The neurofibromatosis 2 tumor suppressor protein interacts with hepatocyte growth factor-regulated tyrosine kinase substrate

The neurofibromatosis 2 tumor suppressor protein schwannomin/merlin is commonly mutated in schwannomas and meningiomas. Schwannomin, a member of the 4.1 family of proteins, which are known to link the cytoskeleton to the plasma membrane, has little known function other than its ability to suppress tumor growth. Using yeast two-hybrid interaction cloning, we identified the HGF-regulated tyrosine kinase substrate (HRS) as a schwannomin interactor. We verified the interaction by both immunoprecipitation of endogenous HRS with endogenous schwannomin in vivo as well as by using bacterially purified HRS and schwannomin in vitro. We narrowed the regions of interaction to include schwannomin residues 256-579 and HRS residues from 480 to the end of either of two HRS isoforms. Schwannomin molecules with a L46R, L360P, L535P or Q538P missense mutation demonstrated reduced affinity for HRS binding. As HRS is associated with early endosomes and may mediate receptor translocation to the lysosome, we demonstrated that schwannomin and HRS co-localize at endosomes using the early endosome antigen 1 in STS26T Schwann cells by indirect immunofluorescence. The identification of schwannomin as a HRS interactor implicates schwannomin in HRS-mediated cell signaling.     

The NF2 interactor, hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), associates with merlin in the "open" conformation and suppresses cell growth and motility

The neurofibromatosis 2 tumor suppressor protein, merlin or schwannomin, functions as a negative growth regulator; however, its mechanism of action is not known. In an effort to determine how merlin regulates cell growth, we analyzed a recently identified novel merlin interactor, hepatocyte growth factor-regulated tyrosine kinase substrate (HRS). We demonstrate that regulated overexpression of HRS in rat schwannoma cells results in similar effects as overexpression of merlin, including growth inhibition, decreased motility and abnormalities in cell spreading. Previously, we showed that merlin forms an intramolecular association between the N- and C-termini and exists in "open" and "closed" conformations. Merlin interacts with HRS in the unfolded, or open, conformation. This HRS binding domain maps to merlin residues 453-557. Overexpression of C-terminal merlin has no effect on HRS function, arguing that merlin binding to HRS does not negatively regulate HRS growth suppressor activity. These results suggest the possibility that merlin and HRS may regulate cell growth in schwannoma cells through interacting pathways.     

Hepatocyte growth factor-regulated tyrosine kinase substrate (Hgs) is involved in BMP signaling through phosphorylation of SMADS and TAK1 in early mouse embryo

Hepatocyte growth factor-regulated tyrosine kinase substrate that is encoded by Hgs promotes degradation of ubiquitinated signaling molecule in the early endosome. We previously reported that a targeted mutation in Hgs results in embryonic lethality soon after gastrulation in the mouse. Here, we report that downstream target genes for BMP signaling were highly down-regulated in the Hgs mutant embryos. We also showed that Hgs is required for phosphorylation of SMAD1/5/8 and TAK1/p38 to transduce BMP signaling. Furthermore, we found that HGS functions to localize TAK1 in early endosome for its activation. These results suggest that HGS is critical to localize TAK1 to early endosome for transducing BMP signaling for proper development. Our data revealed a new mechanism to modify BMP signaling by Hgs during early mouse development.     

