The human topoisomerase I-and p53-binding protein topors contains a highly conserved, N-terminal C3HC4-type RING domain that is homologous to the RING domains of known E3 ubiquitin ligases. We demonstrate that topors functions in vitro as a RING-dependent E3 ubiquitin ligase with the E2 enzymes UbcH5a, UbcH5c, and UbcH6 but not with UbcH7, CDC34, or UbcH2b. Additional studies indicate that a conserved tryptophan within the topors RING domain is required for ubiquitination activity. Furthermore, both in vitro and cellular studies implicate p53 as a ubiquitination substrate for topors. Similar to MDM2, overexpression of topors results in a proteasome-dependent decrease in p53 protein expression in a human osteosarcoma cell line. These results are similar to the recent finding that a Drosophila topors orthologue ubiquitinates the Hairy transcriptional repressor and suggest that topors functions as a ubiquitin ligase for multiple transcription factors.Topors was originally discovered in a screen for proteins that bind to the N terminus of topoisomerase I (1) and was also identified in a screen for proteins that interact with p53 (denoted p53BP3) (2). Furthermore, topors was identified in an assay for RING domain proteins that are expressed in normal lung tissue (denoted LUN) (3). While topors is widely expressed in normal human tissues, topors mRNA and protein levels are commonly decreased or undetectable in colon adenocarcinomas and cell lines (4). The topors protein contains an N-terminal C3HC4-type RING domain that is conserved in orthologues from various species (5) and is closely related in sequence to the RING domains of known E3 ubiquitin ligases such as the herpesvirus protein ICP0 and Cbl (6, 7). Recently, a Drosophila topors orthologue was shown to interact physically and genetically with the Hairy transcriptional repressor (8). Furthermore, the Drosophila topors protein was shown to ubiquitinate Hairy in vitro and to decrease expression of an epitope-tagged Hairy protein in co-transfection studies (8).Topors is also known to associate with promyelocytic leukemia (PML) 1 nuclear bodies in the nuclei of exponentially growing cells (5, 9). Treatment with transcriptional inhibitors or with the topoisomerase I-targeting drug camptothecin results in rapid dispersion of topors to the nucleoplasm, suggesting that topors is involved in the cellular response to transcriptional perturbation (5).To gain insight into the role of the topors and the conserved RING domain, we determined whether topors functions as a ubiquitin ligase. Our results indicate that topors acts as a RING domain-dependent, E3 ubiquitin ligase with specific E2 enzymes. Similar to the E3 ubiquitin ligase Cbl, a conserved tryptophan within the topors RING domain is required for ubiquitin ligase activity. Furthermore, additional in vitro and cellular studies implicate p53 as a ubiquitination substrate for topors. EXPERIMENTAL PROCEDURESExpression Plasmids-A plasmid expressing a GST-N-terminal topors fusion protein (pGEX-topors) was constructed using...

Mutations in LRRK2 (leucine-rich repeat kinase 2) have been identified as major genetic determinants of Parkinson's disease (PD). The most prevalent mutation, G2019S, increases LRRK2's kinase activity, therefore understanding the sites and substrates that LRRK2 phosphorylates is critical to understanding its role in disease aetiology. Since the physiological substrates of this kinase are unknown, we set out to reveal potential targets of LRRK2 G2019S by identifying its favored phosphorylation motif. A non-biased screen of an oriented peptide library elucidated F/Y-x-T-x-R/K as the core dependent substrate sequence. Bioinformatic analysis of the consensus phosphorylation motif identified several novel candidate substrates that potentially function in neuronal pathophysiology. Peptides corresponding to the most PD relevant proteins were efficiently phosphorylated by LRRK2 in vitro. Interestingly, the phosphomotif was also identified within LRRK2 itself. Autophosphorylation was detected by mass spectrometry and biochemical means at the only F-x-T-x-R site (Thr 1410) within LRRK2. The relevance of this site was assessed by measuring effects of mutations on autophosphorylation, kinase activity, GTP binding, GTP hydrolysis, and LRRK2 multimerization. These studies indicate that modification of Thr1410 subtly regulates GTP hydrolysis by LRRK2, but with minimal effects on other parameters measured. Together the identification of LRRK2's phosphorylation consensus motif, and the functional consequences of its phosphorylation, provide insights into downstream LRRK2-signaling pathways.

