Expression of the ectodomain-releasing protease ADAM17 is directly regulated by the osteosarcoma and bone-related transcription factor RUNX2.

Osteoblast differentiation is controlled by transcription factor RUNX2 which temporally activates or represses several bone-related genes, including those encoding extracellular matrix proteins or factors that control cell-cell, and cell-matrix interactions. Cell-cell communication in the many skeletal pericellular micro-niches is critical for bone development and involves paracrine secretion of growth factors and morphogens. This paracrine signaling is in part regulated by "A Disintegrin And Metalloproteinase" (ADAM) proteins. These cell membrane-associated metalloproteinases support proteolytic release ("shedding") of protein ectodomains residing at the cell surface. We analyzed microarray and RNA-sequencing data for Adam genes and show that Adam17, Adam10, and Adam9 are stimulated during BMP2 mediated induction of osteogenic differentiation and are robustly expressed in human osteoblastic cells. ADAM17, which was initially identified as a tumor necrosis factor alpha (TNFα) converting enzyme also called (TACE), regulates TNFα-signaling pathway, which inhibits osteoblast differentiation. We demonstrate that Adam17 expression is suppressed by RUNX2 during osteoblast differentiation through the proximal Adam17 promoter region (-0.4 kb) containing two functional RUNX2 binding motifs. Adam17 downregulation during osteoblast differentiation is paralleled by increased RUNX2 expression, cytoplasmic-nuclear translocation and enhanced binding to the Adam17 proximal promoter. Forced expression of Adam17 reduces Runx2 and Alpl expression, indicating that Adam17 may negatively modulate osteoblast differentiation. These findings suggest a novel regulatory mechanism involving a reciprocal Runx2-Adam17 negative feedback loop to regulate progression through osteoblast differentiation. Our results suggest that RUNX2 may control paracrine signaling through regulation of ectodomain shedding at the cell surface of osteoblasts by directly suppressing Adam17 expression.

KCTD5 and Ubiquitin Proteasome Signaling Are Required for Helicobacter pylori Adherence.

In order to establish infection, bacterial pathogens modulate host cellular processes by using virulence factors, which are delivered from the bacteria to the host cell leading to cellular reprogramming. In this context, several pathogens regulate the ubiquitin proteasome system in order to regulate the cellular effectors required for their successful colonization and persistance. In this study, we investigated how Helicobacter pylori affect the ubiquitination of the host proteins to achieve the adherence to the cells, using AGS gastric epithelial cells cultured with H. pylori strains, H. pylori 26695 and two isogenic mutants H. pylori cag::cat and vacA::apha3, to characterize the ability of H. pylori to reprogram the ubiquitin proteasome systems. The infection assays suggest that the ubiquitination of the total proteins does not change when cells were co-culture with H. pylori. We also found that the proteasome activity is necessary for H. pylori adhesion to AGS cells and the adherence increases when the level of KCTD5, an adaptor of Cullin-3, decrease. Moreover, we found that KCTD5 is ubiquitinated and degraded by the proteasome system and that CagA and VacA played no role on reducing KCTD5 levels. Furthermore, H. pylori impaired KCTD5 ubiquitination and did not increase global proteasome function. These results suggest that H. pylori affect the ubiquitin-proteasome system (UPS) to facilitate the adhesion of this microorganism to establish stable colonization in the gastric epithelium and improve our understanding of how H. pylori hijack host systems to establish the adherence.

Wnt/ß-Catenin Signaling Activates Expression of the Bone-Related Transcription Factor RUNX2 in Select Human Osteosarcoma Cell Types.

