Browse
Submit Data
Databases
API
Help

Dataset Information

15 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Kinase activity-independent regulation of cyclin pathway by GRK2 is essential for zebrafish early development.

ABSTRACT: G protein-coupled receptor (GPCR) kinases (GRKs) are known as a family of serine/threonine kinases that function as key regulators of GPCRs, as well as other types of receptors. Extensive studies of GRKs at the cellular and organismal levels have led to a consensus that GRK-catalyzed phosphorylation of receptors is the primary mechanism underlying their physiological functions. Here, we report that down-regulation of GRK2 in zebrafish embryos with GRK2 morpholino results in developmental early arrest and, interestingly, that this arrest can be rescued by exogenous expression of a GRK2 kinase-dead mutant, K220R. A physical interaction between GRK2 and cyclin B1 regulator patched homolog 1 (PTCH1), stimulated by Hedgehog (Hh), rather than GRK2-mediated phosphorylation of downstream targets, appears as the underlying mechanism. We identify residues 262-379 as the PTCH1-binding region (BP). Interaction of GRK2, K220R, and BP with PTCH1 reduces the association of PTCH1 with cyclin B1 and disrupts PTCH1-mediated inhibition of cyclin B1 nuclear translocation, whereas the PTCH1-binding deficient GRK2 mutant (Delta312-379) does not. Cell cycle and cell proliferation assays show that overexpressing PTCH1 remarkably inhibited cell growth and this effect could be attenuated by GRK2, K220R, or BP, but not Delta312-379. In vivo studies show that BP, as well as the nuclear-localizing cyclin B1 mutant, is effective in rescuing the early arrest phenotype in GRK2 knockdown embryos, but Delta312-379 is not. Our data thus reveal a novel kinase activity-independent function for GRK and establish a role for GRK2 as a cell-cycle regulator during early embryonic development.

SUBMITTER: Jiang X

PROVIDER: S-EPMC2700943 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Identification of 315 genes essential for early zebrafish development.

Project description:We completed a large insertional mutagenesis screen in zebrafish to identify genes essential for embryonic and early larval development. We isolated 525 mutants, representing lesions in approximately 390 different genes, and we cloned the majority of these. Here we describe 315 mutants and the corresponding genes. Our data suggest that there are roughly 1,400 embryonic-essential genes in the fish. Thus, we have mutations in approximately 25% of these genes and have cloned approximately 22% of them. Re-screens of our collection to identify mutants with specific developmental defects suggest that approximately 50 genes are essential for the development of some individual organs or cell types. Seventy-two percent of the embryonic-essential fish genes have homologues in yeast, 93% have homologues in invertebrates (fly or worm), and 99% have homologues in human. Yeast and worm orthologues of genes that are essential for early zebrafish development have a strong tendency to be essential for viability in yeast and for embryonic development in the worm. Thus, the trait of being a genetically essential gene is conserved in evolution. This mutant collection should be a valuable resource for diverse studies of cell and developmental biology.

| S-EPMC516474 | biostudies-literature

EIF3D promotes gallbladder cancer development by stabilizing GRK2 kinase and activating PI3K-AKT signaling pathway.

Project description:Recent evidence suggests that dysregulated eIF3d expression may be critical in various genetic disorders as well as cancer. In this study, we observed that EIF3d levels increased in gallbladder cancer (GBC) samples compared with non-tumor tissue. High eIF3d levels were associated with advanced tumor stage and metastasis and were correlated with poor prognosis in 92 patients with GBC. Depletion of EIF3d in GBC cell lines inhibited cell proliferation, colony formation and metastasis and induced apoptosis and cell cycle arrest in vitro and in vivo. In contrast, ectopic expression of eIF3d had the opposite effects. Moreover, in this study, we revealed that a novel non-translational factor function of eIF3d mediated its protumoral effects. In details, eIF3d stabilizes GRK2 protein by blocking ubiquitin-mediated degradation, consequently activates PI3K/Akt signaling, and promotes GBC cell proliferation and migration. In conclusion, eIF3d promotes GBC progression mainly via eIF3d-GRK2-AKT axis and it may be used as a prognostic factor. The therapeutic targeting of eIF3d-GRK2 axis may be a potential treatment approach for GBC.

| S-EPMC5520919 | biostudies-literature

Microarray gene expression profiling of kinase-dependent and kinase-independent effects of GRK2

Project description:The ubiquitously expressed G-protein-coupled receptor kinase 2 (GRK2, ADRBK1) is an indispensable kinase involved in growth, differentiation and development. Exaggerated GRK2 activity plays a major pathophysiological role in the development of cardiovascular diseases such as heart failure and hypertension. GRK2 exerts its functions by kinase-dependent and kinase-independent effects. To assess the differential impact of GRK2 on cellular signalling we established HEK cell clones with over-expression of comparable protein levels of GRK2 or the kinase-deficient GRK2-K220R mutant, respectively. HEK cells were either cultured in vitro or expanded in vivo, in immunodeficient NOD.Scid mice to discriminate between in vitro and in vivo effects of GRK2. Whole genome microarray gene expression profiling was performed of cultured HEK cells and of NOD.Scid mouse-expanded HEK clones. As an additional control, cells were re-cultured in vitro after expansion in NOD.Scid mice.