The NKX3.1 gene located at 8p21.2 encodes a homeodomaincontaining transcription factor that acts as a haploinsufficient tumor suppressor in prostate cancer. Diminished protein expression of NKX3.1 has been observed in prostate cancer precursors and carcinomas. TOPORS is a ubiquitously expressed E3 ubiquitin ligase that can ubiquitinate tumor suppressor p53. Here we report interaction between NKX3.1 and TOPORS. NKX3.1 can be ubiquitinated by TOPORS in vitro and in vivo, and overexpression of TOPORS leads to NKX3.1 proteasomal degradation in prostate cancer cells. Conversely, small interfering RNA-mediated knockdown of TOPORS leads to an increased steady-state level and prolonged half-life of NKX3.1. These data establish TOPORS as a negative regulator of NKX3.1 and implicate TOPORS in prostate cancer progression.Prostate cancer is the second leading cause of cancer deaths and the most frequently diagnosed malignancy in American men (1). Prostate carcinomas are considered to arise from cancer precursors including prostatic intraepithelial neoplasia (PIN) 2 and proliferative inflammatory atrophy (2). Molecular alterations, including hereditary and somatic gene mutations, gene deletions, gene amplification, chromosomal rearrangements, as well as epigenetic changes, have been implicated in prostate cancer initiation and progression (3).The NK class homeobox gene NKX3.1 has been studied extensively over the past decade for its roles in prostate development and carcinogenesis (4). The expression of the murine Nkx3.1 gene is androgen-dependent and is restricted largely to prostate epithelial cells in adults (5-7). Deletion of Nkx3.1 by gene targeting leads to prostate ductal morphological defects, as well as prostatic dysplasia and hyperplasia that resembles human PIN (8 -10). Interestingly, heterozygous Nkx3.1 mice also develop hyperplasia and PIN-like lesions (8). The human NKX3.1 gene maps to 8p21.2 within a region where loss of heterozygosity occurs in PIN and is common in prostate carcinomas; however, no mutations have been found in the coding region of the NKX3.1 allele remaining (11,12). In light of these observations, NKX3.1 has been proposed to function as a haploinsufficient tumor suppressor. In support of a dose-dependent growth regulatory function of NKX3.1, reduced but not complete loss of NKX3.1 protein expression is now well documented in most human prostate cancer samples in a manner inversely correlated with Gleason score (13) and also with disease progression (14). Diminished NKX3.1 expression is thought to be an early event in prostate carcinogenesis, and reduced NKX3.1 immunostaining was observed in most proliferative inflammatory atrophy and PIN lesions analyzed (13). The diminished NKX3.1 expression may be partly attributed to selective CpG methylation of the NKX3.1 promoter in some prostate cancer cases (15). Nevertheless, quantitative analyses of mRNA and protein levels of NKX3.1 in prostate carcinoma lesions revealed a lack of concordance between mRNA and protein levels (13). In particular, decre...

Parkinson's disease (PD) is the second most prevalent neurodegenerative disorder in humans, and has a relatively poorly understood etiology. Linkage analysis studies in families with PD identified several mutations in the leucine-rich repeat kinase 2 gene (LRRK2) [1,2]. Moreover, epidemiological studies have shown that these mutations are the most prevalent cause of the autosomal form of the disorder, with high penetrance of certain mutations [3]. The similarity in age of onset and clinical symptoms between familial and idiopathic forms may also provide insights into the pathways involved in sporadic cases of PD.LRRK2 Mutations in leucine-rich repeat kinase 2 (LRRK2) comprise the leading cause of autosomal dominant Parkinson's disease, with age of onset and symptoms identical to those of idiopathic forms of the disorder. Several of these pathogenic mutations are thought to affect its kinase activity, so understanding the roles of LRRK2, and modulation of its kinase activity, may lead to novel therapeutic strategies for treating Parkinson's disease. In this study, highly purified, baculovirus-expressed proteins have been used, for the first time providing large amounts of protein that enable a thorough enzymatic characterization of the kinase activity of LRRK2. Although LRRK2 undergoes weak autophosphorylation, it exhibits high activity towards the peptidic substrate LRRKtide, suggesting that it is a catalytically efficient kinase. We have also utilized a time-resolved fluorescence resonance energy transfer (TR-FRET) assay format (LanthaScreen TM ) to characterize LRRK2 and test the effects of nonselective kinase inhibitors. Finally, we have used both radiometric and TR-FRET assays to assess the role of clinical mutations affecting LRRK2's kinase activity. Our results suggest that only the most prevalent clinical mutation, G2019S, results in a robust enhancement of kinase activity with LRRKtide as the substrate. This mutation also affects binding of ATP to LRRK2, with wild-type binding being tighter (K m,app of 57 lm) than with the G2019S mutant (K m,app of 134 lm). Overall, these studies delineate the catalytic efficiency of LRRK2 as a kinase and provide strategies by which a therapeutic agent for Parkinson's disease may be identified.Abbreviations COR, C-terminus of Roc; FRET, fluorescence resonance energy transfer; GST, glutathione S-transferase; LRRK2, leucine-rich repeat kinase 2; LRRK2-FL, full-length leucine-rich repeat kinase 2; PD, Parkinson's disease; Roc, Ras of complex; TR-FRET, time-resolved fluorescence resonance energy transfer; 4E-BP, eukaryotic initiation factor 4E-binding protein.

TOPORS is the first example of a protein with both ubiquitin and SUMO-1 E3 ligase activity and has been implicated as a tumor suppressor in several different malignancies. To gain insight into the cellular role of TOPORS, a proteomic screen was performed to identify candidate sumoylation substrates. The results indicate that many of the putative substrates are involved in chromatin modification or transcriptional regulation. Transfection studies confirmed mammalian Sin3A as a sumoylation substrate for TOPORS. These findings suggest that TOPORS may function as a tumor suppressor by regulating mSin3A and other proteins involved in chromatin modification.