Rafaela Cañete-Soler

PyK2 and FAK connections to p190Rho guanine nucleotide exchange factor regulate RhoA activity, focal adhesion formation, and cell motility

Integrin binding to matrix proteins such as fibronectin (FN) leads to formation of focal adhesion (FA) cellular contact sites that regulate migration. RhoA GTPases facilitate FA formation, yet FA-associated RhoA-specific guanine nucleotide exchange factors (GEFs) remain unknown. Here, we show that proline-rich kinase-2 (Pyk2) levels increase upon loss of focal adhesion kinase (FAK) in mouse embryonic fibroblasts (MEFs). Additionally, we demonstrate that Pyk2 facilitates deregulated RhoA activation, elevated FA formation, and enhanced cell proliferation by promoting p190RhoGEF expression. In normal MEFs, p190RhoGEF knockdown inhibits FN-associated RhoA activation, FA formation, and cell migration. Knockdown of p190RhoGEF-related GEFH1 does not affect FA formation in FAK−/− or normal MEFs. p190RhoGEF overexpression enhances RhoA activation and FA formation in MEFs dependent on FAK binding and associated with p190RhoGEF FA recruitment and tyrosine phosphorylation. These studies elucidate a compensatory function for Pyk2 upon FAK loss and identify the FAK–p190RhoGEF complex as an important integrin-proximal regulator of FA formation during FN-stimulated cell motility.

Aldolases A and C Are Ribonucleolytic Components of a Neuronal Complex That Regulates the Stability of the Light-Neurofilament mRNA

A 68 nucleotide segment of the light neurofilament (NF-L) mRNA, spanning the translation termination signal, participates in regulating the stability of the transcript in vivo. Aldolases A and C, but not B, interact specifically with this segment of the transcript in vitro. Aldolases A and C are glycolytic enzymes expressed in neural cells, and their mRNA binding activity represents a novel function of these isozymes. This unsuspected new activity was first uncovered by Northwestern blotting of a brainstem/spinal cord cDNA library. It was confirmed by two-dimensional fractionation of mouse brain cytosol followed by Northwestern hybridization and protein sequencing. Both neuronal aldolases interact specifically with the NF-L but not the heavy neurofilament mRNA, and their binding to the transcript excludes the poly(A)-binding protein (PABP) from the complex. Constitutive ectopic expression of aldolases A and C accelerates the decay of a neurofilament transgene (NF-L) driven by a tetracycline inducible system. In contrast, mutant transgenes lacking mRNA sequence for aldolase binding are stabilized. Our findings strongly suggest that aldolases A and C are regulatory components of a light neurofilament mRNA complex that modulates the stability of NF-L mRNA. This modulation likely involves endonucleolytic cleavage and a competing interaction with the PABP. Interactions of aldolases A and C in NF-L expression may be linked to regulatory pathways that maintain the highly asymmetrical form and function of large neurons.

Stability Determinants Are Localized to the 3′-Untranslated Region and 3′-Coding Region of the Neurofilament Light Subunit mRNA Using a Tetracycline-inducible Promoter

The tetracycline-responsive expression system of Bujard was used to compare rates of decay of wild-type and mutant neurofilament (NF) light subunit (NF-L) mRNAs. Optimal conditions for activation and inactivation of the target transgene were determined using a luciferase reporter gene. Analyses of mRNA stability were thereupon conducted on cells that were doubly transfected with transactivator and inducible target genes and derived from pooled clones of transfected cells. Rates of mRNA decay were compared upon inactivation of the transgenes after high levels of mRNA had been induced. Deletion of the 445-nucleotide (nt) 3′-untranslated region (3′-UTR) (L/+++−) or 527 nt of the 3′-coding region (3′-CR) (L/++−+) increased the stability of NF-L mRNA compared with the full-length (L/++++) transcript in neuronal (N2a and P19 cells) and non-neuronal (L cells) lines. Deletion of both the 3′-UTR and 3′-CR (L/++−−) led to a further stabilization of the transcript. A major stability determinant was then localized to a 68-nt sequence that forms the junction between the 3′-CR and 3′-UTR of NF-L and is the binding site of a unique ribonucleoprotein complex (Cañete-Soler, R., Schwartz, M. L., Hua, Y., and Schlaepfer, W. W. (1998) J. Biol. Chem. 273, 12655–12661). The studies establish a novel system for mapping determinants of mRNA stability and have applied the system to localize determinants that regulate the stability of the NF-L mRNA.

3′ Untranslated Region in a Light Neurofilament (NF-L) mRNA Triggers Aggregation of NF-L and Mutant Superoxide Dismutase 1 Proteins in Neuronal Cells

The pathogenesis of neurodegenerative diseases is believed to involve abnormal aggregation of proteins, but the mechanisms initiating protein aggregation are unclear. Here we report a novel phenomenon that could be instrumental in triggering protein aggregation in neurodegenerative diseases. We show that the 3′ untranslated region (3′UTR) of a light neurofilament (NF-L) transcript enhances the reactivity of its own translated product and leads to loss of solubility and aggregation of NF-L protein and to coaggregation of mutant superoxide dismutase 1 (SOD1) protein. Full-length mouse NF-L cDNAs, with and without NF-L 3′UTR, were fused to the C terminus of a green fluorescent protein (GFP) reporter gene, and the GFP-tagged NF-L proteins were examined in transfected Neuro2a cells. The GFP-tagged NF-L protein expressed from the transgene containing NF-L 3′UTR, but not from the transgene lacking NF-L 3′UTR, colocalizes with endogenous heavy neurofilament protein and, at high-level expression, leads to loss of solubility and aggregation of GFP-tagged NF-L protein. Aggregation of GFP-tagged NF-L protein triggers coaggregation and loss of solubility of coexpressed DsRed-tagged mutant (G93A) SOD1 protein but not wild-type SOD1 protein. Deletional mutagenesis maps the RNA sequence causing aggregation of GFP-tagged NF-L protein to the proximal 45 nucleotides of NF-L 3′UTR. This is the site of a major destabilizing element in NF-L RNA and binding site for RNA-binding proteins. Our findings support a working model whereby NF-L RNA, or cognate RNA-binding factors, enhances the reactivity of NF-L protein and provides a triggering mechanism leading to aggregation of NF-L and other proteins in neurodegenerative diseases.

