Expression of the A-raf proto-oncogene in the normal adult and embryonic mouse.

We have determined the expression pattern of the A-raf proto-oncogene in the embryonic and adult mouse. Western blot analysis of protein lysates from tissues of adult mice show that p69A-raf is ubiquitously expressed, but that levels of expression vary among different tissues. To determine the cell-specific expression pattern of A-raf, we generated transgenic mice expressing the beta-galactosidase reporter gene from the A-raf promoter. We show that A-raf expression is highly specific within a given tissue, and we identify cell types expressing this gene in the adult testis, epididymis, vas deferens, seminal vesicle, ovary, oviduct, bladder, kidney, intestine, heart, spleen, thymus, and cerebellum. In the embryo, ubiquitous expression of the reporter gene is observed, but the highest levels of expression are specifically detected in the embryonic heart at stages 9.5-11.5 days post-coitum.     

Embryonic Expression of Vasoactive Intestinal Peptide (VIP) and VIP Receptor Genes

Abstract: Vasoactive intestinal peptide (VIP) exhibits pronounced effects on the growth rate of cultured mouse embryonic day (E) 9.5 embryos and acts in tissue culture as a potent glial mitogen and neuron survival factor. However, previous studies using immunohistochemistry or in situ hybridization in the rat have not revealed the presence and location of VIP or VIP mRNA in the early developing embryo CNS. Using a sensitive in situ hybridization assay with a ³³P-labeled riboprobe, we show here that the VIP gene is expressed at least as early as E11 in the mouse hindbrain. Northern blot analysis on RNA from brain dissected from mouse embryos beginning at E14 confirmed that a correct-size mRNA for VIP was present by E14 and at later time points. Expression of the VIP2 receptor gene was also detected by northern analysis in E14 mouse brains. These studies support the hypothesis that VIP produced by the embryo exerts important effects on embryonic nervous system development.     

The molecular scaffold KSR1 regulates the proliferative and oncogenic potential of cells

The specificity of signaling through mitogen-activated protein kinase pathways has been attributed to both the control of intensity and duration of signaling and the actions of protein scaffolds. Here we demonstrate that the molecular scaffold KSR1 regulates the intensity and duration of ERK activation to modulate a cell's proliferative and oncogenic potential. Deletion of KSR1 eliminates the prolonged phase of ERK activation induced by platelet-derived growth factor and blocks Ras(V12)-induced transformation. The introduction of KSR1 into KSR1(-/-) mouse embryo fibroblasts causes a concentration-dependent increase in signaling and transformation, to a maximum at 14 times the wild-type KSR1 expression levels, but inhibits these responses at higher expression levels. An increase in KSR1 expression to levels that are optimal for signaling leads to a threefold increase in proliferative capacity and is coincident with the level of KSR1 expression that maximally associates with all members of the Raf/MEK/ERK cascade. These data reveal that cells contain a reserve proliferative capacity that is accessible by the optimal expression of a noncatalytic signaling component and that altering the expression level of a molecular scaffold can modulate the actions of growth factors and oncogenes.     

Caspase-dependent cleavage disrupts the ERK cascade scaffolding function of KSR1

Kinase suppressor of Ras 1 (KSR1) is a protein scaffold that facilitates ERK cascade activation at the plasma membrane, a critical step in the signal transduction process that allows cells to respond to survival, proliferative, and differentiative cues. Here, we report that KSR1 undergoes caspase-dependent cleavage in apoptotic cells and that cleavage destroys the scaffolding function of the full-length KSR1 protein and generates a stable C-terminal fragment that can inhibit ERK activation. KSR1 is cleaved in response to multiple apoptotic stimuli and occurs in vivo during the involution of mouse mammary tissues, a morphogenic process requiring cellular apoptosis. In addition, we find that in comparison with KSR1(-/-) mouse embryonic fibroblasts expressing wild type KSR1 (WT-KSR1), cells expressing a cleavage-resistant KSR1 protein (DEVA-KSR1) exhibit reduced apoptotic signaling in response to tumor necrosis factor-alpha/cycloheximide treatment. The effect of DEVA-KSR1 expression was found to correlate with increased levels of active phosphoERK and could be significantly reversed by treating cells with the MEK inhibitor U0126. In contrast, reduced phosphoERK levels and enhanced apoptotic signaling were observed in cells constitutively expressing the C-terminal KSR1 fragment (CTF-KSR1). Moreover, we find that cleavage of WT-KSR1 correlates with a dramatic reduction in active phosphoERK levels. These findings identify KSR1 as a caspase target and suggest that cleavage of the KSR1 scaffold represents another mechanism whereby caspases down-regulate ERK survival signaling to promote cellular apoptosis.     