Osteosarcoma is the most common malignant bone tumor in children and adolescents. Metastasis and poor responsiveness to chemotherapy in osteosarcoma correlates with over-expression of the runt-related transcription factor RUNX2, which normally plays a key role in osteogenic lineage commitment, osteoblast differentiation, and bone formation. Furthermore, WNT/ß-catenin signaling is over-activated in osteosarcoma and promotes tumor progression. Importantly, the WNT/ß-catenin pathway normally activates RUNX2 gene expression during osteogenic lineage commitment. Therefore, we examined whether the WNT/ß-catenin pathway controls the tumor-related elevation of RUNX2 expression in osteosarcoma. We analyzed protein levels and nuclear localization of ß-catenin and RUNX2 in a panel of human osteosarcoma cell lines (SAOS, MG63, U2OS, HOS, G292, and 143B). In all six cell lines, ß-catenin and RUNX2 are expressed to different degrees and localized in the nucleus and/or cytoplasm. SAOS cells have the highest levels of RUNX2 protein that is localized in the nucleus, while MG63 cells have the lowest RUNX2 levels which is mostly localized in the cytoplasm. Levels of ß-catenin and RUNX2 protein are enhanced in HOS, G292, and 143B cells after treatment with the GSK3ß inhibitor SB216763. Furthermore, small interfering RNA (siRNA)-mediated depletion of ß-catenin inhibits RUNX2 expression in G292 cells. Thus, WNT/ß-catenin activation is required for RUNX2 expression in at least some osteosarcoma cell types, where RUNX2 is known to promote expression of metastasis related genes. J. Cell. Biochem. 118: 3662-3674, 2017. © 2017 Wiley Periodicals, Inc.

Mitotic Inheritance of mRNA Facilitates Translational Activation of the Osteogenic-Lineage Commitment Factor Runx2 in Progeny of Osteoblastic Cells.

Epigenetic mechanisms mediate the acquisition of specialized cellular phenotypes during tissue development, maintenance and repair. When phenotype-committed cells transit through mitosis, chromosomal condensation counteracts epigenetic activation of gene expression. Subsequent post-mitotic re-activation of transcription depends on epigenetic DNA and histone modifications, as well as other architecturally bound proteins that "bookmark" the genome. Osteogenic lineage commitment, differentiation and progenitor proliferation require the bone-related runt-related transcription factor Runx2. Here, we characterized a non-genomic mRNA mediated mechanism by which osteoblast precursors retain their phenotype during self-renewal. We show that osteoblasts produce maximal levels of Runx2 mRNA, but not protein, prior to mitotic cell division. Runx2 mRNA partitions symmetrically between daughter cells in a non-chromosomal tubulin-containing compartment. Subsequently, transcription-independent de novo synthesis of Runx2 protein in early G1 phase results in increased functional interactions of Runx2 with a representative osteoblast-specific target gene (osteocalcin/BGLAP2) in chromatin. Somatic transmission of Runx2 mRNAs in osteoblasts and osteosarcoma cells represents a versatile mechanism for translational rather than transcriptional induction of this principal gene regulator to maintain osteoblast phenotype identity after mitosis.

The cancer-related transcription factor Runx2 modulates cell proliferation in human osteosarcoma cell lines.

Runx2 regulates osteogenic differentiation and bone formation, but also suppresses pre-osteoblast proliferation by affecting cell cycle progression in the G(1) phase. The growth suppressive potential of Runx2 is normally inactivated in part by protein destabilization, which permits cell cycle progression beyond the G(1)/S phase transition, and Runx2 is again up-regulated after mitosis. Runx2 expression also correlates with metastasis and poor chemotherapy response in osteosarcoma. Here we show that six human osteosarcoma cell lines (SaOS, MG63, U2OS, HOS, G292, and 143B) have different growth rates, which is consistent with differences in the lengths of the cell cycle. Runx2 protein levels are cell cycle-regulated with respect to the G(1)/S phase transition in U2OS, HOS, G292, and 143B cells. In contrast, Runx2 protein levels are constitutively expressed during the cell cycle in SaOS and MG63 cells. Forced expression of Runx2 suppresses growth in all cell lines indicating that accumulation of Runx2 in excess of its pre-established levels in a given cell type triggers one or more anti-proliferative pathways in osteosarcoma cells. Thus, regulatory mechanisms controlling Runx2 expression in osteosarcoma cells must balance Runx2 protein levels to promote its putative oncogenic functions, while avoiding suppression of bone tumor growth.

