Recruitment of the inhibitor Cand1 to the cullin substrate adaptor site mediates interaction to the neddylation site.

Cand1 inhibits cullin RING ubiquitin ligases by binding unneddylated cullins. The Cand1 N-terminus blocks the cullin neddylation site, whereas the C-terminus inhibits cullin adaptor interaction. These Cand1 binding sites can be separated into two functional polypeptides which bind sequentially. C-terminal Cand1 can directly bind to unneddylated cullins in the nucleus without blocking the neddylation site. The smaller N-terminal Cand1 cannot bind to the cullin neddylation region without C-terminal Cand1. The separation of a single cand1 into two independent genes represents the in vivo situation of the fungus Aspergillus nidulans, where C-terminal Cand1 recruits smaller N-terminal Cand1 in the cytoplasm. Either deletion results in an identical developmental and secondary metabolism phenotype in fungi, which resembles csn mutants deficient in the COP9 signalosome (CSN) deneddylase. We propose a two-step Cand1 binding to unneddylated cullins which initiates at the adaptor binding site and subsequently blocks the neddylation site after CSN has left.

The COP9 signalosome mediates transcriptional and metabolic response to hormones, oxidative stress protection and cell wall rearrangement during fungal development.

The COP9 signalosome complex (CSN) is a crucial regulator of ubiquitin ligases. Defects in CSN result in embryonic impairment and death in higher eukaryotes, whereas the filamentous fungus Aspergillus nidulans survives without CSN, but is unable to complete sexual development. We investigated overall impact of CSN activity on A. nidulans cells by combined transcriptome, proteome and metabolome analysis. Absence of csn5/csnE affects transcription of at least 15% of genes during development, including numerous oxidoreductases. csnE deletion leads to changes in the fungal proteome indicating impaired redox regulation and hypersensitivity to oxidative stress. CSN promotes the formation of asexual spores by regulating developmental hormones produced by PpoA and PpoC dioxygenases. We identify more than 100 metabolites, including orsellinic acid derivatives, accumulating preferentially in the csnE mutant. We also show that CSN is required to activate glucanases and other cell wall recycling enzymes during development. These findings suggest a dual role for CSN during development: it is required early for protection against oxidative stress and hormone regulation and is later essential for control of the secondary metabolism and cell wall rearrangement.

Suppressor of cytokine signalling (SOCS) 1 and 3 enhance cell adhesion and inhibit migration towards the chemokine eotaxin/CCL11.

Suppressors of cytokine signalling (SOCS) proteins regulate signal transduction, but their role in responses to chemokines remains poorly understood. We report that cells expressing SOCS1 and 3 exhibit enhanced adhesion and reduced migration towards the chemokine CCL11. Focal adhesion kinase (FAK) and the GTPase RhoA, control cell adhesion and migration and we show the presence of SOCS1 or 3 regulates expression and tyrosine phosphorylation of FAK, while also enhancing activation of RhoA. Our novel findings suggest that SOCS1 and 3 may control chemotaxis and adhesion by significantly enhancing both FAK and RhoA activity, thus localizing immune cells to the site of allergic inflammation.

SOCS3 tyrosine phosphorylation as a potential bio-marker for myeloproliferative neoplasms associated with mutant JAK2 kinases.

JAK2 V617F, identified in the majority of patients with myeloproliferative neoplasms, tyrosine phosphorylates SOCS3 and escapes its inhibition. Here, we demonstrate that the JAK2 exon 12 mutants described in a subset of V617F-negative MPN cases, also stabilize tyrosine phosphorylated SOCS3. SOCS3 tyrosine phosphorylation was also observed in peripheral blood mononuclear cells and granulocytes isolated from patients with JAK2 H538QK539L or JAK2 F537-K539delinsL mutations. JAK kinase inhibitors, which effectively inhibited the proliferation of cells expressing V617F or K539L, also caused a dose-dependent reduction in both mutant JAK2 and SOCS3 tyrosine phosphorylation. We propose, therefore, that SOCS3 tyrosine phosphorylation may be a novel bio-marker of myeloproliferative neoplasms resulting from a JAK2 mutation and a potential reporter of effective JAK2 inhibitor therapy currently in clinical development.

An eight-subunit COP9 signalosome with an intact JAMM motif is required for fungal fruit body formation.

Fruit body formation in filamentous fungi is a complex and yet hardly understood process. We show here that protein turnover control is crucial for Aspergillus nidulans development. Deletion of genes encoding COP9 signalosome (CSN) subunits 1, 2, 4, or 5 resulted in identical blocks in fruit body formation. The CSN multiprotein complex controls ubiquitin-dependent protein degradation in eukaryotes. Six CSN subunits interacted in a yeast two-hybrid analysis, and the complete eight-subunit CSN was recruited by a functional tandem affinity purification tag fusion of subunit 5 (CsnE). The tagged CsnE was unable to recruit any CSN subunit in a strain deleted for subunit 1 or subunit 4. Mutations in the JAMM metalloprotease core of CsnE resulted in mutant phenotypes identical to those of csn deletion strains. We propose that a correctly assembled CSN including a functional JAMM links protein turnover to fungal sexual development.

Modulation of GAPDH expression and cellular localization after vaccinia virus infection of human adherent monocytes.

Vaccinia virus is able to replicate in many cell types and is known to modulate apoptosis in infected cells. In this study, expression of apoptosis-related genes was screened in human adherent monocytes after vaccinia infection using a DNA array. A marked increase of the key glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was found. Increased expression and nuclear translocation of GAPDH have recently been reported to participate in apoptosis of many cell types. To confirm the array results, levels of GAPDH mRNA were estimated by RT-PCR, showing an increase at 4 h p.i. followed by a slight decrease, which correlated with the viral anti-apoptotic E3L gene transcript levels. Subcellular localization of the enzyme in human monocytes was examined by Western blot and immunostaining of the infected cells. Both experiments revealed accumulation of GAPDH in the nucleus at 14 h p.i., which was completely suppressed at 24 h p.i. This might indicate GAPDH as a novel target for vaccinia anti-apoptotic modulation.