6 research outputs found

    The ePHD protein SPBP interacts with TopBP1 and together they co-operate to stimulate Ets1-mediated transcription

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    SPBP (Stromelysin-1 PDGF responsive element binding protein) is a ubiquitously expressed 220 kDa nuclear protein shown to enhance or repress the transcriptional activity of various transcription factors. A yeast two-hybrid screen, with the extended plant homeodomain (ePHD) of SPBP as bait, identified TopBP1 (topoisomerase II β-binding protein 1) as a candidate interaction partner of SPBP. TopBP1 has eight BRCA1 carboxy-terminal (BRCT) domains and is involved in DNA replication, DNA damage responses and in the regulation of gene expression. The interaction between SPBP and TopBP1 was confirmed in vitro and in vivo, and was found to be mediated by the ePHD domain of SPBP and the BRCT6 domain of TopBP1. Both SPBP and TopBP1 enhanced the transcriptional activity of Ets1 on the c-myc P1P2- and matrix metalloproteinase-3 (MMP3) promoters. Together they displayed a more than additive effect. Both proteins were associated with these promoters. The involvement of TopBP1 was dependent on the serine 1159 phosphorylation site, known to be important for transcriptional activation. Depletion of endogenous SPBP by siRNA treatment reduced MMP3 secretion by 50% in phorbol ester-stimulated human fibroblasts. Taken together, our results show that TopBP1 and SPBP interact physically and functionally to co-operate as co-activators of Ets1

    Serine residue 115 of MAPK-activated protein kinase MK5 is crucial for its PKA-regulated nuclear export and biological function

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    The mitogen-activated protein kinase-activated protein kinase-5 (MK5) resides predominantly in the nucleus of resting cells, but p38MAPK, extracellular signal-regulated kinases-3 and -4 (ERK3 and ERK4), and protein kinase A (PKA) induce nucleocytoplasmic redistribution of MK5. The mechanism by which PKA causes nuclear export remains unsolved. In the study reported here we demonstrated that Ser-115 is an in vitro PKA phosphoacceptor site, and that PKA, but not p38MAPK, ERK3 or ERK4, is unable to redistribute MK5 S115A to the cytoplasm. However, the phosphomimicking MK5 S115D mutant resides in the cytoplasm in untreated cells. While p38MAPK, ERK3 and ERK4 fail to trigger nuclear export of the kinase dead T182A and K51E MK5 mutants, S115D/T182A and K51E/S115D mutants were able to enter the cytoplasm of resting cells. Finally, we demonstrated that mutations in Ser-115 affect the biological properties of MK5. Taken together, our results suggest that Ser-115 plays an essential role in PKA-regulated nuclear export of MK5, and that it also may regulate the biological functions of MK5

    Alpha-mannosidosis: Correlation between phenotype, genotype and mutant MAN2B1 subcellular localisation Inherited metabolic diseases

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    License: Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0)Background: Alpha-mannosidosis is caused by mutations in MAN2B1, leading to loss of lysosomal alpha-mannosidase activity. Symptoms include intellectual disabilities, hearing impairment, motor function disturbances, facial coarsening and musculoskeletal abnormalities. Methods: To study the genotype-phenotype relationship for alpha-mannosidosis 66 patients were included. Based on the predicted effect of the mutations and the subcellular localisation of mutant MAN2B1 in cultured cells, the patients were divided into three subgroups. Clinical and biochemical data were collected. Correlation analyses between each of the three subgroups of genotype/ subcellular localisation and the clinical and biochemical data were done to investigate the potential relationship between genotype and phenotype in alpha-mannosidosis. Statistical analyses were performed using the SPSS software. Analyses of covariance were performed to describe the genotype-phenotype correlations. The phenotype parameters were modelled by the mutation group and age as a covariate. P values of <0.05 were considered as statistically significant. Results: Complete MAN2B1 genotypes were established for all patients. We found significantly higher scores in the Leiter-R test, lower concentrations of CSF-oligosaccharides, higher point scores in the Bruininks-Oseretsky Test of Motor Proficiency subtests (BOT-2); Upper limb coordination and Balance, and a higher FVC% in patients in subgroup 3, harbouring at least one variant that allows localisation of the mutant MAN2B1 protein to the lysosomes compared to subgrou 2 and/or subgroup 1 with no lysosomal localization of the mutant MAN2B1 protein. Conclusion: Our results indicate a correlation between the MAN2B1 genotypes and the cognitive function, upper limb coordination, balance, FVC% and the storage of oligosaccharides in CSF. This correlation depends on the subcellular localisation of the mutant MAN2B1 protein
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