RESUMEN
Polo family kinase 4 (Plk4) is required for mitotic progression, and is haploinsufficient for tumor suppression and timely hepatocyte polarization in regenerating liver. At the same time, recent evidence suggests that Plk4 expression may have a role in clinical cancer progression, although the mechanisms are not clear. Here we identify a gene expression pattern predictive of reduced motility in Plk4(+/-) murine embryonic fibroblasts (MEFs) and validate this prediction with functional assays of cell spreading, migration and invasion. Increased Plk4 expression enhances cell spreading in Plk4(+/-) MEFs and migration in human embryonic kidney 293T cells, and increases invasion by DLD-1 colon cancer cells. Plk4 depletion impairs invasion of wild-type MEFs and suppresses invasion by MDA-MB231 breast cancer cells. Cytoskeletal reorganization and development of polarity are impaired in Plk4-deficient cells that have been stimulated to migrate. Endogenous Plk4 phosphorylated at the autophosphorylation site S305 localizes to the protrusions of motile cells, coincident with the RhoA GEF Ect2, GTP-bound RhoA and the RhoA effector mDia. Taken together, our findings reveal an unexpected activity of Plk4 that promotes cell migration and may underlie an association between increased Plk4 expression, cancer progression and death from metastasis in solid tumor patients.
Asunto(s)
Movimiento Celular/genética , Proliferación Celular/genética , Proteínas Serina-Treonina Quinasas/fisiología , Animales , Adhesión Celular/genética , Células Cultivadas , Progresión de la Enfermedad , Embrión de Mamíferos , Células HEK293 , Células HeLa , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Metástasis de la Neoplasia , Neoplasias/genética , Neoplasias/patologíaRESUMEN
The Arabidopsis thaliana MSH2 (AtMSH2) gene encodes a protein that belongs to a family of highly conserved proteins (MutS homologues (MSH)) involved in DNA mismatch repair. Sequence analysis strongly suggests that this single copy gene is indeed a homologue of MSH2, a gene known to play a central role in eukaryotic mismatch repair. In this report, we show that the AtMSH2 protein has functional attributes characteristic of previously described mismatch repair proteins. First, over-expression of this protein in Escherichia coli leads to a mutator phenotype similar to that reported previously for known functional homologues. Second, gel retardation assays revealed that the AtMSH2 protein has a 10-fold greater affinity for DNA containing a single pair of mismatched nucleotides versus perfectly matched DNA. These results provide experimental evidence that AtMSH2 is indeed a functional homologue of MutS.
Asunto(s)
Adenosina Trifosfatasas , Proteínas de Arabidopsis , Arabidopsis/genética , Proteínas de Unión al ADN , Proteínas de Escherichia coli , Mutación , Proteínas de Plantas/genética , Proteínas Bacterianas/genética , Disparidad de Par Base , Western Blotting , Clonación Molecular , ADN/metabolismo , Reparación del ADN , Electroforesis en Gel de Poliacrilamida , Inhibidores Enzimáticos/farmacología , Escherichia coli/metabolismo , Immunoblotting , Proteína MutS de Unión a los Apareamientos Incorrectos del ADN , Proteína 2 Homóloga a MutS , Fenotipo , Plásmidos/metabolismo , Unión Proteica , Proteínas Recombinantes de Fusión/metabolismo , Rifampin/farmacología , TemperaturaRESUMEN
We expressed the wild-type cry3Aa3 and cry3Ca1 Bacillus thuringiensis genes, which code for insecticidal proteins, in an Escherichia coli expression system. Highly purified preparations of the soluble delta-endotoxins were used to perform comparative bioassays with third-instar larvae of the Colorado potato beetle (CPB). Acute mortality data showed that Cry3Ca1 (LD(50) = 320.1 ng) was 2-fold more toxic than Cry3Aa3 (LD(50) = 672.9 ng). We also compared the chronic effects of sublethal doses of these toxins by measuring the consumption of untreated foliage and monitoring survival and development for 6 days after intoxication. No significant additional mortality was recorded, but we found that surviving larvae fed Cry3Ca1 consumed foliage at a slower rate than the larvae fed Cry3Aa3, suggesting more damage to their digestive epithelium. This study, the first assessment of the toxicity of cry3Ca1 in third-instar CPB, suggests cry3Ca1 will prove useful for the control of this important insect pest.
Asunto(s)
Bacillus thuringiensis/química , Proteínas Bacterianas/toxicidad , Toxinas Bacterianas , Escarabajos , Endotoxinas/toxicidad , Animales , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Secuencia de Bases , Clonación Molecular , Escarabajos/crecimiento & desarrollo , Cartilla de ADN , Electroforesis en Gel de Poliacrilamida , Endotoxinas/genética , Endotoxinas/aislamiento & purificación , Escherichia coli/genética , Proteínas Hemolisinas , Larva/efectos de los fármacos , Larva/crecimiento & desarrollo , Dosificación Letal Mediana , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/toxicidadRESUMEN
The cry genes that code for the insecticidal crystal proteins of Bacillus thuringiensis (B.t.) have been widely used to develop insect-resistant transgenic plants. The cry3Ca1 gene has been reported to code for a crystal protein which is particularly potent against the Colorado potato beetle (CPB). To explore the biotechnological potential of cry3Ca1, we introduced this gene into transgenic potato plants under the control of the CaMV 35S promoter. In the resulting transformants, the cry3-Ca1 gene was very poorly expressed. In fact, no full-length transcript (2300 nt) could be detected. Instead, only short transcripts of approximately 1100 nt were observed. Analysis of these short transcripts by Northern hybridization, RT-PCR as well as by cloning and sequencing showed that they resulted from premature polyadenylation. These processing events occurred at four sites within the cry3Ca1 coding region (at positions 652, 669, 914 and 981 relative to the translation start site). The sites at which premature polyadenylation took place were not those that showed the highest degree of identity to the canonical AAUAAA motif. Together with other recent data, our findings suggest that premature polyadenylation is an important mechanism which can contribute to the poor expression of transgenes in a foreign host.