Nerina Gnesutta

A role for the Ras signalling pathway in synaptic transmission and long-term memory.

Members of the Ras subfamily of small guanine-nucleotide-binding proteins are essential for controlling normal and malignant cell proliferation as well as cell differentiation. The neuronal-specific guanine-nucleotide-exchange factor, Ras-GRF/CDC25Mm (refs 2,3,4), induces Ras signalling in response to Ca2+ influx and activation of G-protein-coupled receptors in vitro, suggesting that it plays a role in neurotransmission and plasticity in vivo. Here we report that mice lacking Ras-GRF are impaired in the process of memory consolidation, as revealed by emotional conditioning tasks that require the function of the amygdala; learning and short-term memory are intact. Electrophysiological measurements in the basolateral amygdala reveal that long-term plasticity is abnormal in mutant mice. In contrast, Ras-GRF mutants do not reveal major deficits in spatial learning tasks such as the Morris water maze, a test that requires hippocampal function. Consistent with apparently normal hippocampal functions, Ras-GRF mutants show normal NMDA (N-methyl-D-aspartate) receptor-dependent long-term potentiation in this structure. These results implicate Ras-GRF signalling via the Ras/MAP kinase pathway in synaptic events leading to formation of long-term memories.

The Serine/Threonine Kinase PAK4 Prevents Caspase Activation and Protects Cells from Apoptosis

The serine/threonine kinase PAK4 was identified first as an effector molecule for the Rho GTPase Cdc42. PAK4 differs from other members of the PAK family both in sequence and function. Previously we have shown that an important function of this kinase is to mediate the induction of filopodia in response to activated Cdc42. Studies with a constitutively active PAK4 mutant have shown that it also has a role in promoting anchorage-independent growth, an important hallmark of oncogenic transformation. Here we show that another function of PAK4 is to protect cells against apoptotic cell death. Expression of wild-type or constitutively active PAK4 delays the onset of apoptosis in response to tumor necrosis factor α stimulation, UV irradiation, and serum starvation. Consistent with an antiapoptotic function, expression of PAK4 leads to an increase in phosphorylation of the proapoptotic protein Bad and an inhibition of caspase activation.

The Promiscuous Life of Plant NUCLEAR FACTOR Y Transcription Factors

The CCAAT box is one of the most common cis-elements present in eukaryotic promoters and is bound by the transcription factor NUCLEAR FACTOR Y (NF-Y). NF-Y is composed of three subunits, NF-YA, NF-YB, and NF-YC. Unlike animals and fungi, plants have significantly expanded the number of genes encoding NF-Y subunits. We provide a comprehensive classification of NF-Y genes, with a separation of closely related, but distinct, histone fold domain proteins. We additionally review recent experiments that have placed NF-Y at the center of many developmental stress-responsive processes in the plant lineage.

Sequence-Specific Transcription Factor NF-Y Displays Histone-like DNA Binding and H2B-like Ubiquitination

The sequence-specific transcription factor NF-Y binds the CCAAT box, one of the sequence elements most frequently found in eukaryotic promoters. NF-Y is composed of the NF-YA and NF-YB/NF-YC subunits, the latter two hosting histone-fold domains (HFDs). The crystal structure of NF-Y bound to a 25 bp CCAAT oligonucleotide shows that the HFD dimer binds to the DNA sugar-phosphate backbone, mimicking the nucleosome H2A/H2B-DNA assembly. NF-YA both binds to NF-YB/NF-YC and inserts an α helix deeply into the DNA minor groove, providing sequence-specific contacts to the CCAAT box. Structural considerations and mutational data indicate that NF-YB ubiquitination at Lys138 precedes and is equivalent to H2B Lys120 monoubiquitination, important in transcriptional activation. Thus, NF-Y is a sequence-specific transcription factor with nucleosome-like properties of nonspecific DNA binding and helps establish permissive chromatin modifications at CCAAT promoters. Our findings suggest that other HFD-containing proteins may function in similar ways.

Cloning and characterization of mouse UBPy, a deubiquitinating enzyme that interacts with the ras guanine nucleotide exchange factor CDC25(Mm)/Ras-GRF1.

We used yeast “two-hybrid” screening to isolate cDNA-encoding proteins interacting with the N-terminal domain of the Ras nucleotide exchange factor CDC25Mm. Three independent overlapping clones were isolated from a mouse embryo cDNA library. The full-length cDNA was cloned by RACE-polymerase chain reaction. It encodes a large protein (1080 amino acids) highly homologous to the human deubiquitinating enzyme hUBPy and contains a well conserved domain typical of ubiquitin isopeptidases. Therefore we called this new protein mouse UBPy (mUBPy). Northern blot analysis revealed a 4-kilobase mRNA present in several mouse tissues and highly expressed in testis; a good level of expression was also found in brain, where CDC25Mm is exclusively expressed. Using a glutathione S-transferase fusion protein, we demonstrated an “in vitro” interaction between mUBPy and the N-terminal half (amino acids 1–625) of CDC25Mm. In addition “in vivo” interaction was demonstrated after cotransfection in mammalian cells. We also showed that CDC25Mm, expressed in HEK293 cells, is ubiquitinated and that the coexpression of mUBPy decreases its ubiquitination. In addition the half-life of CDC25Mm protein was considerably increased in the presence of mUBPy. The specific function of the human homolog hUBPy is not defined, although its expression was correlated with cell proliferation. Our results suggest that mUBPy may play a role in controlling degradation of CDC25Mm, thus regulating the level of this Ras-guanine nucleotide exchange factor.

