate its role in checkpoint function and chromosome instability, we studied the consequences of over-expression of mouse Usp44 in non-transformed murine embryonic fibroblasts. Here we show that cells with high Usp44 are prone to chromosome segregation errors and aneuploidization. We find that high Usp44 promotes association of Mad2 with Cdc20 and reinforces the mitotic checkpoint. Surprisingly, the APC/C-Cdc20 substrate cyclin B1 is stabilized in G2 when Usp44 is over-expressed, but is degraded with normal kinetics once cells enter mitosis. Furthermore, we show that USP44 expression is elevated in subset of T-cell leukemias. These data are consistent with an important role for USP44 in regulating Cdc20-APC/C activity and suggest that high levels of this enzyme may contribute to the pathogenesis of T-ALL.

UCHL1 is a biomarker of aggressive multiple myeloma required for disease progression