Fanconi anemia (FA) 1 is a genetic disease of cancer susceptibility marked by congenital defects, bone marrow failure, and myeloid leukemia (1-4). To date at least seven complementation groups have been defined (5, 6). Six genes accounting for six groups have been cloned (7-15). However, all the gene products resemble no known proteins and have no identifiable functional protein motif.Cells derived from patients with the disease exhibit characteristic hypersensitivity caused by DNA cross-linking agents and generalized decreased survival (16 -20). In addition, a well described G 2 phase cell cycle delay has also been described that is thought to be secondary to a defective S or G 2 checkpoint (21-23). However, no defined biochemical mechanism for this hypersensitivity has been elucidated. Patient and cellular phenotypes across all the complementation groups are similar, suggesting an interrelatedness or cooperativity between the FA proteins.This cooperativity has been borne out by work we have done in showing binding of FancA and FancC in a protein complex in both nucleus and cytoplasm (24 -26). Recent work has found the FancG and FancF proteins in the complex as well (27)(28)(29)(30). A large complex is suggested by our recent work 2 , and binding does not occur in any of the complementation groups except the FA-D group.One clue to FA function lies in the study of the FancA protein, which contains a classic bipartite nuclear localization signal and is phosphorylated. FancA nuclear localization, phosphorylation, and binding to FancC are abolished in all complementation groups except the FA-D group (24 -26, 29). This suggests that a nuclear event is critical to the normal function of the FA proteins, and the aberrant protein in the FA-D group may have a role downstream of the FA complex in the nucleus.To date, other than the alteration of binding, nuclear localization, and FancA phosphorylation in mutant FA complementation groups, no other consistent biochemical change has been described, although some have described variations in FancC levels in the cell cycle (31,32). In our study, we have found that the FA proteins not only reside in the nucleus but also are closely associated with the nuclear matrix and chromatin.The nuclear matrix is a loose mechanical framework of proteins that has also been implicated in enzymatic activities in the regulation of transcription, replication, and DNA repair. The nuclear matrix is intimately associated with chromatin. BRG and brm, the human swi-snf homologues, are examples of nuclear matrix proteins involved in transcriptional regulation that change phosphorylation state, shift to chromatin, and become distinct from the nuclear matrix during mitosis (33-35). Immunofluorescence of FancA reveals a similar shift away from the condensed chromosomes of mitosis, suggesting that they also interact with chromatin and the nuclear matrix. Our studies demonstrate that the FA proteins associate with chromatin and the nuclear matrix in an inducible fashion. MATERIALS AND METHODSCell Cultu...

Fanconi anemia (FA) 1 is a genetic disease of cancer susceptibility marked by congenital defects, bone marrow failure, and myeloid leukemia (1-4). To date at least 11 complementation groups have been defined (5-7). Eight genes have been cloned (8 -17). However, the gene products resemble no known proteins and have few identifiable functional protein motifs. One exception is the recently cloned FANCL gene, which contains the ubiquitin ligase motif. In addition, the FANCD1 gene has been identified as BRCA2, one of the familial breast cancer genes.Cells derived from patients with the FA exhibit characteristic hypersensitivity caused by DNA cross-linking agents and generalized decreased survival (18 -22). However, no defined biochemical mechanism for this hypersensitivity has been elucidated, although studies have implicated cytokine dysregulation, excessive, oxidative damage, defects in DNA repair, and lack of cell cycle control (23-27). Patient and cellular phenotypes across all the complementation groups are similar, suggesting an inter-relatedness or cooperativity between the FA proteins.This cooperativity has been borne out by work we have done in showing binding of FANCA and FANCC in a protein complex in both nucleus and cytoplasm (28 -30). Recent work has found the FANCE, FANCF, FANCG, and FANCL proteins in the complex as well (31-34). A large complex is suggested by our recent work (35), and binding does not occur in any of the complementation groups except the FA-D1, D2, I, and J groups (7).One clue to FA function lies in the study of the FANCA protein, which contains a classic bipartite nuclear localization signal and is phosphorylated. Generally, FANCA nuclear localization, phosphorylation, and binding to FANCC are abolished in all complementation groups except the FA-D1 and D2 groups (28 -30, 33). This suggests that a nuclear event is critical to the normal function of the FA proteins, and the aberrant proteins in the FA-D groups may have a role downstream of the FA complex in the nucleus. However, some have found FANCA point mutants that are expressed, translocated to the nucleus, and are phosphorylated to some extent. Some of these mutants are of intermediate MMC sensitivity (36).Over the years, little information has been found that addresses the regulation of the FA proteins. mRNA or protein levels change little in response to DNA damage or the cell cycle. Our recent work has revealed that at least a subset of the FA proteins resides in the nucleus bound to chromatin, where increased protein binding occurs in response to DNA damage (37). One of the non-core complex FA proteins, FANCD2, becomes monoubiquitinated in response to DNA damage (38).In addition, we have shown during the cell cycle that the FA proteins detach from chromatin during mitosis, and FANCG becomes phosphorylated while remaining part of the complex (37). One group has demonstrated that FANCG has a isoform seen in asynchronous cells that is phosphatase sensitive (39).In this paper we report the identification of a phosphopeptide from ...