2012-12-06 | GSE42771 | GEO

Microarray gene expression profiling of kinase-dependent and kinase-independent effects of GRK2

2012-12-06 | E-GEOD-42771 | biostudies-arrayexpress

Kcnh1 voltage-gated potassium channels are essential for early zebrafish development.

Project description:The Kcnh1 gene encodes a voltage-gated potassium channel highly expressed in neurons and involved in tumor cell proliferation, yet its physiological roles remain unclear. We have used the zebrafish as a model to analyze Kcnh1 function in vitro and in vivo. We found that the kcnh1 gene is duplicated in teleost fish (i.e. kcnh1a and kcnh1b) and that both genes are maternally expressed during early development. In adult zebrafish, kcnh1a and kcnh1b have distinct expression patterns but share expression in brain and testis. Heterologous expression of both genes in Xenopus oocytes revealed a strong conservation of characteristic functional properties between human and fish channels, including a unique sensitivity to intracellular Ca(2+)/calmodulin and modulation of voltage-dependent gating by extracellular Mg(2+). Using a morpholino antisense approach, we demonstrate a strong kcnh1 loss-of-function phenotype in developing zebrafish, characterized by growth retardation, delayed hindbrain formation, and embryonic lethality. This late phenotype was preceded by transcriptional up-regulation of known cell-cycle inhibitors (p21, p27, cdh2) and down-regulation of pro-proliferative factors, including cyclin D1, at 70% epiboly. These results reveal an unanticipated basic activity of kcnh1 that is crucial for early embryonic development and patterning.

| S-EPMC3471731 | biostudies-literature

Ribosomal biogenesis genes play an essential and p53-independent role in zebrafish pancreas development.

Project description:Mutations in the human Shwachman-Bodian-Diamond syndrome (SBDS) gene cause defective ribosome assembly and are associated with exocrine pancreatic insufficiency, chronic neutropenia and skeletal defects. However, the mechanism underlying these phenotypes remains unclear. Here we show that knockdown of the zebrafish sbds ortholog fully recapitulates the spectrum of developmental abnormalities observed in the human syndrome, and further implicate impaired proliferation of ptf1a-expressing pancreatic progenitor cells as the basis for the observed pancreatic phenotype. It is thought that diseases of ribosome assembly share a p53-dependent mechanism. However, loss of p53 did not rescue the developmental defects associated with loss of zebrafish sbds. To clarify the molecular mechanisms underlying the observed organogenesis defects, we performed transcriptional profiling to identify candidate downstream mediators of the sbds phenotype. Among transcripts displaying differential expression, functional group analysis revealed marked enrichment of genes related to ribosome biogenesis, rRNA processing and translational initiation. Among these, ribosomal protein L3 (rpl3) and pescadillo (pes) were selected for additional analysis. Similar to knockdown of sbds, knockdown or mutation of either rpl3 or pes resulted in impaired expansion of pancreatic progenitor cells. The pancreatic phenotypes observed in rpl3- and pes-deficient embryos were also independent of p53. Together, these data suggest novel p53-independent roles for ribosomal biogenesis genes in zebrafish pancreas development.

| S-EPMC3413166 | biostudies-literature

UHRF1 phosphorylation by cyclin A2/cyclin-dependent kinase 2 is required for zebrafish embryogenesis.

Project description:Ubiquitin-like, containing PHD and RING finger domains 1 (uhrf1) is regulated at the transcriptional level during the cell cycle and in developing zebrafish embryos. We identify phosphorylation as a novel means of regulating UHRF1 and demonstrate that Uhrf1 phosphorylation is required for gastrulation in zebrafish. Human UHRF1 contains a conserved cyclin-dependent kinase 2 (CDK2) phosphorylation site at Ser-661 that is phosphorylated in vitro by CDK2 partnered with cyclin A2 (CCNA2), but not cyclin E. An antibody specific for phospho-Ser-661 recognizes UHRF1 in both mammalian cancer cells and in nontransformed zebrafish cells, but not in zebrafish bearing a mutation in ccna2. Depleting Uhrf1 from zebrafish embryos by morpholino injection causes arrest before gastrulation and early embryonic death. This phenotype is rescued by wild-type UHRF1, but not by UHRF1 in which the phospho-acceptor site is mutated, demonstrating that UHRF1 phosphorylation is essential for embryogenesis. UHRF1 was detected in the nucleus and cytoplasm, whereas nonphosphorylatable UHRF1 is unable to localize to the cytoplasm, suggesting the importance of localization in UHRF1 function. Together, these data point to an essential role for UHRF1 phosphorylation by CDK/CCNA2 during early vertebrate development.

| S-EPMC3248904 | biostudies-literature

Cyclin-dependent kinase 16/PCTAIRE kinase 1 is activated by cyclin Y and is essential for spermatogenesis.