Desarrollo de los Miembros en los Vertebrados / Limb Development in Vertebrates

Los miembros de los vertebrados son estructuras complejas con tres ejes a considerar, proximal-distal, anterior-posterior y dorsal-ventral. La batería de genes involucrados en la formación de estas estructuras está bastante conservada en la evolución. El esbozo del miembro está compuesto de células mesenquimáticas indiferenciadas que derivan del mesodermo lateral somático cubiertas por ectodermo. La cresta apical ectodérmica es un centro productor de señales para el desarrollo y se ubica en el margen distal del esbozo de miembro. La zona de progreso esta a continuación y permite el crecimiento del miembro. El tipo de estructuras formadas a lo largo del eje proximal distal es especificado por los genes Hox. La proteína Sonic Hedgehog está involucrada en la regulación de la actividad de un segundo centro de señales conocido como zona de actividad polarizante. Los miembros también tienen una polaridad dorso ventral. La proteína WNT7A secretada desde el ectodermo dorsal, instruye a las células mesenquimales circundantes a diferenciarse en estructuras dorsales mientras que Engrailed 1 expresado en el ectodermo ventral, inhibe la expresión de WNT7A en esta zona del esbozo de miembro promoviendo la formación de estructuras ventrales.

Members of vertebrates are complex structures with three lines to consider, proximal-distal, anterior-posterior and dorsal-ventral. The battery of genes involved in the formation of these structures is well conserved in evolution. The outline of the member is composed of undifferentiated mesenchymal cells derived from somatic lateral mesoderm covered by ectoderm. The apical ectodermal ridge is a signal producing center for development and is located in the distal margin of the outline of a member. The area of progress is below and allows the growth of the member. The type of structures formed along the proximal distal axis is specified by Hox genes. Sonic Hedgehog protein is involved in regulating the activity of a second signaling center known as the zone of polarizing activity. Members also have a dorsal ventral polarity. The Wnt protein secreted from the dorsal ectoderm, instructs the surrounding mesenchymal cells to differentiate into dorsal structures whereas Engrailed 1 expressed in the ventral ectoderm, inhibit the expression of WNT7A outline in this membership area promoting the formation of ventral structures.

Involvement of TACE/ADAM17 and ADAM10 in etoposide-induced apoptosis of germ cells in rat spermatogenesis.

Germ cell apoptosis is important to regulate sperm production in the mammalian testis, but the molecular mechanisms underlying apoptosis are still poorly understood. We have recently shown that in vitro, etoposide induces upregulation of TACE/ADAM17 and ADAM10, two membrane-bound extracellular metalloproteases. Here we show that in vivo these enzymes are involved in etoposide-, but not in heat shock-, induced apoptosis in rat spermatogenesis. Germ cell apoptosis induced by DNA damage was associated with an increase in protein levels and cell surface localization of TACE/ADAM17 and ADAM10. On the contrary, apoptosis of germ cells induced by heat stress, another cell death stimulus, did not change levels or localization of these proteins. Pharmacological in vivo inhibition of TACE/ADAM17 and ADAM10 prevents etoposide-induced germ cell apoptosis. Finally, Gleevec (STI571) a pharmacological inhibitor of p73, a master gene controlling apoptosis induced by etoposide, prevented the increase of TACE/ADAM17 levels. Our results strongly suggest that TACE/ADAM17 participates in in vivo apoptosis of male germ cells induced by DNA damage.

CK2 functionally interacts with AKT/PKB to promote the ß-catenin-dependent expression of survivin and enhance cell survival.

ß-Catenin is crucial in the canonical Wnt signaling pathway. This pathway is up-regulated by CK2 which is associated with an enhanced expression of the antiapoptotic protein survivin, although the underlying molecular mechanism is unknown. AKT/PKB kinase phosphorylates and promotes ß-catenin transcriptional activity, whereas CK2 hyperactivates AKT by phosphorylation at Ser129; however, the role of this phosphorylation on ß-catenin transcriptional activity and cell survival is unclear. We studied in HEK-293T cells, the effect of CK2-dependent hyperactivation of AKT on cell viability, as well as analyzed ß-catenin subcellular localization and transcriptional activity and survivin expression. CK2α overexpression led to an augmented ß-catenin-dependent transcription and protein levels of survivin, and consequently an enhanced resistance to apoptosis. However, CK2α-enhancing effects were reversed when an AKT mutant deficient in Ser129 phosphorylation by CK2 was co-expressed. Therefore, our results strongly suggest that CK2α-specific enhancement of ß-catenin transcriptional activity as well as cell survival may depend on AKT hyperactivation by CK2.