食管鳞状细胞癌中HGF表达与肿瘤血管形成的关系

目的研究肝细胞生长因子(hepatocyte growth factor,HGF)在食管鳞癌中的表达及其与肿瘤血管形成之间的关系。方法应用免疫组化PV两步法检测64例食管鳞癌组织及20例癌旁正常组织中HGF及CD34的表达情况,并计数微血管密度(microvessel density,MVD)。结果 HGF在食管鳞癌中的表达(76.6%)高于癌旁正常组织(15.0%,P<0.05),且与肿瘤直径、分化程度及淋巴结转移有关;食管鳞癌组织的MVD值(38.87±17.55)/mm2明显高于癌旁正常组织的MVD值(15.10±3.76)/mm2(P<0.05),肿瘤直径>5 cm,有淋巴结转移及侵袭较深的肿瘤MVD值高(P<0.05);食管鳞癌中HGF阳性组MVD值为(42.25±16.50)/mm2,高于HGF阴性组MVD值(27.89±16.83)/mm2(P=0.005),且MVD值与HGF的表达之间具有相关性。结论 HGF的高表达与食管鳞癌的恶性生物学行为及肿瘤血管生成关系密切,HGF可作为食管鳞癌生物治疗的潜在靶点。     

髓系来源的抑制性细胞在肿瘤进展过程中的研究

进展性肿瘤显著的特性是逃避机体免疫系统的监视、躲避免疫系统的杀伤、抑制自体免疫系统。肿瘤在进展过程中一群髓系来源的抑制性细胞（MDSC）在肿瘤微环境、引流区淋巴结大量聚集,其能够促进肿瘤的发展同时抑制机体的免疫作用。其机制概括为：髓系细胞向正常树突状细胞或巨噬细胞的分化受阻,分泌促进肿瘤生长的细胞因子,参与肿瘤血管的形成,诱导调节性T淋巴细胞（Treg）的产生,从而抑制了T淋巴细胞的抗肿瘤作用。     

Functional analysis of a mutant form of the receptor tyrosine kinase Tie2 causing venous malformations

Tie2 is expressed predominantly in endothelial cells and is required for blood vessel formation and maintenance. A missense mutation resulting in an R to W substitution in the kinase domain of Tie2 co-segregates with an autosomal dominantly inherited form of vascular dysmorphogenesis, venous malformation (VM). The mechanism by which this activating mutation leads to vessel dysmorphogenesis in VM is not known. Here we examined Tie2 activation status in VM and found activated receptor in lesional and non-lesional vessels. To gain insight into functional effects of VM mutant Tie2, wild-type and R849W mutant receptor were expressed in cultured human venous endothelial cells. Mutant Tie2 was constitutively phosphorylated in endothelial cells in vivo and caused a marked suppression of apoptosis. The anti-apoptotic kinase Akt was constitutively activated in cells expressing mutant receptor. Dominant-negative Akt inhibited the pro-survival activity of mutant Tie2. Migration of smooth muscle cells induced by conditioned medium from cells expressing mutant receptor was similar to that from cells expressing wild-type receptor. These data suggest that a primary effect of R849W Tie2 in VM is to allow survival of mural cell poor vessels via ligand-independent Tie2 activation of Akt and endothelial survival, rather than to directly induce formation of dysmorphogenic vessels.     

A kinase shRNA screen links LATS2 and the pRB tumor suppressor

pRB-mediated inhibition of cell proliferation is a complex process that depends on the action of many proteins. However, little is known about the specific pathways that cooperate with the Retinoblastoma protein (pRB) and the variables that influence pRB's ability to arrest tumor cells. Here we describe two shRNA screens that identify kinases that are important for pRB to suppress cell proliferation and pRB-mediated induction of senescence markers. The results reveal an unexpected effect of LATS2, a component of the Hippo pathway, on pRB-induced phenotypes. Partial knockdown of LATS2 strongly suppresses some pRB-induced senescence markers. Further analysis shows that LATS2 cooperates with pRB to promote the silencing of E2F target genes, and that reduced levels of LATS2 lead to defects in the assembly of DREAM (DP, RB [retinoblastoma], E2F, and MuvB) repressor complexes at E2F-regulated promoters. Kinase assays show that LATS2 can phosphorylate DYRK1A, and that it enhances the ability of DYRK1A to phosphorylate the DREAM subunit LIN52. Intriguingly, the LATS2 locus is physically linked with RB1 on 13q, and this region frequently displays loss of heterozygosity in human cancers. Our results reveal a functional connection between the pRB and Hippo tumor suppressor pathways, and suggest that low levels of LATS2 may undermine the ability of pRB to induce a permanent cell cycle arrest in tumor cells.     

