2', 3'-Dideoxycytidine represses thymidine kinases 1 and 2 expression in T-lymphoid cells

In vitro culture of H9 human lymphoid cells in the presence of 5.0 microM dideoxycytidine (ddC), for about 40-45 days, selected cells (H9-ddC cells), which were resistant to the drug and cross-resistant to AZT (zidovudine) and 5-fluoro-2'-deoxyuridine (FdUR). The major mechanism of cross-resistance to AZT and FdUR in these cells was low cellular activity of thymidine kinase (TK). To explore molecular mechanisms of the reduced TK activity in H9-ddC cells, the mRNA expression of TK1 and TK2 and western blot analysis of TK1 protein were performed. RT-PCR analysis revealed that in H9-ddC cells the expression of both TK1 and TK2 mRNA was reduced to 27.1% and 79.4%, respectively. The reduced TK1 gene expression was confirmed by an absence of a detectable TK1 protein band in western blot of H9-ddC cells. These results demonstrate that long-term treatment of H9 cells in the presence of ddC down-regulated TK1 and TK2 gene expression and reduced the expression and activity of TK in the resistant cells.     

Docosahexaenoic acid inhibits cancer cell growth via p27Kip1, CDK2, ERK1/ERK2, and retinoblastoma phosphorylation

Docosahexaenoic acid (DHA), a PUFA of the n-3 family, inhibited the growth of FM3A mouse mammary cancer cells by arresting their progression from the late-G(1) to the S phase of the cell cycle. DHA upregulated p27(Kip1) levels by inhibiting phosphorylation of mitogen-activated protein (MAP) kinases, i.e., ERK1/ERK2. Indeed, inhibition of ERK1/ERK2 phosphorylation by DHA, U0126 [chemical MAPK extracellularly signal-regulated kinase kinase (MEK) inhibitor], and MEK(SA) (cells expressing dominant negative constructs of MEK) resulted in the accumulation of p27(Kip1). MAP kinase (MAPK) inhibition by DHA did not increase p27(Kip1) mRNA levels. Rather, this fatty acid stabilized p27(Kip1) contents and inhibited MAPK-dependent proteasomal degradation of this protein. DHA also diminished cyclin E phosphorylation, cyclin-dependent kinase-2 (CDK2) activity, and phosphorylation of retinoblastoma protein in these cells. Our study shows that DHA arrests cell growth by modulating the phosphorylation of cell cycle-related proteins.     

Extracellular signal-regulated kinase 1/2 mediates survival, but not axon regeneration, of adult injured central nervous system neurons in vivo

Neurotrophins play important roles in the response of adult neurons to injury. The intracellular signaling mechanisms used by neurotrophins to regulate survival and axon growth in the mature CNS in vivo are not well understood. The goal of this study was to define the role of the extracellular signal-regulated kinases 1/2 (Erk1/2) pathway in the survival and axon regeneration of adult rat retinal ganglion cells (RGCs), a prototypical central neuron population. We used recombinant adeno-associated virus (AAV) to selectively transduce RGCs with genes encoding constitutively active or wild-type mitogen-activated protein kinase kinase 1 (MEK1), the upstream activator of Erk1/2. In combination with anterograde and retrograde tracing techniques, we monitored neuronal survival and axon regeneration in vivo. MEK1 gene delivery led to robust and selective transgene expression in multiple RGC compartments including cell bodies, dendrites, axons and targets in the brain. Furthermore, MEK1 activation induced in vivo phosphorylation of Erk1/2 in RGC bodies and axons. Quantitative analysis of cell survival demonstrated that Erk1/2 activation promoted robust RGC neuroprotection after optic nerve injury. In contrast, stimulation of the Erk1/2 pathway was not sufficient to induce RGC axon growth beyond the lesion site. We conclude that the Erk1/2 pathway plays a key role in the survival of axotomized mammalian RGCs in vivo, and that activation of other signaling components is required for axon regeneration in the growth inhibitory CNS environment.     