Effects of prenyl pyrophosphates on the binding of S-Ras proteins with KSR

BALB/3T3 cells were transformed by transfection with DNA encoding the mutated ras(Q(61)K) from shrimp Penaeus japonicus (Huang et al., 2001. J. Exp. Zool. 289:441-448). On a Western blot, the kinase suppressor of Ras (KSR) in the membrane fraction was expressed at slightly reduced level as compared to that of the untransformed cells. To understand this in more detail, the interaction of the bacterially expressed shrimp Ras (S-Ras) with KSR was investigated using KSR purified from mice brains. SDS-polyacrylamide gel electrophoresis and Western blot analysis revealed that the monomers of the purified KSR have a relative molecular mass of 60,000. Purified KSR was found to bind with digoxigenylated S-ras-encoding fusion protein (Dig-S-Ras) with high affinity in the absence of ATP, and the binding activity of KSR was sustained upon phosphorylation of Dig-S-Ras with mitogen-activated protein kinase (MAPK). The association of purified KSR with S-Ras was confirmed. Differences between the effects of farnesyl pyrophosphate and geranylgeranyl pyrophosphate on the binding of S-Ras with the purified KSR were assessed. Densitometer analysis revealed that at nanogram concentration, farnesyl pyrophosphate inhibited the binding of S-Ras with KSR competently, but geranylgeranyl pyrophosphate did not. The present study provides the evidence that decrease of the concentration of farnesyl pyrophosphate to sub-microgram levels lower the affinity of Ras proteins with KSR in the signaling pathway.     

Kinase Suppressor of Ras 2 (KSR2) Regulates Tumor Cell Transformation via AMPK

Kinase suppressor of Ras 1 (KSR1) and KSR2 are scaffolds that promote extracellular signal-regulated kinase (ERK) signaling but have dramatically different physiological functions. KSR2(-/-) mice show marked deficits in energy expenditure that cause obesity. In contrast, KSR1 disruption has inconsequential effects on development but dramatically suppresses tumor formation by activated Ras. We examined the role of KSR2 in the generation and maintenance of the transformed phenotype in KSR1(-/-) mouse embryo fibroblasts (MEFs) expressing activated Ras(V12) and in tumor cell lines MIN6 and NG108-15. KSR2 rescued ERK activation and accelerated proliferation in KSR1(-/-) MEFs. KSR2 expression alone induced anchorage-independent growth and synergized with the transforming effects of Ras(V12). Similarly, RNA interference (RNAi) of KSR2 in MIN6 and NG108-15 cells inhibited proliferation and colony formation, with concomitant defects in AMP-activated protein kinase (AMPK) signaling, nutrient metabolism, and metabolic capacity. While constitutive activation of AMPK was sufficient to complement the loss of KSR2 in metabolic signaling and anchorage-independent growth, KSR2 RNAi, MEK inhibition, and expression of a KSR2 mutant unable to interact with ERK demonstrated that mitogen-activated protein (MAP) kinase signaling is dispensable for the transformed phenotype of these cells. These data show that KSR2 is essential to tumor cell energy homeostasis and critical to the integration of mitogenic and metabolic signaling pathways.     

