K63-specific deubiquitination by two JAMM/MPN+ complexes: BRISC-associated Brcc36 and proteasomal Poh1.

An unusual deubiquitinating (DUB) activity exists in HeLa cell extracts that is highly specific for cleaving K63-linked but not K48-linked polyubiquitin chains. The activity is insensitive to both N-ethyl-maleimide and ubiquitin aldehyde, indicating that it lacks an active site cysteine residue, and gel filtration experiments show that it resides in a high molecular weight (approximately 600 kDa) complex. Using a biochemical approach, we found that the K63-specific DUB activity co-fractionated through seven chromatographic steps with three multisubunit complexes: the 19S (PA700) portion of the 26S proteasome, the COP9 signalosome (CSN) and a novel complex that includes the JAMM/MPN+ domain-containing protein Brcc36. When we analysed the individual complexes, we found that the activity was intrinsic to PA700 and the Brcc36 isopeptidase complex (BRISC), but that the CSN-associated activity was due entirely to an interaction with Brcc36. None of the complexes cleave K6, K11, K29, K48 or alpha-linked polyubiquitin, but they do cleave K63 linkages within mixed-linkage chains. Our results suggest that specificity for K63-linked polyubiquitin is a common property of the JAMM/MPN+ family of DUBs.

Protein pyrophosphorylation by inositol pyrophosphates is a posttranslational event.

In a previous study, we showed that the inositol pyrophosphate diphosphoinositol pentakisphosphate (IP(7)) physiologically phosphorylates mammalian and yeast proteins. We now report that this phosphate transfer reflects pyrophosphorylation. Thus, proteins must be prephosphorylated by ATP to prime them for IP(7) phosphorylation. IP(7) phosphorylates synthetic phosphopeptides but not if their phosphates have been masked by methylation or pyrophosphorylation. Moreover, IP(7) phosphorylated peptides are more acid-labile and more resistant to phosphatases than ATP phosphorylated peptides, indicating a different type of phosphate bond. Pyrophosphorylation may represent a novel mode of signaling to proteins.

Mac-2-binding protein is a diagnostic marker for biliary tract carcinoma.

BACKGROUND: Biliary tract carcinoma is a deadly disease, accounting for nearly 4500 malignancy-related deaths each year in the United States. Early detection has the potential to improve survival for patients with biliary tract malignancies, enabling curative surgical resection. Early detection approaches would benefit from an accurate, minimally invasive diagnostic test. To identify novel diagnostic markers, the authors recently completed a comprehensive proteomic study of bile samples from patients with biliary carcinoma. One of the proteins identified by tandem mass spectrometry was Mac-2-binding protein (Mac-2BP). The authors evaluated the performance of Mac-2BP and its ligand, galectin-3, as diagnostic markers for patients with biliary carcinoma. METHODS: Levels of Mac-2BP, galectin-3, and CA19-9 were measured using an enzyme-linked immunosorbent assay (ELISA) in bile samples from patients with biliary tract carcinoma (n = 26), benign biliary conditions (n = 32), and primary sclerosing cholangitis (n = 20). Serum levels of Mac-2BP and galectin-3 also were determined using ELISA. Mac-2BP tissue expression was investigated by immunohistochemical methods using a biliary carcinoma tissue microarray. RESULTS: Biliary Mac-2BP levels were elevated by a factor of approximately 3 in the biliary carcinoma group compared with the group of patients who had PSC or another type of nonneoplastic biliary disease. In contrast, Mac-2BP levels were not elevated in serum samples from patients with biliary carcinoma. According to the immunohistochemical analysis, Mac-2BP was expressed in 34 of 36 patients (94.4%) with biliary tract carcinoma. As a diagnostic marker for biliary carcinoma, Mac-2BP levels were as accurate as biliary CA19-9 levels, with an area under the curve (AUC) of 0.70 on receiver operator characteristic analysis. The use of both of these bile markers in combination, however, led to significantly better diagnostic accuracy compared with the accuracy achieved using CA19-9 alone (AUC, 0.75; P < 0.001). Serum and biliary galectin-3 levels did not differ in the biliary carcinoma group relative to the control groups. CONCLUSIONS: Biliary Mac-2BP levels, especially when used in conjunction with biliary CA19-9 levels, showed promise as a novel diagnostic marker for biliary tract carcinoma.