Origins, lineage-specific expansions, and multiple losses of tyrosine kinases in eukaryotes

Tyrosine kinases are important components of metazoan signaling pathways, and their mutant forms are implicated in various malignancies. Searching the sequences from the genomes of 28 eukaryotes and the GenBank, we found tyrosine kinases not only in metazoans but also in the green algae Chlamydomonas reinhardtii, the potato late blight pathogen Phytophthora infestans, and the protozoan pathogen Entamoeba histolytica, contrary to the current view that tyrosine kinases are animal-specific. Based on a phylogenetic analysis, we divided this gene family into 43 subfamilies and found that at least 19 tyrosine kinases were likely present in the common ancestor of chordates, arthropods, and nematodes. Interestingly, most of the subfamilies have conserved domain organizations among subfamily members but have undergone different degrees of expansion during the evolution of metazoans. In particular, a large number of duplications occurred in the lineage leading to the common ancestor of Tagifugu and mammals after its split from the Ciona lineage about 450 to 550 MYA. The timing of expansion coincides with proposed large-scale duplication event in the chordate lineage. Furthermore, gene losses have occurred in most subfamilies. Interestingly, different subfamilies have similar net gain rates in the chordates studied. However, the tyrosine kinases in mouse and human or in fruit fly and mosquito mostly have a one-to-one relationship between species, indicating that static periods of 90 Myr or longer in tyrosine kinase evolution have followed large expansion events.     

Polymorphisms of CYP1a1, CYP2e1, GSTM1, GSTT1, and TP53 genes in Amerindians

Polymorphisms at the TP53, cytochrome P‐450 (CYP), and glutathione S‐transferase (GST) genes are related to cancer susceptibility and present high diversity in allele frequencies among ethnic groups. This study concerns the CYP2E1, GSTM1, and GSTT1 polymorphisms in seven Amerindian populations (Xavante, Guarani, Aché, Wai Wai, Zoró, Surui, and Gavião). Polymorphic sites at CYP1A1 and TP53 were also studied in the Aché and Guarani tribes and compared with previous results about these systems already obtained in the other populations. The CYP2E1*5B haplotype showed, respectively, the highest and the lowest frequencies already observed in human groups. High frequencies of CYP1A1*2A and CYP1A1*2C alleles and mostly low values of GSTM1*0/*0 and GSTT1*0/*0 genotypes were observed. These data may be interpreted as being due to genetic drift or selection for these high‐frequency CYP1A1 alleles and against GST null genotypes during America's colonization. Intrapopulation diversity varied from 0.19 (Guarani) to 0.38 (Surui), and 90% of the total diversity was due to the variability within populations. The relationships between these Amerindians and with other ethnic groups were evaluated based on D_A distances and the neighbor‐joining method. Low correlation was observed between genetic relationships and geographic distances or linguistic groups. In the TP53 comparison with other ethnic groups, Amerindians clustered together and then joined Chinese populations. The cluster analysis seems to indicate that the Aché tribe might descend from a Gê group that could have first colonized that Paraguayan region, but had also assimilated some amount of the Guarani gene pool, maybe through intertribal admixture. Am J Phys Anthropol 119:249–256, 2002. © 2002 Wiley‐Liss, Inc.     

MMK2, a novel alfalfa MAP kinase, specifically complements the yeast MPK1 function

Mitogen-activated protein (MAP) kinases are serine/threonine protein kinases that are activated in response to a variety of stimuli. Here we report the isolation of an alfalfa cDNA encoding a functional MAP kinase, termedMMK2. The predicted amino acid sequence ofMMK2 shares 65% identity with a previously identified alfalfa MAP kinase, termedMMK1. Both alfalfa cDNA clones encode functional kinases when expressed in bacteria, undergoing autophosphorylation and activation to phosphorylate myelin basic protein in vitro. However, only MMK2 was able to phosphorylate a 39 kDa protein from the detergent-resistant cytoskeleton of carrot cells. The distinctiveness ofMMK2 was further shown by complementation analysis of three different MAP kinase-dependent yeast pathways; this revealed a highly specific replacement of the yeastMPK1 (SLT2) kinase byMMK2, which was found to be dependent on activation by the upstream regulators of the pathway. These results establish the existence of MAP kinases with different characteristics in higher plants, suggesting the possibility that they could mediate different cellular responses.     

