Protein kinases that phosphorylate 14-3-3 isoforms

14 -3 -3 is an abundant, predominantly phospho-binding protein, intimately involved in the regulation of many diverse signal transduction events including cell cycle regulation, nucleo-cytoplasmic targeting of essential transcription factors and regulation of catecholamine synthesis. The 14 -3 -3 family consists of 7 isoforms (denoted a, ß, y, 8, c, rl and in mammals and shows a degree of isoform specificity in binding target proteins. 14 -3 -3 is phosphorylated in an isoform specific manner, for example SDK1 /PKD phosphorylates 14 -3 -3 11,13 and (, but not cs and T. Our laboratory has previously identified in vivo 14 -3 -3 phosphorylation sites, S185 and S233. Phosphorylation of S233 by the serine /threonine protein kinase Casein kinase la (CK1 a) was shown to negatively affect the interaction with Raf kinase. The group of Gotoh have recently shown that phosphorylation of S185 by the stress activated kinase c -Jun NH2- terminal kinase (JNK) negatively effects the interaction with Bax. During studies in our laboratory that identified CKla as a 14 -3 -3 kinase, several other proteins co- purified through four steps of chromatography, including centaurin -a1 and CPI -17, suggesting a protein complex - these interactions have subsequently been characterised. CK1 has a potential phosphorylation dependent 14 -3 -3 binding site within the same region previously shown to be the interaction site for centaurin -al and the aim of this investigation was to examine the possible interaction.14 -3 -3 was found to associate with CK1 isoforms, with CKIa being studied further to reveal an interaction through serine residues 218 and 242 in a phosphorylation dependent manner, in vivo. Centaurin -a1 was found to interact with a region corresponding to residues 214 -226, only if S218 was in a dephosphorylated state, suggesting a possible regulatory mechanism. Mutagenesis of CKIa suggests that S242 is a high affinity binding site for 14 -3 -3, with S218 being of lower affinity. Investigations to identify possible kinase(s) responsible for phosphorylation of CK1 showed that stimulation of PKA can increase CKla:14 -3 -3 association in cells, but PKA does not appear to phosphorylate CKIa in vitro.As phosphorylation of 14 -3 -3 itself is an important regulatory mechanism, attempts were made to produce antibodies to phosphorylated S185 and S233 on 14 -3- 3. A phospho -specific antibody to S185 was successful, but antibodies to a- phospho- S233 had no preference to the phosphorylation state of 14 -3 -3, although they were of high selectivity and affinity for 14 -3 -3 isoforms.The kinase BCR (Breakpoint cluster region) is an important, but poorly understood protein that has been shown to associate with and phosphorylate 14 -3 -3. Investigations showed that BCR phosphorylates 14 -3 -3 on Ser233 in vitro. Additionally, BCR is shown to associate with another two isoforms of 14 -3 -3 (e and rl) both in vitro and in vivo. However 14 -3 -3 a did not associate with BCR in vitro. BCR selectively phosphorylates 14 -3 -3 r more than , in contrast to CK1.In summary, Interactions with CK1 and 14 -3 -3 are characterised in detail and a possible regulatory mechanism discussed for CK1:centaurin- a1:14 -3 -3 interaction. Further insights into BCR signalling are revealed by identification of the phosphorylation site on 14 -3 -3 that has been shown to negatively affect binding to Raf kinase

Resource: A multi‐species multi‐timepoint transcriptome database and webpage for the pineal gland and retina

The website and database https://snengs.nichd.nih.gov provides RNA sequencing data from multi-species analysis of the pineal glands from zebrafish (Danio rerio), chicken (White Leghorn), rat (Rattus novegicus), mouse (Mus musculus), rhesus macaque (Macaca mulatta), and human (Homo sapiens); in most cases, retinal data are also included along with results of the analysis of a mixture of RNA from tissues. Studies cover day and night conditions; in addition, a time series over multiple hours, a developmental time series and pharmacological experiments on rats are included. The data have been uniformly re-processed using the latest methods and assemblies to allow for comparisons between experiments and to reduce processing differences. The website presents search functionality, graphical representations, Excel tables, and track hubs of all data for detailed visualization in the UCSC Genome Browser. As more data are collected from investigators and improved genomes become available in the future, the website will be updated. This database is in the public domain and elements can be reproduced by citing the URL and this report. This effort makes the results of 21st century transcriptome profiling widely available in a user-friendly format that is expected to broadly influence pineal research.Fil: Chang, Eric. National Instituto of Child Health & Human Development; Estados UnidosFil: Fu, Cong. National Instituto of Child Health & Human Development; Estados UnidosFil: Coon, Steven L.. National Instituto of Child Health & Human Development; Estados UnidosFil: Alon, Shahar. No especifíca;Fil: Bozinoski, Marjan. No especifíca;Fil: Breymaier, Matthew. National Instituto of Child Health & Human Development; Estados UnidosFil: Bustos, Diego Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Clokie, Samuel J.. National Instituto of Child Health & Human Development; Estados UnidosFil: Gothilf, Yoav. No especifíca;Fil: Esnault, Caroline. National Instituto of Child Health & Human Development; Estados UnidosFil: Iuvone, P. Michael. Emory University School of Medicine; Estados UnidosFil: Mason, Christopher E.. No especifíca;Fil: Ochocinska, Margaret J.. National Instituto of Child Health & Human Development; Estados UnidosFil: Tovin, Adi. No especifíca;Fil: Wang, Charles. Loma Linda University; Estados UnidosFil: Xu, Pinxian. No especifíca;Fil: Zhu, Jinhang. No especifíca;Fil: Dale, Ryan. National Instituto of Child Health & Human Development; Estados UnidosFil: Klein, David C.. National Instituto of Child Health & Human Development; Estados Unido