Identification of Constitutive and Ras-Inducible Phosphorylation Sites of KSR: Implications for 14-3-3 Binding, Mitogen-Activated Protein Kinase Binding, and KSR Overexpression

Genetic and biochemical studies have identified kinase suppressor of Ras (KSR) to be a conserved component of Ras-dependent signaling pathways. To better understand the role of KSR in signal transduction, we have initiated studies investigating the effect of phosphorylation and protein interactions on KSR function. Here, we report the identification of five in vivo phosphorylation sites of KSR. In serum-starved cells, KSR contains two constitutive sites of phosphorylation (Ser297 and Ser392), which mediate the binding of KSR to the 14-3-3 family of proteins. In the presence of activated Ras, KSR contains three additional sites of phosphorylation (Thr260, Thr274, and Ser443), all of which match the consensus motif (Px[S/T]P) for phosphorylation by mitogen-activated protein kinase (MAPK). Further, we find that treatment of cells with the MEK inhibitor PD98059 blocks phosphorylation of the Ras-inducible sites and that activated MAPK associates with KSR in a Ras-dependent manner. Together, these findings indicate that KSR is an in vivo substrate of MAPK. Mutation of the identified phosphorylation sites did not alter the ability of KSR to facilitate Ras signaling in Xenopus oocytes, suggesting that phosphorylation at these sites may serve other functional roles, such as regulating catalytic activity. Interestingly, during the course of this study, we found that the biological effect of KSR varied dramatically with the level of KSR protein expressed. In Xenopus oocytes, KSR functioned as a positive regulator of Ras signaling when expressed at low levels, whereas at high levels of expression, KSR blocked Ras-dependent signal transduction. Likewise, overexpression of Drosophila KSR blocked R7 photoreceptor formation in the Drosophila eye. Therefore, the biological function of KSR as a positive effector of Ras-dependent signaling appears to be dependent on maintaining KSR protein expression at low or near-physiological levels.     

Caveolin-1 Is Required for Kinase Suppressor of Ras 1 (KSR1)-Mediated Extracellular Signal-Regulated Kinase 1/2 Activation,H-RasV12-Induced Senescence,and Transformation

The molecular scaffold kinase suppressor of Ras 1 (KSR1) regulates the activation of the Raf/MEK/extracellular signal-regulated kinase (ERK) signal transduction pathway. KSR1 disruption in mouse embryo fibroblasts (MEFs) abrogates growth factor-induced ERK activation, H-Ras^V12-induced replicative senescence, and H-Ras^V12-induced transformation. Caveolin-1 has been primarily described as a major component of the coating structure of caveolae, which can serve as a lipid binding adaptor protein and coordinates the assembly of Ras, Raf, MEK, and ERK. In this study, we show that KSR1 interacts with caveolin-1 and is responsible for MEK and ERK redistribution to caveolin-1-rich fractions. The interaction between KSR1 and caveolin-1 is essential for optimal activation of ERK as a KSR1 mutant unable to interact with caveolin-1 does not efficiently mediate growth factor-induced ERK activation at the early stages of pathway activation. Furthermore, abolishing the KSR1–caveolin-1 interaction increases growth factor demands to promote H-Ras^V12-induced proliferation and has adverse effects on H-Ras^V12-induced cellular senescence and transformation. These data show that caveolin-1 is necessary for optimal KSR1-dependent ERK activation by growth factors and oncogenic Ras.     

Expression of low density lipoprotein receptor-related protein 4 (Lrp4) gene in the mouse germ cells

The low density lipoprotein receptor-related protein 4 gene (Lrp4) was identified by subtractive screening of cDNAs of the migratory primordial germ cells (PGCs) of E8.5-9.5 embryo and E3.5 blastocysts. Lrp4 is expressed in PGCs in the hindgut and the dorsal mesentery of E9.5 embryos, and in germ cells in the genital ridges of male and female E10.5-13.5 embryos. Lrp4 is also expressed in spermatogonia of the neonatal and adult testes and in the immature oocytes and follicular cells of the adult ovary. The absence of Lrp4 expression in the blastocyst, embryonic stem cells and embryonic germ cells suggests the Lrp4 is a molecular marker that distinguishes the germ cells from embryo-derived pluripotent stem cells.     