The Mck1 GSK-3 kinase inhibits the activity of Clb2-Cdk1 post-nuclear division

The glycogen synthase kinase-3 homolog, Mck1, has been implicated in many cellular functions, from sporulation to calcium stress response in budding yeast. Here, we report a novel function for Mck1 in the inhibition of Clb2-Cdk1 activity post nuclear division. Clb2-Cdk1, the major mitotic cyclin-Cdk complex in yeast, accumulates before anaphase and must be inhibited in telophase for cells to exit mitosis and enter into the next cell cycle. We show that the mck1Δ mutant is highly sensitive to increased Clb2-Cdk1 activity caused either by overexpression of Clb2 or the Cdk1-activating phosphatase Mih1. Deletion of the Cdk1 inhibitory kinase, SWE1, in combination with a mck1Δ mutant results in a synthetic growth defect, suggesting that Mck1 and Swe1 function in parallel pathways to inhibit Clb2-Cdk1. We find that mck1Δ strains have a delay in mitotic exit as well as elevated levels of Clb2-Cdk1 activity post-nuclear division. Using a co-immunoprecipitation assay, we identify a physical interaction between Mck1 and both Clb2 and Mih1. Finally, we demonstrate that phosphorylation of purified Clb2 by Cdk1 is inhibited by catalytically active Mck1 but not catalytically inactive Mck1 in vitro. We propose that Mck1 inhibits the activity of Clb2-Cdk1 via interaction with Clb2. The mammalian glycogen synthase kinase-3 homolog has been implicated in cyclin inhibition, suggesting a conserved cell cycle function for both yeast and mammalian glycogen synthase kinases.     

The Mck1 GSK-3 kinase inhibits the activity of Clb2-Cdk1 post-nuclear division

The glycogen synthase kinase-3 homolog, Mck1, has been implicated in many cellular functions, from sporulation to calcium stress response in budding yeast. Here, we report a novel function for Mck1 in the inhibition of Clb2-Cdk1 activity post nuclear division. Clb2-Cdk1, the major mitotic cyclin-Cdk complex in yeast, accumulates before anaphase and must be inhibited in telophase for cells to exit mitosis and enter into the next cell cycle. We show that the mck1Δ mutant is highly sensitive to increased Clb2-Cdk1 activity caused either by overexpression of Clb2 or the Cdk1-activating phosphatase Mih1. Deletion of the Cdk1 inhibitory kinase, SWE1, in combination with a mck1Δ mutant results in a synthetic growth defect, suggesting that Mck1 and Swe1 function in parallel pathways to inhibit Clb2-Cdk1. We find that mck1Δ strains have a delay in mitotic exit as well as elevated levels of Clb2-Cdk1 activity post-nuclear division. Using a co-immunoprecipitation assay, we identify a physical interaction between Mck1 and both Clb2 and Mih1. Finally, we demonstrate that phosphorylation of purified Clb2 by Cdk1 is inhibited by catalytically active Mck1 but not catalytically inactive Mck1 in vitro. We propose that Mck1 inhibits the activity of Clb2-Cdk1 via interaction with Clb2. The mammalian glycogen synthase kinase-3 homolog has been implicated in cyclin inhibition, suggesting a conserved cell cycle function for both yeast and mammalian glycogen synthase kinases.     

Rod outer segment membrane guanylate cyclase type 1 (ROS-GC1) gene: Structure,organization and regulation by phorbol ester,a protein kinase C activator

At present there are two recognized members of the ROS-GC subfamily of membrane guanylate cyclases. They are ROS-GC1 and ROS-GC2. A distinctive feature of this family is that its members are not switched on by the extracellular peptide hormones; instead, they are modulated by intracellular Ca2+ signals, consistent to their linkage with phototransduction. An intriguing feature of ROS-GC1, which distinguishes it from ROS-GC2, is that it has two Ca2+ switches. One switch inhibits the enzyme at micromolar concentrations of Ca2+, as in phototransduction; the other, stimulates. The stimulatory switch, most likely, is linked to retinal synaptic activity. Thus, ROS-GC1 is linked to both phototransduction and the synaptic activity. The present study describes (1) the almost complete structural identity of 18.5 kb ROS-GC1 gene; (2) its structural organization: the gene is composed of 20 exons and 19 introns with classical GT/AG boundaries; (3) the activity of the ROS-GC1 promoter assayed through luciferase reporter in COS cells; and (4) induction of the gene by phorbol ester, a protein kinase C (PKC) activator. The co-presence of PKC and ROS-GC1 in photoreceptors suggests that regulation of the ROS-GC1 gene by PKC might be a physiologically relevant phenomenon.     