Phosphorylation of AKT/PKB by CK2 is necessary for the AKT-dependent up-regulation of ß-catenin transcriptional activity.

ß-Catenin is a key protein in the canonical Wnt signaling pathway and in many cancers alterations in transcriptional activity of its components are observed. This pathway is up-regulated by the protein kinase CK2, but the underlying mechanism of this change is unknown. It has been demonstrated that CK2 hyperactivates AKT/PKB by phosphorylation at Ser129, and AKT phosphorylates ß-catenin at Ser552, which in turn, promotes its nuclear localization and transcriptional activity. However, the consequences of CK2-dependent hyperactivation of AKT on ß-catenin activity and cell viability have not been evaluated. We assessed this regulatory process by manipulating the activity of CK2 and AKT through overexpression of wild-type, constitutively active and dominant negative forms of these proteins as well as analyzing ß-catenin-dependent transcriptional activity, survivin expression and viability in HEK-293T cells. We observed that CK2α overexpression up-regulated the ß-catenin transcriptional activity, which correlated to an increased nuclear localization of ß-catenin as well as survivin expression. Importantly, these effects were strongly reversed when an AKT-S129A mutant was co-expressed in the same cells, followed by a significant decrease in cell viability but no changes in ß-catenin stability. Taken together, the data suggest that the CK2α-dependent up-regulation of ß-catenin activity requires phosphorylation of AKT in human embryonic kidney cells.

TACE/ADAM17 is involved in germ cell apoptosis during rat spermatogenesis.

The pathways leading to male germ cell apoptosis in vivo are poorly understood, but are highly relevant for the comprehension of sperm production regulation by the testis. In this work, we show the evidence of a mechanism where germ cell apoptosis is induced through the inactivation and shedding of the extracellular domain of KIT (c-kit) by the protease TACE/a disintegrin and metalloprotease 17 (ADAM17) during the first wave of spermatogenesis in the rat. We show that germ cells undergoing apoptosis lacked the extracellular domain of the KIT receptor. TACE/ADAM17, a membrane-bound metalloprotease, was highly expressed in germ cells undergoing apoptosis as well. On the contrary, cell surface presence of ADAM10, a closely related metalloprotease isoform, was not associated with apoptotic germ cells. Pharmacological inhibition of TACE/ADAM17, but not ADAM10, significantly prevented germ cell apoptosis in the male pubertal rat. Induction of TACE/ADAM17 by the phorbol-ester phorbol 12-myristate 13-acetate (PMA) induced germ cell apoptosis, which was prevented when an inhibitor of TACE/ADAM17 was present in the assay. Ex-vivo rat testis culture showed that PMA induced the cleavage of the KIT extracellular domain. Isolation of apoptotic germ cells showed that even though protein levels of TACE/ADAM17 were higher in apoptotic germ cells than in nonapoptotic cells, the contrary was observed for ADAM10. These results suggest that TACE/ADAM17 is one of the elements triggering physiological germ cell apoptosis during the first wave of spermatogenesis.

Impaired cell cycle regulation of the osteoblast-related heterodimeric transcription factor Runx2-Cbfbeta in osteosarcoma cells.

Bone formation and osteoblast differentiation require the functional expression of the Runx2/Cbfbeta heterodimeric transcription factor complex. Runx2 is also a suppressor of proliferation in osteoblasts by attenuating cell cycle progression in G(1). Runx2 levels are modulated during the cell cycle, which are maximal in G(1) and minimal beyond the G(1)/S phase transition (S, G(2), and M phases). It is not known whether Cbfbeta gene expression is cell cycle controlled in preosteoblasts nor how Runx2 or Cbfbeta are regulated during the cell cycle in bone cancer cells. We investigated Runx2 and Cbfbeta gene expression during cell cycle progression in MC3T3-E1 osteoblasts, as well as ROS17/2.8 and SaOS-2 osteosarcoma cells. Runx2 protein levels are reduced as expected in MC3T3-E1 cells arrested in late G(1) (by mimosine) or M phase (by nocodazole), but not in cell cycle arrested osteosarcoma cells. Cbfbeta protein levels are cell cycle independent in both osteoblasts and osteosarcoma cells. In synchronized MC3T3-E1 osteoblasts progressing from late G1 or mitosis, Runx2 levels but not Cbfbeta levels are cell cycle regulated. However, both factors are constitutively elevated throughout the cell cycle in osteosarcoma cells. Proteasome inhibition by MG132 stabilizes Runx2 protein levels in late G(1) and S in MC3T3-E1 cells, but not in ROS17/2.8 and SaOS-2 osteosarcoma cells. Thus, proteasomal degradation of Runx2 is deregulated in osteosarcoma cells. We propose that cell cycle control of Runx2 gene expression is impaired in osteosarcomas and that this deregulation may contribute to the pathogenesis of osteosarcoma.