小鼠BTB/锌指结构新基因Bsg6的克隆及表达谱分析

Bsg6 (brain specific gene 6) 是用消减差异筛选的方法克隆的小鼠头部特异表达 新基因. Bsg6基因cDNA长3 871 bp,编码一个670个氨基酸残基的蛋白,GenBank 登录号AY635051,位于小鼠第4号染色体,由2个外显子构成. Bsg6蛋白含有一个N端BTB（Broad complex, Tramtrack, and Bric a brac）结构域和两个C端C２Ｈ２型锌指结构域. 小鼠Bsg6蛋白与其在人类和鸡中同源蛋白的同源性分别为86.2%和79.1%. Bsg6在小鼠胚胎中的表达具有一定动态性,在E8.5的小鼠胚胎中,Bsg6主要在前脑和神经管表达. 在E9.5的小鼠胚胎中,Bsg6的表达明显增强并主要集中在前脑的端脑部. Bsg6在E10.5小鼠胚胎端脑的表达出现了下降,但是在中脑和后脑的表达增加,此外,Bsg6 mRNA的表达还出现在肢芽和尾部. 在HH10期的鸡胚中,Bsg6主要在头部和神经管前端表达. Northern杂交结果显示,Bsg6在很多小鼠成体组织中没有表达,但是在破骨细胞瘤中高表达. Bsg6的表达谱提示,Bsg6可能是在器官形成期对脑的发育起到重要作用的转录因子,而且其表达受到严格的调控,此外Bsg6还能与肿瘤的发生有关.     

一个新的人类乙酰转移酶基因hNATL的克隆与表达谱研究

从小鼠cDNA序列入手,依据同源分析的电子克隆方法得到一个包含N末端乙酰转移酶结构域的人类新基因hNATL(Human NAT Like)。hNATL的cDNA长1803bp,编码区621bp,Genbank 登陆号AY632082。hNATL编码的蛋白长206个氨基酸残基,包含有N乙酰转移酶结构域。hNATL 基因定位于人染色体17q25．2区,包括6个外显子和5个内含子。基因表达汇编分析结果显示, hNATL在人脑和生殖腺中高表达。Northern杂交显示,在成年小鼠中hNATL在心脏,脾脏和卵巢中出现表达。整体原位杂交的结果显示,hNATL特异表达于E7．5和E8．5的小鼠胚胎脑部和HH10期鸡胚胎的头部。上述结果提示,hNATL对胚胎期脑的发育和成体中重要器官行使正常功能发挥十分重要的作用。     

Characterization of mouse orthologue of ELOVL4: genomic organization and spatial and temporal expression

Mutations in ELOVL4 are associated with dominant macular degeneration (adMD/STGD3). This gene is highly expressed in the retina and is conserved through evolution. Here we report the genomic organization of the mouse orthologue of ELOVL4 and its temporal and spatial expression. A significant amount of ELOVL4 mRNA expression is detected in the adult retina, brain, skin, testis, and lens. During development, expression is first noted at embryonic day 7 (E7). A significant level of the mRNA is observed both in brain and in eyes at postnatal day 1 (P1), after which levels decrease in the brain and increase in the retina until they stabilize at P30. ELOVL4 protein is evident in the ocular tissues by E10.5 and becomes restricted predominantly to the photoreceptor layer in the mature retina. These observations suggest that ELOVL4 may play an important role in embryonic development and in maintaining normal physiology of retina and brain at later stages of development.     

Mouse very long-chain Acyl-CoA synthetase 3/fatty acid transport protein 3 catalyzes fatty acid activation but not fatty acid transport in MA-10 cells