Cloning,expression and genomic structure of a novel human GNB2L1 gene,which encodes a receptor of activated protein kinase C (RACK)*

During a large-scale screen of a human fetal brain cDNA library, a novel human gene GNB2L1 encoding a novel RACK (receptor of activated protein kinase C) protein was isolated and sequenced. The cDNA is 1142 bp long and has a predicted open reading frame encoding 316 aa. The predicted protein shows higher similarity to rat RACK1 and many RACK proteins of different organisms including Drosophila, C. elegans, mouse, rat, human, C. fasciculata, zebrafish, A. thaliana, S. cerevisiae and so on, suggesting it is conserved during evolution. The gene was mapped to human chromosome 5q35.3, the telomer position of chromosome 5q, in which the disease gene for early-onset primary congenital lymphedema was mapped. Also, 5q35.3 is a frequently reported location for cytogenetic and molecular abnormalities in renal cell carcinomas. The gene has 8 exons and 7 introns. It is expressed ubiquitously in many human tissues detected by northern blot analysis and RT-PCR.     

Amplification and overexpression of oncogene Mdm2 and orphan receptor gene Nr1h4 in immortal PRKDC knockout cells

DNA-dependent protein kinase (DNA-PK) is required for the repair of double strand DNA breaks by nonhomologous DNA end joining. The catalytic subunit of DNA-PK, PRKDC, may also be involved in repair-related or separate cell signaling pathways. To learn more about the cellular function of DNA-PK under normal physiological conditions, we identified genes that are differentially expressed between an immortalized wild-type mouse fibroblast cell line and its DNA-PK-deficient counterpart (Prkdc -/-). The proto-oncogene Mdm2 and the farnesoid X receptor gene Nrlh4 were overexpressed in the DNA-PK-deficient cell line. We show that in the DNA-PK-deficient cell line the genes for both Mdm2 and Nrlh4 are amplified to a degree that could account for most, if not all, of their increased expression. Other genes were strongly downregulated in the DNA-PK-deficient cell line, but this opposite expression pattern was not due to gene amplification in the wild-type cells. None of these genes was differentially expressed in DNA-PK-containing and DNA-PK-deficient primary mouse embryo fibroblasts. Our results suggest a model in which DNA-PK indirectly affects the cellular gene expression profile through its caretaker role and by preventing gene amplification.     

Clathrin-independent endocytosis used by the IL-2 receptor is regulated by Rac1, Pak1 and Pak2

There are several endocytic pathways, which are either dependent on or independent of clathrin. This study focuses on a poorly characterized mechanism-clathrin- and caveolae-independent endocytosis-used by the interleukin-2 receptor beta (IL-2R beta). We address the question of its regulation in comparison with the clathrin-dependent pathway. First, we show that Ras-related C3 botulinum toxin substrate 1 (Rac1) is specifically required for IL-2R beta entry, and we identify p21-activated kinases (Paks) as downstream targets. By RNA interference, we show that Pak1 and Pak2 are both necessary for IL-2R beta uptake, in contrast to the clathrin-dependent route. We observe that cortactin, a partner of actin and dynamin-two essential endocytic factors-is required for IL-2R beta uptake. Furthermore, we find that cortactin acts downstream from Paks, suggesting control of its function by these kinases. Thus, we describe a cascade composed of Rac1, Paks and cortactin specifically regulating IL-2R beta internalization. This study indicates Paks as the first specific regulators of the clathrin-independent endocytosis pathway.     