Modulation of M2-type pyruvate kinase activity by the cytoplasmic PML tumor suppressor protein

The promyelocytic leukemia (PML) tumor suppressor protein accumulates in PML nuclear bodies (PML-NBs), and can induce growth arrest, cellular senescence and apoptosis. PML has also been localized in the cytoplasm, although its function in this localization remains elusive. A general property of primary cancers is their high glycolytic rate which results from increased glucose consumption. However, the mechanism by which cancer cells up-regulate glycolysis is not well understood. Here, we have shown that cytoplasmic PML (cPML) directly interacts with M2-type pyruvate kinase (PKM2), a key regulator of carbon fate. PKM2 determines the proportion of carbons derived from glucose that are used for glycolytic energy production. Over-expression of PML-2KA mutant in the cytoplasm, which was generated by mutagenesis of the nuclear localization signals of PML, in MCF-7 breast cancer cells suppressed PKM2 activity and the accumulation of lactate. PKM2 exists in either an active tetrameric form which has high affinity for its substrate phosphoenolpyruvate (PEP) or a less active dimeric form which has low affinity for its substrate. Over-expression of PML-2KA suppressed the activity of the tetrameric form of PKM2, but not the dimeric form. Our findings suggest that cPML plays a role in tumor metabolism through its interaction with PKM2.     

Mutational analysis of the SH2-kinase linker region of Bruton's tyrosine kinase defines alternative modes of regulation for cytoplasmic tyrosine kinase families

Bruton's tyrosine kinase (Btk) plays critical roles in B cell development and activation. Mutations of Btk cause X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency in mice. An Src homology domain 2-kinase linker region exists in all Src, Abl, ZAP70/Syk and Btk/Tec non-receptor tyrosine kinase families. Missense mutations in the Btk linker region can cause XLA, supporting an essential role for this protein segment. We investigated the regulatory role of the linker region in Btk function by mutational analysis. XLA-causing mutations L369F and R372G abolished Btk-mediated calcium response without affecting Btk protein stability and kinase activity significantly. Although mutation of a well-conserved tryptophan (W260A) in the linker region of the Src family kinase Hck has been shown to cause a hyperactive kinase, an analogous mutation in Btk (W395A) dramatically decreased Btk kinase activity. Tyrosine phosphorylation in the linker region was previously shown to regulate the function of Abl and ZAP70/Syk kinases. Even though tyrosine phosphorylation was detected on tyrosine 375 in the Btk linker region, no significant alteration was observed in Btk-signaling activity and biological function when this tyrosine was mutated in DT-40 cells or in Y375F knock-in mice. Our data and previous studies suggest that each cytoplasmic tyrosine kinase family has evolved a unique strategy to utilize the linker region to regulate the function of the enzyme.     

A functional complex is formed in human T lymphocytes between the protein tyrosine phosphatase CD45, the protein tyrosine kinase p56lck and pp32, a possible common substrate.

In vitro protein kinase assays of CD45 immunoprecipitates prepared from digitonin lysates of resting human T lymphocytes resulted in exclusive tyrosine phosphorylation of a 32-kDa protein (pp32). Reprecipitation of the in vitro phosphorylated proteins and Western blot analysis of whole CD45 immunoprecipitates employing antisera specifically directed at different protein tyrosine kinases demonstrated that the p56lck protein tyrosine kinase was responsible for in vitro phosphorylation of pp32. Since in vitro kinase assays of p56lck immunoprecipitates also resulted in phosphorylation of pp32, the present data strongly suggest that a functional complex is formed between CD45, p56lck and pp32. Such a notion is supported by the findings that phosphorylation of pp32 by p56lck correlated with expression of the CD45 molecules and that in vitro phosphorylated pp32 was completely dephosphorylated by purified CD45.     