Differential localization of alpha' and beta subunits of protein kinase CK2 during rat spermatogenesis.

Protein kinase CK2 is a serine/threonine kinase expressed in organisms from yeast to human and is composed of a catalytic subunit (alpha or alpha') and a regulatory subunit (beta) forming a holoenzyme with the possible subunit combinations alpha(2)beta(2), alpha'(2)beta(2), or alphaalpha'beta(2). This kinase has been shown to be involved in embryonic development and gametogenesis. We have studied the expression of the CK2alpha' and CK2beta subunits during the first wave of spermatogenesis and in adult testis in the rat. Western blot analyses have demonstrated that both CK2alpha' and CK2beta are expressed in testes from birth to adulthood. A more detailed study of the protein localization of CK2alpha' and CK2beta by immunohistochemistry suggests that CK2alpha' and CK2beta are localized in the nuclei of Sertoli cells in 5-day-old rats, whereas they appear to have a cytoplasmic localization in older animals. In adult testes, CK2alpha' and CK2beta subunits are present in spermatocytes. Both subunits exhibit a similar expression pattern with the highest level in spermatocytes at stages VIII-XIV. Interestingly, CK2beta is highly expressed in spermatogonia, whereas CK2alpha' is barely detectable. Mature epididymal spermatozoa express CK2alpha' in the acrosome and CK2beta in the flagellum. This new evidence therefore indicates that protein kinase CK2 has a possible role at various stages during mammalian spermatogenesis, a process that involves proliferation, meiosis, apoptosis, and differentiation. CK2 might thus emerge as a new pivotal control enzyme at various levels in mammalian spermatogenesis.

The CK1 gene family: expression patterning in zebrafish development.

Protein kinase CK1 is a ser/thr protein kinase family which has been identified in the cytosol cell fraction, associated with membranes as well as in the nucleus. Several isoforms of this gene family have been described in various organisms: CK1alpha, CK1beta, CK1delta, CK1epsilon and CK1gamma. Over the last decade, several members of this family have been involved in development processes related to wnt and sonic hedgehog signalling pathways. However, there is no detailed temporal information on the CK1 family in embryonic stages, even though orthologous genes have been described in several different vertebrate species. In this study, we describe for the first time the cloning and detailed expression pattern of five CK1 zebrafish genes. Sequence analysis revealed that zebrafish CK1 proteins are highly homologous to other vertebrate orthologues. Zebrafish CK1 genes are expressed throughout development in common and different territories. All the genes studied in development show maternal and zygotic expression with the exception of CK1epsilon. This last gene presents only a zygotic component of expression. In early stages of development CK1 genes are ubiquitously expressed with the exception of CK1epsilon. In later stages the five CK1 genes are expressed in the brain but not in the same way. This observation probably implicates the CK1 family genes in different and also in redundant functions. This is the first time that a detailed comparison of the expression of CK1 family genes is directly assessed in a vertebrate system throughout development.

Chemical dissection of the APC Repeat 3 multistep phosphorylation by the concerted action of protein kinases CK1 and GSK3.