A cluster of five cell wall-associated receptor kinase genes,Wak1–5, are expressed in specific organs of Arabidopsis

WAK1 (wall-associated kinase 1) is a cytoplasmic serine/threonine kinase that spans the plasma membrane and extends into the extracellular region to bind tightly to the cell wall. The Wak1 gene was mapped and found to lie in a tight cluster of five highly similar genes (Wak1–5) within a 30 kb region. All of the Wak genes encode a cytoplasmic serine/threonine protein kinase, a transmembrane domain, and an extracytoplasmic region with several epidermal growth factor (EGF) repeats. The extracellular regions also contain limited amino acid identities to the tenascin superfamily, collagen, or the neurexins. RNA blot analysis with gene-specific probes revealed that Wak1, Wak3 and Wak5 are expressed primarily in leaves and stems of Arabidopsis. Wak4 mRNA is only detected in siliques, while Wak2 mRNA is found in high levels in leaves and stems, and in lower levels in flowers and siliques. A trace amount of Wak2 can also be detected in roots. Wak1 is induced by pathogen infection and salicylic acid or its analogue INA and is involved in the plant's response, and Wak2, Wak3 and Wak5 also can be greatly induced by salicylic acid or INA. The WAK proteins have the potential to serve as both linkers of the cell wall to the plasma membrane and as signaling molecules, and since Wak expression is organ-specific and the isoforms vary significantly in the cell wall associated domain this family of proteins may be involved in cell wall-plasma membrane interactions that direct fundamental processes in angiosperms.     

非小细胞肺癌中Livin和JNK1的表达及相关性研究

目的探讨在非小细胞肺癌（non small cell lung cancer,NSCLC）中的Livin、JNKI蛋白的表达及相关性。方法采用免疫组织化学（二步法）检测187例NSCLC肺癌组织Livin、JNK1[包括总JN1蛋白（t-JNK1）,磷酸化JNK1（p-JNK1）]蛋白的表达情况,采用Spearman等级相关分析探讨其相关性。结果Livin主要在胞浆中定位表达,在肺癌中的表达阳性率明显高于肺良性病变对照组;在鳞癌中表达阳性率明显高于腺癌和其他癌类（P〈0．05）。其在肺癌表达的积分明显高于良性对照组（P〈0．05）,其在鳞癌中表达的积分明显高于腺癌及其他癌类组织。t-JNK1、p-JNK1在肺癌细胞上及在假复层纤毛柱状上皮均可见表达,主要在胞浆中定位表达,t—JNKl未见核内定位表达,而p-JNKl偶见核内定位表达。t—JNK1在肺癌组表达阳性率明显低于对照组（P〈0．05）,t-JNKl在肺癌中表达的积分低于对照组;而p-JNK1在肺癌组表达阳性率明显高于对照组（P〈0．05）。p-JNK1在肺癌中表达的积分高于对照组,但两者无统计学意义（P〉0．05）。Livin与t-JNK1呈现显著的负相关,Livin与p-JNK1呈现显著的正相关。结论Livin在肺癌中高表达,其在鳞癌中表达高于腺癌和其他类型癌。t-JNK1在肺癌中低表达,而p-JNK1在肺癌中高表达。Livin与p-JNK1表达正相关,推测Livin可促JNK1蛋白磷酸化。     

Characterization,subcellular localization and nuclear targeting of casein kinase 2 from Zea mays

We have isolated and characterized the genomic clone of maize casein kinase 2 (CK2) subunit using the previously described CK2-1 cDNA clone as a probe. The genomic clone is 7.5 kb long and contains 10 exons, separated by 9 introns of different size, two larger than 1.5 kb and the others around 100–150 bp. The sequence of the exons is 100% homologous to the sequence of the CK2-1 cDNA. Southern hybridization of total genomic DNA from maize embryos with CK2 cDNA indicated that the CK2-1 gene is part of a multigenic family. We also isolated a new embryo cDNA clone coding for an CK2-2 subunit. We studied the regulation of the enzyme in embryos at the mRNA level, at the protein level and by activity testing. By using immunocytochemistry the CK2 protein was localized in several types of cells of mature embryos. Particularly strong signals were visible in the cytoplasm of epidermis and meristematic cells. Decoration of nuclei of root cortex and scutellum cells was also observed suggesting that CK2 can shift from the cytoplasm into nuclei in specific cell types. We examined whether CK2 contained specific protein domains which actively target the protein to the nucleus by using in-frame fusions of the maize CK2 subunit to the reporter gene encoding -glucuronidase (GUS) which were assayed in transiently transformed onion epidermal cells. Analysis of chimeric constructs identified one region containing a nuclear localization signal (NLS) that is highly conserved in other CK2 proteins.     