Proportion of necrosis in transplanted murine adenocarcinomas and its relationship to tumor growth

Percentage of necrosis has been measured in 30 murine adenocarcinoma transplants of known volume. It was found that changes in necrotic proportion during the growth of these particular tumors agree with the concept of a core of necrosis surrounded by a viable shell of constant thickness. This means that viable and necrotic fractions were proportional to the surface area of the tumor, which is consistent with the frequently observed superficial distribution of nutrient-supplying arteries. According to this model a plot of 1n tumor volume versus necrotic fraction is sigmoid and, if tumor growth is directly related to viable fraction, such a plot is not compatible with popular mathematical growth equations. However, the equation of von Bertalanffy gives a reasonable fit to the data here for total tumor growth, suggesting that growth too was proportional to surface area and in accord with a surface-related viable fraction and blood supply. An appropriate physiological explanation is given of necrosis development resulting from a superficial nutrient supply.     

EphA2受体与恶性肿瘤关系的研究进展

<正>促红细胞生成素产生肝细胞受体(erythropoietin producing hepatocyte receptor,Eph)家族是己知最大的受体酪氨酸激酶(receptor tyrosine kinase,RTK)亚家族之一,现有14个成员,其配体名为Ephrin,即Eph家族受体相互作用蛋白,现有8个成员。Eph受体为跨膜Ⅰ型糖蛋白,包括三个结构域,即胞外配体结合区、胞内具有酪氨酸激酶活性的功能区和连接这两个区的由疏水氨基酸组成的跨膜区。Eph与Ephrin可互为受体和配     

Genomic structure and alternative splicing of the insulin receptor tyrosine kinase substrate of 53-kDa protein

Insulin receptor tyrosine kinase substrate of 53-kDa protein (IRSp53) is now known to be a key factor in cytoskeleton reorganization. The human IRSp53 was identified as a binding partner with DRPLA protein, a product of the gene responsible for a neurodegenerative disorder, dentatorubral pallidoluysian atrophy, as well as a binding partner with brain-specific angiogenesis inhibitor 1. Previous studies identified at least four isoforms (L-, M-, S- and T-forms) in human, where 511 amino acid residues from the N-terminus were identical, followed by unique sequences of 9–41 amino acid residues. As each isoform had a distinct function, the unique sequences at the C-terminus had a vital role in its function. Here we report that these isoforms were indeed generated by alternative splicing, which was established by experimental and computational studies on human and rodent genomes. Previous biochemical reports suggested that rodents may lack one of the isoforms (L-form). This study solved this issue, as a nucleotide substitution occurred at a splice donor site followed by a large deletion in the rodent genome compared with human, which made the generation of the L-form impossible. This study also revealed overlapping of the IRSp53 and AATK genes coded for by complementary strands.Accession numbers: AB104726, AB104729, AB105194, AB105195, AB105196     

Brain levels of cytoplasmic casein kinase 2 and its substrate proteins in Alzheimer's disease

Mental Health Research Center, Russian Academy of Medical Sciences, Moscow. (Presented by Academician of the Russian Academy of Medical Sciences I. P. Ashmarin.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 113, No. 6, pp. 602–603, June, 1992.     

己烯雌酚对肿瘤生长,转移的影响及机制探讨

目的：研究己烯雌酚（ＤＥＳ）对实验性肿瘤转移的影响。方法：利用自发性Ｌｅｗｉｓ肺癌（ＬＬＣ）及实验性Ｂ１６黑色素瘤（Ｂ１６Ｍ）转移模型研究ＤＥＳ对肿瘤生长、转移的影响。结果：发现预先用ＤＥＳ处理的小鼠对其移植的ＬＬＣ生长和自发性肺转移均有抑制作用，且ＤＥＳ剂量与肿瘤的生长、转移呈直线负相关（ｒ〉０．８０，Ｐ〈０．０１）；Ｂ１６Ｍ实验性肺转移也明显减少；小鼠接种ＬＬＣ后给予ＤＥＳ对ＬＬＣ也有抑制作用     