A crucial event in machinery controlled by Wnt signaling is the association of beta-catenin with the adenomatous polyposis coli (APC) protein, which is essential for the degradation of beta-catenin and requires the multiple phosphorylation of APC at six serines (1501, 1503, 1504, 1505, 1507, and 1510) within its repeat three (R3) region. Such a phosphorylation is believed to occur by the concerted action of two protein kinases, CK1 and GSK3, but its mechanistic aspects are a matter of conjecture. Here, by combining the usage of variably phosphorylated peptides reproducing the APC R3 region and Edman degradation assisted localization of residues phosphorylated by individual kinases, we show that the process is initiated by CK1, able to phosphorylate S1510 and S1505, both specified by non-canonical determinants. Phosphorylation of S1505 primes subsequent phosphorylation of S1501 by GSK3. In turn, phospho-S1501 triggers the hierarchical phosphorylation of S1504 and S1507 by CK1. Once phosphorylated, S1507 primes the phosphorylation of both S1510 and S1503 by CK1 and GSK3, respectively, thus completing all six phosphorylation steps. Our data also rule out the intervention of CK2 despite the presence of a potential CK2 phosphoacceptor site, S1510LDE, in the R3 repeat. S1510 is entirely unaffected by CK2, while it is readily phosphorylated even in the unprimed peptide by CK1delta but not by CK1gamma. This discloses a novel motif significantly different from non-canonical sequences phosphorylated by CK1 in other proteins, which appears to be specifically recognized by the delta isoform of CK1.

Cloning of hif-1alpha and hif-2alpha and mRNA expression pattern during development in zebrafish.

Hypoxia-inducible factors (HIFs) regulate gene expression in response to hypoxia and in vertebrates they are known to participate in several developmental processes, including angiogenesis, vasculogenesis, heart and central nervous system development. Over the last decade, major progress in unraveling the molecular mechanisms that mediate regulation of HIF proteins by oxygen tension has been reported, but our knowledge on their developmental regulation during embryogenesis in model organisms is limited. Expression of hif-1alpha and hif-2alpha genes has been characterized during normal mouse development and they were found to be expressed from stages E7.5, later in E9.5 and E15.5 in several different tissues such as the brain, heart and blood vessels. However, there is no detailed temporal information on their expression at other embryonic stages, even though orthologous genes have been described in several different vertebrate species. In this study, we describe the cloning and detailed expression pattern of zebrafish hif-1alpha and hif-2alpha genes. Sequence analysis revealed that zebrafish Hif proteins are highly homologous to other vertebrate orthologues. Zebrafish hif-1alpha and hif-2alpha are both expressed throughout development in discrete territories in a dynamic pattern. Interestingly, in the notochord the expression of hif-1alpha is switched off, while hif-2alpha transcription is turned on, signifying that the two genes might have partially overlapping, although non-redundant functions in development. This is the first time that a detailed comparison of the expression of hif-1alpha and hif-2alpha is directly assessed in a vertebrate model system throughout development.

The CK1 gene family: expression patterning in zebrafish development

Protein kinase CK1 is a ser/thr protein kinase family which has been identified in the cytosol cell fraction, associated with membranes as well as in the nucleus. Several isoforms of this gene family have been described in various organisms: CK1 , CK1á, CK1δ, CK1å and CK1γ. Over the last decade, several members of this family have been involved in development processes related to wnt and sonic hedgehog signalling pathways. However, there is no detailed temporal information on the CK1 family in embryonic stages, even though orthologous genes have been described in several different vertebrate species. In this study, we describe for the first time the cloning and detailed expression pattern of five CK1 zebrafish genes. Sequence analysis revealed that zebrafish CK1 proteins are highly homologous to other vertebrate orthologues. Zebrafish CK1 genes are expressed throughout development in common and different territories. All the genes studied in development show maternal and zygotic expression with the exception of CK1å. This last gene presents only a zygotic component of expression. In early stages of development CK1 genes are ubiquitously expressed with the exception of CK1å. In later stages the five CK1 genes are expressed in the brain but not in the same way. This observation probably implicates the CK1 family genes in different and also in redundant functions. This is the first time that a detailed comparison of the expression of CK1 family genes is directly assessed in a vertebrate system throughout development.

A noncanonical sequence phosphorylated by casein kinase 1 in beta-catenin may play a role in casein kinase 1 targeting of important signaling proteins.

