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1.
Oncogene ; 38(8): 1324-1339, 2019 02.
Article in English | MEDLINE | ID: mdl-30659267

ABSTRACT

Copy number gains, point mutations and epigenetic silencing events are increasingly observed in genes encoding elements of the Ras/Raf/MEK/ERK signaling axis in human breast cancer. The three Raf kinases A-Raf, B-Raf, and Raf-1 have an important role as gatekeepers in ERK pathway activation and are often dysregulated by somatic alterations of their genes or by the aberrant activity of receptor tyrosine kinases (RTKs) and Ras-GTPases. B-Raf represents the most potent Raf isoform and a critical effector downstream of RTKs and RAS proteins. Aberrant RTK signaling is mimicked by the polyoma middle T antigen (PyMT), which activates various oncogenic signaling pathways, incl. the RAS/ERK axis, in a similar manner as RTKs in human breast cancer. Mammary epithelial cell directed expression of PyMT in mice by the MMTV-PyMT transgene induces mammary hyperplasia progressing over adenoma to metastatic breast cancer with an almost complete penetrance. To understand the functional role of B-Raf in this model for luminal type B breast cancer, we crossed MMTV-PyMT mice with animals that either lack B-Raf expression in the mammary gland or express the signaling impaired B-RafAVKA mutant. The AVKA mutation prevents phosphorylation of T599 and S602 in the B-Raf activation loop and thereby activation of the kinase by upstream signals. We demonstrate for the first time that B-Raf expression and activation is important for tumor initiation in vivo as well as for lung metastasis. Isogenic tumor cell lines generated from conditional Braf knock-out or knock-in mice displayed a reduction in EGF-induced ERK pathway activity as well as in proliferation and invasive growth in three-dimensional matrigel cultures. Our results suggest that B-Raf, which has been hardly studied in the context of breast cancer, represents a critical effector of the PyMT oncoprotein and invite for an assessment of its functional role in human breast cancer.


Subject(s)
Breast Neoplasms/genetics , Cell Transformation, Neoplastic/genetics , Mammary Neoplasms, Animal/genetics , Proto-Oncogene Proteins B-raf/genetics , Animals , Breast Neoplasms/pathology , Cell Line, Tumor , Cell Proliferation/genetics , Disease Models, Animal , Disease Progression , Female , Gene Expression Regulation, Neoplastic , Humans , MAP Kinase Signaling System , Mammary Neoplasms, Animal/pathology , Mice , Mice, Knockout , Mutation , Proto-Oncogene Proteins A-raf/genetics , Proto-Oncogene Proteins B-raf/deficiency , Proto-Oncogene Proteins c-raf/genetics
2.
Med Mol Morphol ; 52(3): 156-163, 2019 Sep.
Article in English | MEDLINE | ID: mdl-30499042

ABSTRACT

Most facial bones, including frontal bones, are derived from neural crest cells through intramembranous ossification. Fibroblast growth factor receptor 1 (Fgfr1) plays a pivotal role in craniofacial bone development, and loss of Fgfr1 leads to cleft palate and facial cleft defects in newborn mice. However, the potential role of the Fgfr1 gene in neural crest cell-mediated craniofacial development remains unclear. To investigate the role of Fgfr1 in neural crest cells, we analyzed Wnt1-Cre;Fgfr1flox/flox mice. Our results show that specific knockout of Fgfr1 in neural crest cells induced heterotopic chondrogenesis and osteogenesis at the interface of the anterior portions of frontal bones. We observed that heterotopic bone formation continued through postnatal day 28, whereas heterotopic chondrogenesis lasted only through the embryonic period. In summary, our results indicate that loss of Fgfr1 in neural crest cells leads to heterotopic chondrogenesis and osteogenesis.


Subject(s)
Chondrogenesis , Frontal Bone/growth & development , Neural Crest/growth & development , Osteogenesis , Receptor, Fibroblast Growth Factor, Type 1/metabolism , Animals , Frontal Bone/metabolism , Gene Expression Regulation, Developmental , Mice , Mice, Knockout , Neural Crest/cytology , Neural Crest/metabolism , Receptor, Fibroblast Growth Factor, Type 1/genetics
4.
PLoS Genet ; 10(7): e1004399, 2014 Jul.
Article in English | MEDLINE | ID: mdl-25009998