Human protein reference database as a discovery resource for proteomics.

The rapid pace at which genomic and proteomic data is being generated necessitates the development of tools and resources for managing data that allow integration of information from disparate sources. The Human Protein Reference Database (http://www.hprd.org) is a web-based resource based on open source technologies for protein information about several aspects of human proteins including protein-protein interactions, post-translational modifications, enzyme-substrate relationships and disease associations. This information was derived manually by a critical reading of the published literature by expert biologists and through bioinformatics analyses of the protein sequence. This database will assist in biomedical discoveries by serving as a resource of genomic and proteomic information and providing an integrated view of sequence, structure, function and protein networks in health and disease.

Development of human protein reference database as an initial platform for approaching systems biology in humans.

Human Protein Reference Database (HPRD) is an object database that integrates a wealth of information relevant to the function of human proteins in health and disease. Data pertaining to thousands of protein-protein interactions, posttranslational modifications, enzyme/substrate relationships, disease associations, tissue expression, and subcellular localization were extracted from the literature for a nonredundant set of 2750 human proteins. Almost all the information was obtained manually by biologists who read and interpreted >300,000 published articles during the annotation process. This database, which has an intuitive query interface allowing easy access to all the features of proteins, was built by using open source technologies and will be freely available at http://www.hprd.org to the academic community. This unified bioinformatics platform will be useful in cataloging and mining the large number of proteomic interactions and alterations that will be discovered in the postgenomic era.

Cloning and characterization of PAK5, a novel member of mammalian p21-activated kinase-II subfamily that is predominantly expressed in brain.

The p21-activated kinase (PAK) family of protein kinases has recently attracted considerable attention as an effector of Rho family of small G proteins and as an upstream regulator of MAPK signalling pathways during cellular events such as re-arrangement of the cytoskeleton and apoptosis. We have cloned a novel human PAK family kinase that has been designated as PAK5. PAK5 contains a CDC42/Rac1 interactive binding (CRIB) motif at the N-terminus and a Ste20-like kinase domain at the C-terminus. PAK5 is structurally most related to PAK4 and PAK6 to make up the PAK-II subfamily. We have shown that PAK5 preferentially binds to CDC42 in the presence of GTP and that CRIB motif is essential for this interaction. PAK5 is a functional protein kinase but unlike PAK-I family kinases (PAK1, 2, and 3), the kinase activity of PAK5 does not seem to require the binding of CDC42. Overexpression of PAK5 activates the JNK kinase pathway but not p38 or ERK pathways. PAK5 transcript is predominantly expressed in brain as revealed by Northern blot and in situ hybridization. The expression pattern of PAK5 is distinct from that of PAK4 and PAK6, suggesting a functional division among PAK-II subfamily kinases based on differential tissue distribution.

Cloning of MASK, a novel member of the mammalian germinal center kinase III subfamily, with apoptosis-inducing properties.

We have cloned a novel human GCK family kinase that has been designated as MASK (Mst3 and SOK1-related kinase). MASK is widely expressed and encodes a protein of 416 amino acid residues, with an N-terminal kinase domain and a unique C-terminal region. Like other GCK-III subfamily kinases, MASK does not activate any mitogen-activated protein kinase pathways. Wild type MASK, but not a form lacking the C terminus, exhibits homophilic binding in the yeast two-hybrid system and in coimmunoprecipitation experiments. Additionally, deletion of this C-terminal region of MASK leads to an increased kinase activity toward itself as well as toward an exogenous substrate, myelin basic protein. A potential caspase 3 cleavage site (DESDS) is present in the C-terminal region of MASK, and we show that MASK is cleaved in vitro by caspase 3. Finally, wild type and C-terminally truncated forms of MASK can both induce apoptosis upon overexpression in mammalian cells that is abrogated by CrmA, suggesting involvement of MASK in the apoptotic machinery in mammalian cells.