Purpose: Pancreatic ductal adenocarcinoma (PDAC) is a highly metastatic disease that can be separated into distinct subtypes based on molecular signatures. Identifying PDAC subtype-specific therapeutic vulnerabilities is necessary to develop precision medicine approaches to treat PDAC. Experimental Design: A total of 56 PDAC liver metastases were obtained from the UNMC Rapid Autopsy Program and analyzed with quantitative proteomics. PDAC subtypes were identified by principal component analysis based on protein expression profiling. Proteomic subtypes were further characterized by the associated clinical information, including but not limited to survival analysis, drug treatment response, and smoking and drinking status. Results: Over 3,960 proteins were identified and used to delineate four distinct PDAC microenvironment subtypes: (i) metabolic; (ii) progenitor-like; (iii) proliferative; and (iv) inflammatory. PDAC risk factors of alcohol and tobacco consumption correlate with subtype classifications. Enhanced survival is observed in FOLFIRINOX treated metabolic and progenitor-like subtypes compared with the proliferative and inflammatory subtypes. In addition, TYMP, PDCD6IP, ERAP1, and STMN showed significant association with patient survival in a subtype-specific manner. Gemcitabine-induced alterations in the proteome identify proteins, such as serine hydroxymethyltransferase 1, associated with drug resistance. Conclusions: These data demonstrate that proteomic analysis of clinical PDAC liver metastases can identify molecular signatures unique to disease subtypes and point to opportunities for therapeutic development to improve the treatment of PDAC.

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Fanconi anemia (FA) is an autosomal recessive disease marked by congenital defects, bone marrow failure, and high incidence of leukemia and solid tumors. Eight genes have been cloned, with the accompanying protein products participating in at least two complexes, which appear to be functionally dependent upon one another. Previous studies have described chromatin localization of the FA core complex, except at mitosis, which is associated with phosphorylation of the FANCG protein (F. Qiao, A. Moss, and G. M. Kupfer, J. Biol. Chem. 276:23391-23396, 2001). The phosphorylation of FANCG at serine 7 by using mass spectrometry was previously mapped. The purpose of this study was to map the phosphorylation sites of FANCG at mitosis and to assess their functional importance. Reasoning that a potential kinase might be cdc2, which was previously reported to bind to FANCC, we showed that cdc2 chiefly phosphorylated a 14-kDa fragment of the C-terminal half of FANCG. Mass spectrometry analysis demonstrated that this fragment contains amino acids 374 to 504. Kinase motif analysis demonstrated that three amino acids in this fragment were leading candidates for phosphorylation. By using PCR-directed in vitro mutagenesis we mutated S383, S387, and T487 to alanine. Mutation of S383 and S387 abolished the phosphorylation of FANCG at mitosis. These results were confirmed by use of phosphospecific antibodies directed against phosphoserine 383 and phosphoserine 387. Furthermore, the ability to correct FA-G mutant cells of human or hamster (where S383 and S387 are conserved) origin was also impaired by these mutations, demonstrating the functional importance of these amino acids. S387A mutant abolished FANCG fusion protein phosphorylation by cdc2. The FA pathway, of which FANCG is a part, is highly regulated by a series of phosphorylation steps that are important to its overall function.