Polymorphisms in the coding and noncoding regions of murinePgk-1 alleles

The mouse X-linkedPgk-1 gene encodes phosphoglycerate kinase. When transfected into human cells, thePgk-1^b allele causes the appearance of mouse PGK-1^b enzyme activity. We describe here cloning of mousePgk-1^a, an allele ofPgk-1 which encodes an enzyme, PGK-1^a, with distinct electrophoretic mobility. We constructed recombinants between the DNA encodingPgk-1^b andPgk-1^a and transfected these constructs into human to assess the electrophoretic characteristics of each recombinant. In this way the charge variation between the two proteins was localized to exons 4 or 5. Sequencing of these exons revealed a single base-pair difference between the two alleles at codon 155, which predicts the amino acids lysine and threonine in PGK-1^b and PGK-1^a, respectively. A number of other DNA sequence polymorphisms exist betweenPgk-1^b andPgk-1^a including part of an L1 repeated element unique toPgk-1^a. This work was supported by the Medical Research Council of Canada, the National Cancer Institute of Canada, and the Deutsche Forschungsgemeinschaft, SFB 304.     

耐力运动对大鼠骨骼肌ERK1/2活性的影响

目的:探讨耐力运动对大鼠骨骼肌蛋白总量(t-ERK1/2)及磷酸化ERK1/2(p-ERK1/2)及ERK2mRMA表达的影响。方法:SD大鼠随机分为对照组和运动组。运动组分为1h/d和1.5h/d组,共7周,运动结束后24h和48h取材,测定葡萄糖和胰岛素浓度;Westernblot法检测骨骼肌t-ERK1/2、p-ERK1/2蛋白表达;RT-PCR法分析ERK2mRNA表达。结果:与对照组比较,运动组胰岛素浓度降低;各运动组p-ERK1/2升高;1.5h/d-24h和-48h组t-ERK1/2增高;1h/d-24h组与1.5h/d-24h和-48hERK2mRNA表达增高。结论:耐力运动可能通过增加ERK1/2活性,提高大鼠骨骼肌对胰岛素的敏感性。     

CDK1, not ERK1/2 or ERK5, is required for mitotic phosphorylation of BIMEL

The pro-apoptotic BH3 only protein BIM_EL is phosphorylated by ERK1/2 and this targets it for proteasome-dependent degradation. A recent study has shown that ERK5, an ERK1/2-related MAPK, is activated during mitosis and phosphorylates BIM_EL to promote cell survival. Here we show that treatment of cells with nocodazole or paclitaxel does cause phosphorylation of BIM_EL, which is independent of ERK1/2. However, this was not due to ERK5-catalysed phosphorylation, since it was not reversed by the MEK5 inhibitor BIX02189 and proceeded normally in ERK5−/− fibroblasts. Indeed, although ERK5 is phosphorylated at multiple sites in the C-terminal transactivation region during mitosis, these do not include the activation-loop and ERK5 kinase activity does not increase. Mitotic phosphorylation of BIM_EL occurred at proline-directed phospho-acceptor sites and was abolished by selective inhibition of CDK1. Furthermore, cyclin B1 was able to interact with BIM and cyclin B1/CDK1 complexes could phosphorylate BIM in vitro. Finally, we show that CDK1-dependent phosphorylation of BIM_EL drives its polyubiquitylation and proteasome-dependent degradation to protect cells during mitotic arrest. These results provide new insights into the regulation of BIM_EL and may be relevant to the therapeutic use of agents such as paclitaxel.     

Isolation and characterization of a human putative receptor protein kinase cDNA STYK1*

Protein kinases (PKs) represent a well studied but most diverse protein superfamily. The covalent, reversible linkage of phosphate to serine, threonine, and tyrosine residues of substrate proteins by protein kinases is probably ubiquitous cellular mechanism for regulation of physiological processes. It is known to us that most signaling pathways impinge at some point on protein kinases. Here we report a human putative receptor protein kinase cDNA STYK1. The STYK1 cDNA is 2749 base pairs in length and contains an open reading frame encoding 422 amino acids. The STYK1 gene is mapped to human chromosome 12p13 and 11 exons were found. RT-PCR showed that STYK1 is widely expressed in human tissues.