Project description:Cyclin-dependent kinase 16 (CDK16, PCTK1) is a poorly characterized protein kinase, highly expressed in the testis and the brain. Here, we report that CDK16 is activated by membrane-associated cyclin Y (CCNY). Treatment of transfected human cells with the protein kinase A (PKA) activator forskolin blocked, while kinase inhibition promoted, CCNY-dependent targeting of CDK16-green fluorescent protein (GFP) to the cell membrane. CCNY binding to CDK16 required a region upstream of the kinase domain and was found to be inhibited by phosphorylation of serine 153, a potential PKA phosphorylation site. Thus, in contrast to other CDKs, CDK16 is regulated by phosphorylation-controlled cyclin binding. CDK16 isolated from murine testis was unphosphorylated, interacted with CCNY, and exhibited kinase activity. To investigate the function of CDK16 in vivo, we established a conditional knockout allele. Mice lacking CDK16 developed normally, but male mice were infertile. Spermatozoa isolated from their epididymis displayed thinning and elongation of the annulus region, adopted a bent shape, and showed impaired motility. Moreover, CDK16-deficient spermatozoa had malformed heads and excess residual cytoplasm, suggesting a role of CDK16 in spermiation. Thus, CDK16 is a membrane-targeted CDK essential for spermatogenesis.

| S-EPMC3272973 | biostudies-literature

A Kinase-Independent Role for Cyclin-Dependent Kinase 19 in p53 Response.

Project description:The human Mediator complex regulates RNA polymerase II transcription genome-wide. A general factor that regulates Mediator function is the four-subunit kinase module, which contains either cyclin-dependent kinase 8 (CDK8) or CDK19. Whereas CDK8 is linked to specific signaling cascades and oncogenesis, the cellular roles of its paralog, CDK19, are poorly studied. We discovered that osteosarcoma cells (SJSA) are naturally depleted of CDK8 protein. Whereas stable CDK19 knockdown was tolerated in SJSA cells, proliferation was reduced. Notably, proliferation defects were rescued upon the reexpression of wild-type or kinase-dead CDK19. Comparative RNA sequencing analyses showed reduced expression of mitotic genes and activation of genes associated with cholesterol metabolism and the p53 pathway in CDK19 knockdown cells. SJSA cells treated with 5-fluorouracil, which induces metabolic and genotoxic stress and activates p53, further implicated CDK19 in p53 target gene expression. To better probe the p53 response, SJSA cells (shCDK19 versus shCTRL) were treated with the p53 activator nutlin-3. Remarkably, CDK19 was required for SJSA cells to return to a proliferative state after nutlin-3 treatment, and this effect was kinase independent. These results implicate CDK19 as a regulator of p53 stress responses and suggest a role for CDK19 in cellular resistance to nutlin-3.

| S-EPMC5472832 | biostudies-literature

Sufficient numbers of early germ cells are essential for female sex development in zebrafish.

Project description:The sex determination for zebrafish is controlled by a combination of genetic and environmental factors. The determination of sex in zebrafish has been suggested to rely on a mechanism that is affected by germ cell-derived signals. To begin our current study, a simplified and efficient germ cell-specific promoter of the dead end (dnd) gene was identified. Utilizing the metrodinazole (MTZ)/ bacterial nitroreductase (NTR) system for inducible germ cell ablation, several stable Tg (dnd:NTR-EGFP(-3'UTR)) and Tg (dnd:NTR-EGFP(+3'UTR)) zebrafish lines were then generated with the identified promoter. A thorough comparison of the expression patterns and tissue distributions of endogenous dnd and ntr-egfp transcripts in vivo revealed that the identified 2032-bp zebrafish dnd promoter can recapitulate dnd expression faithfully in stable transgenic zebrafish. The correlation between the levels of the germ cell-derived signals and requirement for maintaining the female fate has been also explored with different durations of the MTZ treatments. Our results revealed the decreasing ratios of female presented in the treated transgenic group are fairly associated with the reducing levels of the early germ cell-derived signals. After the juvenile transgenic fish treated with 5 mM MTZ for 20 days, all MTZ-treated transgenic fish exclusively developed into males with subfertilities. Taken together, our results identified here a simplified and efficient dnd promoter, and provide clear evidence indicating that it was not the presence but the sufficiency of signals derived from germ cells that is essential for female sex development in zebrafish. Our model also provides a unique system for sex control in zebrafish studies.

| S-EPMC4332673 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data