Correlation between tumor suppressor inhibitor of growth family member 4 expression and microvessel density in breast cancer

Suppression of cell spreading and migration by inhibitor of growth 4 suggests that its loss may induce metastasis. Inhibitor of growth 4 expression level in 60 breast cancer tissues, 30 normal adjacent tissues, and tissues from patients with benign hyperplasia was determined by real-time polymerase chain reaction, Western blot, and immunohistochemistry. The correlation between inhibitor of growth 4 expression and clinical stage, histologic grade, and microvessel density in breast cancer was analyzed. Inhibitor of growth 4 messenger RNA and protein expression in breast cancer was significantly lower than that observed in adjacent normal and hyperplastic breast tissues (P < .05). Inhibitor of growth 4 expression decreased with increasing clinical stage and histologic grade. Moreover, the presence of lymph node metastasis was correlated with decreased inhibitor of growth 4 messenger RNA expression (P < .01), and a negative correlation was noted between inhibitor of growth 4 protein expression and microvessel density in breast cancer. Inhibitor of growth 4 may represent an important biomarker for assessing the severity of breast cancer. Further studies are required to fully evaluate the diagnostic and possible prognostic value of determining inhibitor of growth 4 levels in breast cancer.     

抑癌因子SOX7在乳腺癌中的表达及其与AXIN2表达的相关性

目的 研究SOX7及AXIN2在乳腺癌中的表达及其相关性.方法 采用免疫组织化学SP法检测104例乳房切片中,SOX7、AXIN2及β-catenin的表达情况,其中74例肿瘤组织,30例正常组织作对照.结果 SOX7在乳腺癌中低表达,且与AXIN2的表达具有正相关性(P=0.001),与β-catenin的表达具有负相关性(P =0.006);SOX7的下调与乳腺癌的分期和分化程度具有相关性(P=0.072;P=0.085),与激素受体和生物标记物无相关性(P =0.059;P =0.438).结论 本研究证实了SOX7在乳腺癌的发生发展中表现为抑癌因子作用,同时也提出了SOX7与AXIN2在乳腺癌中协同表达的现象,并指出SOX7可以作为一种独立预测乳腺癌预后的生物标记物.     

PTEN基因的克隆及其在HepG2细胞中的表达

目的克隆人抑癌基因PTEN全长cDNA,构建其真核表达载体并检测其在人肝癌细胞HepG2中的表达。方法采用RT-PCR法从人正常肝组织中扩增PTEN全长cDNA,将之与pMD18-T Simple Vector连接、测序,获得PTEN基因。将该基因与pcDNA3·1( )载体连接,构建pcDNA3.1-PTEN真核表达载体。用该载体转染HepG2细胞,RT-PCR检测PTEN的表达。结果酶切和测序证实PTEN基因克隆和真核表达载体构建成功。HepG2-PTEN细胞中PTEN mRNA的表达显著高于未转染的HepG2细胞。结论人抑癌基因PTEN在人肝癌细胞系HepG2细胞中能够高效、稳定地表达,为其在肝癌基因治疗研究中的应用奠定了基础。     

The NF2 tumor suppressor gene product, merlin, mediates contact inhibition of growth through interactions with CD44

The neurofibromatosis-2 (NF2) gene encodes merlin, an ezrin-radixin-moesin-(ERM)-related protein that functions as a tumor suppressor. We found that merlin mediates contact inhibition of growth through signals from the extracellular matrix. At high cell density, merlin becomes hypo-phosphorylated and inhibits cell growth in response to hyaluronate (HA), a mucopolysaccharide that surrounds cells. Merlin's growth-inhibitory activity depends on specific interaction with the cytoplasmic tail of CD44, a transmembrane HA receptor. At low cell density, merlin is phosphorylated, growth permissive, and exists in a complex with ezrin, moesin, and CD44. These data indicate that merlin and CD44 form a molecular switch that specifies cell growth arrest or proliferation.