ABSTRACT

Hedgehog (Hh) signaling is essential for normal growth, patterning, and homeostasis of many tissues in diverse organisms, and is misregulated in a variety of diseases including cancer. Cytoplasmic Hedgehog signaling is activated by multisite phosphorylation of the seven-pass transmembrane protein Smoothened (Smo) in its cytoplasmic C-terminus. Aside from a short membrane-proximal stretch, the sequence of the C-terminus is highly divergent in different phyla, and the evidence suggests that the precise mechanism of Smo activation and transduction of the signal to downstream effectors also differs. To clarify the conserved role of G-protein-coupled receptor kinases (GRKs) in Smo regulation, we mapped four clusters of phosphorylation sites in the membrane-proximal C-terminus of Drosophila Smo that are phosphorylated by Gprk2, one of the two fly GRKs. Phosphorylation at these sites enhances Smo dimerization and increases but is not essential for Smo activity. Three of these clusters overlap with regulatory phosphorylation sites in mouse Smo and are highly conserved throughout the bilaterian lineages, suggesting that they serve a common function. Consistent with this, we find that a C-terminally truncated form of Drosophila Smo consisting of just the highly conserved core, including Gprk2 regulatory sites, can recruit the downstream effector Costal-2 and activate target gene expression, in a Gprk2-dependent manner. These results indicate that GRK phosphorylation in the membrane proximal C-terminus is an evolutionarily ancient mechanism of Smo regulation, and point to a higher degree of similarity in the regulation and signaling mechanisms of bilaterian Smo proteins than has previously been recognized.


Subject(s)
Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , G-Protein-Coupled Receptor Kinase 2/genetics , Gene Expression Regulation, Developmental , Receptors, G-Protein-Coupled/metabolism , Animals , Drosophila Proteins/biosynthesis , Drosophila melanogaster/growth & development , G-Protein-Coupled Receptor Kinase 2/biosynthesis , Hedgehog Proteins/genetics , Hedgehog Proteins/metabolism , Humans , Kinesins/metabolism , Mice , Phosphorylation/genetics , Receptors, G-Protein-Coupled/genetics , Signal Transduction/genetics , Smoothened Receptor
5.
Fly (Austin) ; 6(3): 135-41, 2012.
Article in English | MEDLINE | ID: mdl-22653052

ABSTRACT

Hedgehog (Hh) signaling is essential for proper tissue patterning and maintenance and has a substantial impact on human disease. While many of the main components and mechanisms involved in transduction of the Hh signal have been identified, the details of how the pathway functions are continually being refined. One aspect that has attracted much attention recently is the involvement of G-protein-coupled receptor kinases (GRKs) in the pathway. These regulators of G-protein-coupled receptor (GPCR) signaling have an evolutionarily-conserved function in promoting high-threshold Hh target gene expression through regulation of Smoothened (Smo), a GPCR family member that activates intracellular Hh signaling. Several models of how GRKs impact on Smo to increase downstream signaling have been proposed. Recently, we demonstrated that these kinases have surprisingly complex and conflicting roles, acting to limit signaling through the pathway while also promoting Smo activity. In addition to the previously described direct effects of Gprk2 on Smo activation, Gprk2 also indirectly affects Hh signaling by controlling production of the second messenger cyclic AMP to influence Protein kinase A activity.


Subject(s)
Drosophila Proteins/physiology , Drosophila/growth & development , G-Protein-Coupled Receptor Kinases/physiology , Hedgehog Proteins/physiology , Models, Biological , Animals , Cyclic AMP/genetics , Cyclic AMP/metabolism , Cyclic AMP/physiology , Drosophila/genetics , Drosophila/metabolism , Drosophila Proteins/genetics , G-Protein-Coupled Receptor Kinases/genetics , Gene Expression Regulation , Hedgehog Proteins/metabolism , Signal Transduction
6.
Development ; 139(1): 85-94, 2012 Jan.
Article in English | MEDLINE | ID: mdl-22096079

ABSTRACT

G-protein-coupled receptor kinases (GRKs) play a conserved role in Hedgehog (Hh) signaling. In several systems, GRKs are required for efficient Hh target gene expression. Their principal target appears to be Smoothened (Smo), the intracellular signal-generating component of the pathway and a member of the G-protein-coupled receptor (GPCR) protein family. In Drosophila, a GRK called Gprk2 is needed for internalization and downregulation of activated Smo, consistent with the typical role of these kinases in negatively regulating GPCRs. However, Hh target gene activation is strongly impaired in gprk2 mutant flies, indicating that Gprk2 must also positively regulate Hh signaling at some level. To investigate its function in signaling, we analyzed several different readouts of Hh pathway activity in animals or cells lacking Gprk2. Surprisingly, although target gene expression was impaired, Smo-dependent activation of downstream components of the signaling pathway was increased in the absence of Gprk2. This suggests that Gprk2 does indeed play a role in terminating Smo signaling. However, loss of Gprk2 resulted in a decrease in cellular cAMP concentrations to a level that was limiting for Hh target gene activation. Normal expression of target genes was restored in gprk2 mutants by stimulating cAMP production or activating the cAMP-dependent Protein kinase A (Pka). Our results suggest that direct regulation of Smo by Gprk2 is not absolutely required for Hh target gene expression. Gprk2 is important for normal cAMP regulation, and thus has an indirect effect on the activity of Pka-regulated components of the Hh pathway, including Smo itself.