Protein kinase casein kinase 1 (CK1) phosphorylates Ser-45 of beta-catenin, "priming" the subsequent phosphorylation by glycogen synthase-3 of residues 41, 37, and 33. This concerted phosphorylation of beta-catenin signals its degradation and prevents its function in triggering cell division. The sequence around Ser-45 does not conform to the canonical consensus for CK1 substrates, which prescribes either phosphoamino acids or acidic residues in position n-3 from the target serine. However, the beta-catenin sequence downstream from Ser-45 is very similar to a sequence recognized by CK1 in nuclear factor for activated T cells 4. The common features include an SLS motif followed two to five residues downstream by a cluster of acidic residues. Synthetic peptides reproducing residues 38-65 of beta-catenin were assayed with purified rat liver CK1 or recombinant CK1 alpha and CK1 alpha L from zebrafish. The results demonstrate that SLS and acidic cluster motifs are crucial for CK1 recognition. Pro-44 and Pro-52 are also important for efficient phosphorylation. Similar results were obtained with the different isoforms of CK1. Phosphorylation of mutants of full-length recombinant beta-catenin from zebrafish confirmed the importance of the SLS and acidic cluster motifs. A search for proteins with similar motifs yielded, among other proteins, adenomatous polyposis coli, previously found to be phosphorylated by CK1. There is a strong correlation of beta-catenin mutations found in thyroid tumors with the motifs recognized by CK1 in this protein.

Biochemical and cellular characteristics of the four splice variants of protein kinase CK1alpha from zebrafish (Danio rerio).

Protein kinase CK1 (previously known as casein kinase I) conforms to a subgroup of the great protein kinase family found in eukaryotic organisms. The CK1 subgroup of vertebrates contains seven members known as alpha, beta, gamma1, gamma2, gamma3, delta, and epsilon. The CK1alpha gene can generate four variants (CK1alpha, CK1alphaS, CK1alphaL, and CK1alphaLS) through alternate splicing, characterized by the presence or absence of two additional coding sequences. Exon "L" encodes a 28-amino acid stretch that is inserted after lysine 152, in the center of the catalytic domain. The "S" insert encodes 12 amino acid residues and is located close to the carboxyl terminus of the protein. This work reports some biochemical and cellular properties of the four CK1alpha variants found to be expressed in zebrafish (Danio rerio). The results obtained indicate that the presence of the "L" insert affects several biochemical properties of CK1alpha: (a) it increases the apparent Km for ATP twofold, from approximately 30 to approximately 60 microM; (b) it decreases the sensitivity to the CKI-7 inhibitor, raising the I50 values from 113 to approximately 230 microM; (c) it greatly decreases the heat stability of the enzyme at 40 degrees C. In addition, the insertion of the "L" fragment exerts very important effects on some cellular properties of the enzyme. CK1alphaL concentrates in the cell nucleus, excluding nucleoli, while the CK1alpha variant is predominantly cytoplasmic, although some presence is observed in the nucleus. This finding supports the thesis that the basic-rich region found in the "L" insert acts as a nuclear localization signal. The "L" insert-containing variant was also found to be more rapidly degraded (half-life of 100 min) than the CK1alpha variant (half-life of 400 min) in transfected Cos-7 cells.

Insertional mutagenesis in zebrafish rapidly identifies genes essential for early vertebrate development.

To rapidly identify genes required for early vertebrate development, we are carrying out a large-scale, insertional mutagenesis screen in zebrafish, using mouse retroviral vectors as the mutagen. We will obtain mutations in 450 to 500 different genes--roughly 20% of the genes that can be mutated to produce a visible embryonic phenotype in this species--and will clone the majority of the mutated alleles. So far, we have isolated more than 500 insertional mutants. Here we describe the first 75 insertional mutants for which the disrupted genes have been identified. In agreement with chemical mutagenesis screens, approximately one-third of the mutants have developmental defects that affect primarily one or a small number of organs, body shape or swimming behavior; the rest of the mutants show more widespread or pleiotropic abnormalities. Many of the genes we identified have not been previously assigned a biological role in vivo. Roughly 20% of the mutants result from lesions in genes for which the biochemical and cellular function of the proteins they encode cannot be deduced with confidence, if at all, from their predicted amino-acid sequences. All of the genes have either orthologs or clearly related genes in human. These results provide an unbiased view of the genetic construction kit for a vertebrate embryo, reveal the diversity of genes required for vertebrate development and suggest that hundreds of genes of unknown biochemical function essential for vertebrate development have yet to be identified.