The non-receptor tyrosine kinase Src is a critical regulator of cytoskeletal contraction, cell adhesion, and migration. In normal cells, Src activity is stringently controlled by Csk-dependent phosphorylation of Src(Y530), and by Cullin-5-dependent ubiquitinylation, which affects active Src(pY419) exclusively, leading to its degradation by the proteosome. Previous work has shown that Src activity is also limited by Cdk5, a proline-directed kinase, which has been shown to phosphorylate Src(S75). Here we show that this phosphorylation promotes the ubiquitin-dependent degradation of Src, thus restricting the availability of active Src. We demonstrate that Src(S75) phosphorylation occurs in vivo in epithelial cells, and like ubiquitinylation, is associated only with active Src. Preventing Cdk5-dependent phosphorylation of Src(S75), by site-specific mutation of S75 or by Cdk5 inhibition or suppression, increases Src(Y419) phosphorylation and kinase activity, resulting in Src-dependent cytoskeletal changes. In transfected cells, ubiquitinylation of Src(S75A) is about 35% that of wild-type Src-V5, and its half-life is approximately 2.5-fold greater. Cdk5 suppression leads to a comparable decrease in the ubiquitinylation of endogenous Src and a similar increase in Src stability. Together, these findings demonstrate that Cdk5-dependent phosphorylation of Src(S75) is a physiologically significant mechanism of regulating intracellular Src activity.

Ubiquilin-1 (Ubqln1) is a ubiquitin-like protein that has been implicated in Alzheimer’s disease (AD). However, whether Ubqln1 modulates learning and memory and alters AD-like behavior and/or pathology have not been determined in animal models. To understand the function of Ubqln1 in vivo, we previously generated Ubqln1 transgenic (TG) mice that overexpress mouse Ubqln1. With the model, we here characterized the TG mouse cognitive behaviors and found that Ubqln1 TG mice showed better spatial learning and memory capabilities than their wild-type littermates in both radial arm water maze and Y-maze tests. Additionally, we crossed the Ubqln1 TG mice with the AβPPswe/PSEN1dE9 double transgenic AD mouse to generate the AD/Ubqln1 triple TG (AD/TG) mice. Our results suggest that at 12 months of age following the onset of AD, AD/TG mice showed better spatial learning and memory than AD mice. AD/TG mice also exhibited better motor function than AD mice at the same age. Furthermore, compared to AD mice, AD/TG mice showed significant reduction in amyloid-β 40 (Aβ40) and Aβ42 levels in the cerebral cortex and in the hippocampus at the post-onset stage. The number of Aβ plaques was significantly decreased in the cerebral cortex of AD/TG mice at this post-onset stage. Moreover, mature AβPP level in AD/TG hippocampus was lower than that in AD hippocampus. These data not only provide a direct link between overexpression of Ubqln1 and altered learning and memory but also raise the possibility that Ubqln1 is a potential therapeutic target for treating AD and possibly other neurodegenerative disorders.

Oxidative stress aggravates brain injury following ischemia/reperfusion (I/R). We previously showed that ubiquilin-1 (Ubqln1), a ubiquitin-like protein, improves proteostasis and protects brains against oxidative stress and I/R induced brain injury. Here, we demonstrate that a small molecule compound, L-2-oxothiazolidine-4-carboxylic acid (OTC) that functions as a precursor of cysteine, upregulated Ubqln1 and protected cells against oxygen glucose deprivation-induced cell death in neuronal cultures. Further, the administration of OTC either at 1 hour prior to ischemia or 3 hours after the reperfusion significantly reduced brain infarct injury and improved behavioral outcomes in a stroke model. Administration of OTC also increased glutathione (GSH) level and decreased superoxide production, oxidized protein, and neuroinflammation levels in the penumbral cortex after I/R in the stroke mice. Furthermore, I/R reduced both Ubqln1 and the glutathione S-transferase protein levels, whereas OTC treatment restored both protein levels, which was associated with reduced ubiquitin-conjugated protein level. Interestingly, in the Ubqln1 knockout (KO) mice, OTC treatment showed reduced neuroprotection and increased ubiquitinconjugated protein level when compared to the similarly treated non-KO mice following I/R, suggesting that OTC-medicated neuroprotection is, at least partially, Ubqln1-dependent. Thus, OTC is a potential therapeutic agent for stroke and possibly for other neurological disorders and its neuroprotection involves enhanced proteostasis.