Characterization of Ribonucleoprotein Complexes and Their Binding Sites on the Neurofilament Light Subunit mRNA

Levels of neurofilament (NF) gene expression are important determinants of basic neuronal properties, but overexpression can lead to motoneuron degeneration in transgenic mice. In a companion study (Cañete-Soler, R., Schwartz, M. L., Hua, Y., and Schlaepfer, W. W. (1998) J. Biol. Chem. 273, 12650–12654), we show that levels of NF expression are regulated by altering mRNA stability and that stability determinants are present in the 3′-coding region (3′-CR) and 3′-untranslated region (3′-UTR) of the NF light subunit (NF-L) transcript. This study characterizes the ribonucleoprotein complexes that bind to the NF-L mRNA when cytoplasmic brain extracts are incubated with radioactive probes. Gel retardation assays reveal ribonucleoprotein complexes that are selectively competed with poly(C) or poly(U))/poly(A) homoribopolymers and are referred to as C-binding and U/A-binding complexes, respectively. The C-binding complex forms on the proximal 45 nucleotides of 3′-UTR, but its assembly is markedly enhanced by 23 nucleotides of flanking 3′-CR sequence. U/A-binding complexes form at multiple binding sites in the 3′-CR and 3′-UTR. A pattern of reciprocal binding suggests that the C-binding and U/A-binding complexes interact and may compete for common components or binding sites. Cross-linking studies reveal unique polypeptides in the C-binding and U/A-binding complexes. The findings provide the basis for probing mechanisms regulating NF-L mRNA stability and the relationship between NF overexpression and motoneuron degeneration in transgenic mice.

Cytoplasmic retention sites in p190RhoGEF confer anti-apoptotic activity to an EGFP-tagged protein

p190RhoGEF is a large multi-functional protein with guanine nucleotide exchange (GEF) activity. The C-terminal region of p190RhoGEF is a highly interactive domain that binds multiple factors, including proteins with anti-apoptotic activities. We now report that transfection of EGFP-tagged p190RhoGEF protects Neuro 2a cells from stress-induced apoptosis and that anti-apoptotic activity is localized to cytoplasmic retention sequences (CRS-1 and CRS-2) in the C-terminal region of p190RhoGEF. Cytoplasmic retention is conferred to an EGFP fluorescent marker when fused to either CRS-1 or CRS-2. Both cytoplasmic retention and anti-apoptotic activity are lost by deleting CRS-1 and CRS-2 in the p190RhoGEF sequence and can be recovered by restoring either CRS-1 or CRS-2 to the EGFP-tagged sequence. Since the CRS-1 and CRS-2 contain the JIP-1 and 14-3-3 binding sites, we propose that anti-apoptotic activity may be conferred by the binding of p190RhoGEF to JIP-1 or 14-3-3, possibly by altering their interactive properties or nucleocytoplasmic movements. Taken together, our findings support a model whereby multiple interactions of p190RhoGEF confer homeostatic properties to differentiated neurons and may link neuronal homeostasis to the regulation of NF-L expression.

Characterization of the mouse neurofilament light (NF-L) gene promoter by in vitro transcription.

We have used in vitro transcription to access the basic sequences and factors required for the transcription of the mouse neurofilament light promoter (pNF-L) in the absence of chromatin structure. Deletion from -1.7 to -154 results in little change in NF-L promoter activity using nuclear extracts from either brain (expressing) or liver (non-expressing) tissues. Further deletion to -29 results in a gradual five-fold drop in promoter activity in both extracts. Only replacement of the entire -148 to -29 region results in a drop in NF-L promoter activity to basal levels. Thus, the NF-L promoter differs from the mouse NF heavy (NF-H) and mid-sized (NF-M) promoters in that no specific sequence within the immediate upstream NF-L promoter region (-154 to -29) appears to be responsible for enhancement or brain-specific transcription. We show that the order of strength of the three NF promoters is NF-H>NF-M>NF-L and identify sequences that can increase or reduce transcription when placed in front of heterologous NF promoters. We conclude that the NF-L promoter is a modular, weak and promiscuous promoter whose regulation differs from NF-H or NF-M. Our data suggest that chromatin structure may play an important role in the regulation of the NF-L promoter.

The complex relation between genotype and phenotype in motor neuron disease

The success in mapping genetic loci and identifying mutant genes in familial neurodegenerative disease has outpaced our ability to understand the linkage between genotype and phenotype of disease. The results have led to a backlog of genetic information with limited clarification of underlying disease mechanisms. A major dilemma is how mutations in widely expressed proteins lead to degeneration or dysfunction of small subsets of neurons. The problem raises fundamental questions as to the nature and interrelation of pathways that maintain the homeostasis of differentiated neurons. The issue also bears on the pathogenesis of sporadic forms of disease and prospective efficacy of therapeutic applications. This review examines the problem as it relates to motor neuron disease. Ann Neurol 2007