De novo missense variants in FBXW11 cause diverse developmental phenotypes including brain, eye and digit anomalies

The identification of genetic variants implicated in human developmental disorders has been revolutionized by second-generation sequencing combined with international pooling of cases. Here, we describe seven individuals who have diverse yet overlapping developmental anomalies, and who all have de novo missense FBXW11 variants identified by whole exome or whole genome sequencing and not reported in the gnomAD database. Their phenotypes include striking neurodevelopmental, digital, jaw, and eye anomalies, and in one individual, features resembling Noonan syndrome, a condition caused by dysregulated RAS signaling. FBXW11 encodes an F-box protein, part of the Skp1-cullin-F-box (SCF) ubiquitin ligase complex, involved in ubiquitination and proteasomal degradation and thus fundamental to many protein regulatory processes. FBXW11 targets include b-catenin and GLI transcription factors, key mediators of Wnt and Hh signaling, respectively, critical to digital, neurological, and eye development. Structural analyses indicate affected residues cluster at the surface of the loops of the substrate-binding domain of FBXW11, and the variants are predicted to destabilize the protein and/or its interactions. In situ hybridization studies on human and zebrafish embryonic tissues demonstrate FBXW11 is expressed in the developing eye, brain, mandibular processes, and limb buds or pectoral fins. Knockdown of the zebrafish FBXW11 orthologs fbxw11a and fbxw11b resulted in embryos with smaller, misshapen, and underdeveloped eyes and abnormal jaw and pectoral fin development. Our findings support the role of FBXW11 in multiple developmental processes, including those involving the brain, eye, digits, and jaw

Pigment composition and adaptation in free-living and symbiotic strains of Acaryochloris marina

Acaryochloris marina strains have been isolated from several varied locations and habitats worldwide demonstrating a diverse and dynamic ecology. In this study, the whole cell photophysiologies of strain MBIC11017, originally isolated from a colonial ascidian, and the free-living epilithic strain CCMEE5410 are analyzed by absorbance and fluorescence spectroscopy, laser scanning confocal microscopy, sodium dodecyl sulfate polyacrylamide gel electrophoresis and subsequent protein analysis. We demonstrate pigment adaptation in MBIC11017 and CCMEE5410 under different light regimes. We show that the higher the incident growth light intensity for both strains, the greater the decrease in their chlorophyll d content. However, the strain MBIC11017 loses its phycobiliproteins relative to its chlorophyll d content when grown at light intensities of 40 mu E m(-2) s(-1) without shaking and 100 mu E m(-2) s(-1) with shaking. We also conclude that phycobiliproteins are absent in the free-living strain CCMEE5410

Pigment composition and adaptation in free-living and symbiotic strains of Acaryochloris marina

Acaryochloris marina strains have been isolated from several varied locations and habitats worldwide, thus demonstrating a diverse and dynamic ecology. In this study, the whole cell photophysiologies of strain MBIC11017, originally isolated from a colonial ascidian, and the free-living epilithic strain CCMEE5410 are analysed by absorbance and fluorescence spectroscopy, laser scanning confocal microscopy, SDS-PAGE and subsequent protein analysis. We demonstrate pigment adaptation in MBIC11017 and CCMEE5410 under different light regimes. We show that strain MBIC11017 loses its phycobilins relative to its chlorophyll d content when grown at light intensities of 40 μE m-2 s-1 without shaking and 100 μE m-2 s-1 with shaking. Our results indicate the greater the light intensity both strains MBIC11017 and CCMEE5410 are grown at, the greater the decrease in their chlorophyll d content. We also conclude that phycobiliproteins are absent in the free-living strain CCMEE5410

The Lhx9 homeobox gene controls pineal gland development and prevents postnatal hydrocephalus

Lhx9 is a member of the LIM homeobox gene family. It is expressed during mammalian embryogenesis in the brain including the pineal gland. Deletion of Lhx9 results in sterility due to failure of gonadal development. The current study was initiated to investigate Lhx9 biology in the pineal gland. Lhx9 is highly expressed in the developing pineal gland of the rat with transcript abundance peaking early in development; transcript levels decrease postnatally to nearly undetectable levels in the adult, a temporal pattern that is generally similar to that reported for Lhx9 expression in other brain regions. Studies with C57BL/6J Lhx9(−/−) mutant mice revealed marked alterations in brain and pineal development. Specifically, the superficial pineal gland is hypoplastic, being reduced to a small cluster of pinealocytes surrounded by meningeal and vascular tissue. The deep pineal gland and the pineal stalk are also reduced in size. Although the brains of neonatal Lhx9(−/−) mutant mice appear normal, severe hydrocephalus develops in about 70 % of the Lhx9(−/−) mice at 5–8 weeks of age; these observations are the first to document that deletion of Lhx9 results in hydrocephalus and as such indicate that Lhx9 contributes to the maintenance of normal brain structure. Whereas hydrocephalus is absent in neonatal Lhx9(−/−)mutant mice, the neonatal pineal gland in these animals is hypoplastic. Accordingly, it appears that Lhx9 is essential for early development of the mammalian pineal gland and that this effect is not secondary to hydrocephalus