Subject(s)
Cyclic AMP/metabolism , Drosophila melanogaster/physiology , G-Protein-Coupled Receptor Kinase 2/metabolism , Hedgehog Proteins/metabolism , Signal Transduction/physiology , Transcriptional Activation/genetics , Animals , Drosophila Proteins/metabolism , Drosophila melanogaster/enzymology , Fatty Acids, Unsaturated , Plasmids/genetics , Receptors, G-Protein-Coupled/metabolism , Smoothened Receptor , Transcriptional Activation/physiology
7.
Dev Dyn ; 239(11): 2860-74, 2010 Nov.
Article in English | MEDLINE | ID: mdl-20845426

ABSTRACT

Mutations in the gene encoding the T-box transcription factor TBX22 cause X-linked cleft palate and ankyloglossia in humans. Here we show that Tbx22 expression during facial and palatal development is regulated by FGF and BMP signaling. Our results demonstrate that FGF8 induces Tbx22 in the early face while BMP4 represses and thus restricts its expression. This regulation is conserved between chicken and mouse, although the Tbx22-expression patterns differ considerably between these two species. We suggest that these species-specific differences may result at least in part from differences in the spatiotemporal patterns of BMP activity, but we exclude a direct repression of Tbx22 by the BMP-inducible transcriptional repressor MSX1. Together these findings help to integrate Tbx22 into the molecular network of factors regulating facial development.


Subject(s)
Embryo, Mammalian/metabolism , Face/embryology , Palate/embryology , Palate/metabolism , T-Box Domain Proteins/metabolism , Animals , Apoptosis/genetics , Apoptosis/physiology , Bone Morphogenetic Protein 4/genetics , Bone Morphogenetic Protein 4/metabolism , Cell Proliferation , Chick Embryo , Cleft Palate/embryology , Cleft Palate/metabolism , Embryo, Mammalian/ultrastructure , Fibroblast Growth Factor 8/genetics , Fibroblast Growth Factor 8/metabolism , Gene Expression Regulation, Developmental , Immunohistochemistry , In Situ Hybridization , Mice , Mice, Knockout , Microscopy, Atomic Force , Reverse Transcriptase Polymerase Chain Reaction , T-Box Domain Proteins/genetics
8.
Dev Biol ; 337(1): 99-109, 2010 Jan 01.
Article in English | MEDLINE | ID: mdl-19850026

ABSTRACT

The Hedgehog (Hh) signaling pathway plays a conserved and essential role in regulating development and homeostasis of numerous tissues. Cytoplasmic signaling is initiated by Smoothened (Smo), a G-protein-coupled receptor (GPCR) family member, whose levels and activity are regulated by the Hh receptor Patched (Ptc). In response to Hh binding to Ptc, Ptc-mediated repression of Smo is relieved, leading to Smo activation, surface accumulation, and downstream signaling. We find that downregulation of Drosophila Smo protein in Hh-responding imaginal disc cells is dependent on the activity of G-protein-coupled receptor kinase 2 (Gprk2). By analyzing gain- and null loss-of-function phenotypes, we provide evidence that Gprk2 promotes Smo internalization subsequent to its activation, most likely by direct phosphorylation. Ptc-dependent regulation of Smo accumulation is normal in gprk2 mutants, indicating that Gprk2 and Ptc downregulate Smo by different mechanisms. Finally, we show that both Drosophila G-protein-coupled receptor kinase orthologues, Gprk1 and Gprk2, act in a partially redundant manner to promote Hh signaling. Our results suggest that Smo is regulated by distinct Ptc-dependent and Gprk2-dependent trafficking mechanisms in vivo, analogous to constitutive and activity-dependent regulation of GPCRs. G-protein-coupled receptor kinase activity is also important for efficient downstream signaling.


Subject(s)
Drosophila Proteins/genetics , Drosophila Proteins/physiology , G-Protein-Coupled Receptor Kinase 2/physiology , Gene Expression Regulation , Receptors, G-Protein-Coupled/genetics , Animals , Arrestins/physiology , Cells, Cultured , Drosophila melanogaster , G-Protein-Coupled Receptor Kinase 1/physiology , Hedgehog Proteins/physiology , Phosphorylation , Receptors, Cell Surface/physiology , Signal Transduction , Smoothened Receptor , Temperature , beta-Arrestins
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