A Distal CCAAT/NUCLEAR FACTOR Y Complex Promotes Chromatin Looping at the FLOWERING LOCUS T Promoter and Regulates the Timing of Flowering in Arabidopsis

This work demonstrates that chromatin loops form in the promoter of the FLOWERING LOCUS T (FT) gene, an essential integrator of multiple flowering time pathways. These loops bring NF-Y–bound distal regions of the FT promoter into close association with the CONSTANS-bound proximal promoter, providing a recruitment model for photoperiod-dependent flowering. For many plant species, reproductive success relies on the proper timing of flowering, and photoperiod provides a key environmental input. Photoperiod-dependent flowering depends on timely expression of FLOWERING LOCUS T (FT); however, the coordination of various cis-regulatory elements in the FT promoter is not well understood. Here, we provide evidence that long-distance chromatin loops bring distal enhancer elements into close association with the proximal promoter elements bound by CONSTANS (CO). Additionally, we show that NUCLEAR FACTOR Y (NF-Y) binds a CCAAT box in the distal enhancer element and that CCAAT disruption dramatically reduces FT promoter activity. Thus, we propose the recruitment model of photoperiod-dependent flowering where NF-Y complexes, bound at the FT distal enhancer element, help recruit CO to proximal cis-regulatory elements and initiate the transition to reproductive growth.

Death receptor-induced activation of initiator caspase 8 is antagonized by serine/threonine kinase PAK4.

ABSTRACT Normal cell growth requires a precisely controlled balance between cell death and survival. This involves activation of different types of intracellular signaling cascades within the cell. While some types of signaling proteins regulate apoptosis, or programmed cell death, other proteins within the cell can promote survival. The serine/threonine kinase PAK4 can protect cells from apoptosis in response to several different types of stimuli. As is the case for other members of the p21-activated kinase (PAK) family, one way that PAK4 may promote cell survival is by phosphorylating and thereby inhibiting the proapoptotic protein Bad. This leads in turn to the inhibition of effector caspases such as caspase 3. Here we show that in response to cytokines which activate death domain-containing receptors, such as the tumor necrosis factor and Fas receptors, PAK4 can inhibit the death signal by a different mechanism. Under these conditions, PAK4 inhibits apoptosis early in the caspase cascade, antagonizing the activation of initiator caspase 8. This inhibition, which does not require PAK4s kinase activity, may involve inhibition of caspase 8 recruitment to the death domain receptors. This role in regulating initiator caspases is an entirely novel role for the PAK proteins and suggests a new mechanism by which these proteins promote cell survival.

Interactions and CCAAT-Binding of Arabidopsis thaliana NF-Y Subunits

Background NF-Y is a transcription factor that recognizes with high specificity and affinity the widespread CCAAT box promoter element. It is formed by three subunits: NF-YA and the NF-YB/NF-YC- heterodimer containing histone fold domains (HFDs). We previously identified a large NF-Y gene family in Arabidopsis thaliana, composed of 29 members, and characterized their expression patterns in various plant tissues. Methods We used yeast Two-hybrids assays (Y2H), pull-down and Electrophoretic Mobility Shift Assay (EMSA) in vitro experiments with recombinant proteins to dissect AtNF-YB/AtNF-YC interactions and DNA-binding with different AtNF-YAs. Results Consistent with robust conservation within HFDs, we show that heterodimerization is possible among all histone-like subunits, including the divergent and related LEC1/AtNF-YB9 and L1L/AtNF-YB6 required for embryo development. DNA-binding to a consensus CCAAT box was investigated with specific AtNF-YB/AtNF-YC combinations and observed with some, but not all AtNF-YA subunits. Conclusions Our results highlight (i) the conserved heterodimerization capacity of AtNF-Y histone-like subunits, and (ii) the different affinities of AtNF-YAs for the CCAAT sequence. Because of the general expansion of NF-Y genes in plants, these results most likely apply to other species.

The NF-Y/p53 liaison: well beyond repression.

NF-Y is a sequence-specific transcription factor - TF - targeting the common CCAAT promoter element. p53 is a master TF controlling the response to stress signals endangering genome integrity, often mutated in human cancers. The NF-Y/p53 - and p63, p73 - interaction results in transcriptional repression of a subset of genes within the vast NF-Y regulome under DNA-damage conditions. Recent data shows that NF-Y is also involved in pro-apoptotic activities, either directly, by mediating p53 transcriptional activation, or indirectly, by being targeted by a non coding RNA, PANDA. The picture is subverted in cells carrying Gain-of-function mutant p53, through interactions with TopBP1, a protein also involved in DNA repair and replication. In summary, the connection between p53 and NF-Y is crucial in determining cell survival or death.

SCLIP, a microtubule-destabilizing factor, interacts with RasGRF1 and inhibits its ability to promote Rac activation and neurite outgrowth.

RasGRF1 is a neuron-specific guanine nucleotide exchange factor for the small GTPases Ras and Rac. It is implicated in the regulation of memory formation and in the development of tolerance to drug abuse, although the mechanisms have been elucidated only in part. Here we report the isolation, by the yeast two-hybrid screen, of the microtubule-destabilizing factor SCLIP (SCG10-like protein) as a novel RasGRF1-interacting protein. This interaction requires the region spanning the Dblhomology domain of RasGRF1, endowed with catalytic activity on Rac. In search for a possible function we found by biochemical means that SCLIP influences the signaling properties of RasGRF1, greatly reducing its ability to activate the Rac/p38 MAPK pathway, while the Ras/Erk one remains unaffected. Moreover, a potential role is suggested by transfection studies in neuronal PC12 cells in which RasGRF1 induces neurite outgrowth, and coexpression of SCLIP counteracts this effect, causing a dramatic decrease in the percentage of cells bearing neurites, which also appear significantly shortened. This study unveils a physical and functional interaction between RasGRF1 and SCLIP. We suggest that this novel interplay may have possible implications in mechanisms that regulate neuronal morphology and structural plasticity.