The family of proteins that includes very long-chain acyl-CoA synthetases (ACSVL) consists of six members. These enzymes have also been designated fatty acid transport proteins. We cloned full-length mouse Acsvl3 cDNA and characterized its protein product ACSVL3/fatty acid transport protein 3. The predicted amino acid sequence contains two highly conserved motifs characteristic of acyl-CoA synthetases. Northern blot analysis revealed that the mouse Acsvl3 mRNA is highly expressed in adrenal gland, testis, and ovary, with lower expression in the brain of adult mice. A developmental Northern blot revealed that Acsvl3 mRNA levels were significantly higher in embryonic mouse brain (embryonic days 12-14) than in newborn or adult mice, suggesting a possible role in nervous system development. Immunohistochemistry revealed high ACSVL3 expression in adrenal cortical cells, spermatocytes and interstitial cells of the testis, theca cells of the ovary, cerebral cortical neurons, and cerebellar Purkinje cells. Endogenous ACSVL3 was found primarily in mitochondria of MA-10 and Neuro2a cells by both Western blot analysis of subcellular fractions and immunofluorescence analysis. In MA-10 cells, loss-of-function studies using RNA interference confirmed that endogenous ACSVL3 is an acyl-CoA synthetase capable of activating both long-chain (C16:0) and very long-chain (C24:0) fatty acids. However, despite decreased acyl-CoA synthetase activity, initial rates of fatty acid uptake were unaffected by knockdown of Acsvl3 expression in MA-10 cells. These studies cast doubt on the designation of ACSVL3 as a fatty acid transport protein.     

Expression profile of mouse BWF1, a protein with a BEACH domain, WD40 domain and FYVE domain

We isolated a mouse cDNA encoding a protein that contains a BEACH domain, 5 WD40 repeats and a FYVE domain, which we designated as BWF1. The mRNA is approximately 10 kb in size and encodes a protein consisting of 3508 amino acids with a predicted molecular weight of 385 kDa. BWF1 has 45% homology with the Drosophila protein, blue cheese (BCHS). The BWF1 gene consists of 67 exons, which span 270 kb of genomic sequence, and has been mapped to mouse chromosome 5. Northern blot analysis revealed that it was strongly expressed in the liver, moderately in the kidney and testis, and weakly in the brain of adult mice. During the development of the mouse brain, BWF1 mRNA was abundant on embryonic day (E) 14-16; after birth, the level of BWF1 mRNA expression decreased markedly to reach the adult level at postnatal day 3. In situ hybridization analysis revealed that the expressed BWF1 mRNA was restricted to the marginal region both in E14 and E16 embryonic brain, but became diffuse after birth. Confocal microscopy studies of the epitope-tagged BWF1 protein showed that the protein was a cytoplasmic one.     

Expression of the aspartate/glutamate mitochondrial carriers aralar1 and citrin during development and in adult rat tissues.

Aralar1 and citrin are members of the subfamily of calcium-binding mitochondrial carriers and correspond to two isoforms of the mitochondrial aspartate/glutamate carrier (AGC). These proteins are activated by Ca2+ acting on the external side of the inner mitochondrial membrane. Although it is known that aralar1 is expressed mainly in skeletal muscle, heart and brain, whereas citrin is present in liver, kidney and heart, the precise tissue distribution of the two proteins in embryonic and adult tissues is largely unknown. We investigated the pattern of expression of aralar1 and citrin in murine embryonic and adult tissues at the mRNA and protein levels. In situ hybridization analysis indicates that both isoforms are expressed strongly in the branchial arches, dermomyotome, limb and tail buds at early embryonic stages. However, citrin was more abundant in the ectodermal components of these structures whereas aralarl had a predominantly mesenchymal localization. The strong expression of citrin in the liver was acquired postnatally, whereas the characteristic expression of aralar1 in skeletal muscle was detected at E18 and that in the heart began early in development (E11) and was preferentially localized to auricular myocardium in late embryonic stages. Aralar1 was also expressed in bone marrow, T-lymphocytes and macrophages, including Kupffer cells in the liver, indicating that this is the major AGC isoform present in the hematopoietic system. Both aralar1 and citrin were expressed in fetal gut and adult stomach, ovary, testis, and pancreas, but only aralar1 is enriched in lung and insulin-secreting beta cells. These results show that aralar1 is expressed in many more tissues than originally believed and is absent from hepatocytes, where citrin is the only AGC isoform present. This explains why citrin deficiency in humans (type II citrullinemia) only affects the liver and suggests that aralar1 may compensate for the lack of citrin in other tissues.