Kank proteins: a new family of ankyrin-repeat domain-containing proteins

The human Kank gene was found as a candidate tumor suppressor for renal cell carcinoma, and encodes an ankyrin-repeat domain-containing protein, Kank. Here, we report a new family of proteins consisting of three Kank (Kank1)-associated members, Kank2, Kank3 and Kank4, which were found by domain and phylogenetic analyses. Besides the conserved ankyrin-repeat and coiled-coil domains, there was a conserved motif at the N-terminal (KN motif) containing potential motifs for nuclear localization and export signals. Gene expression of these genes was examined by RT-PCR at the mRNA level and by Western blotting and immunostaining at the protein level. Kank family genes showed variations in the expression level among tissues and kidney cell lines. Furthermore, the results of overexpression of these genes in NIH3T3 cells suggest that all of these family proteins have an identical role in the formation of actin stress fibers.     

Aberrant Kank1 expression regulates YAP to promote apoptosis and inhibit proliferation in OSCC

The kidney ankyrin repeat-containing protein 1 (Kank1) gene is one of the most important members of the KANK family. Kank1 has hybridity deletion and promoter methylation in the cancer tissues of the brain, lung, kidney and the corresponding cell lines, leading to downregulation of the gene expression. Meanwhile, Kank1 also plays a key role in the occurrence and development of various types of tumors, suggesting that Kank1 may be an anti-oncogene. However, its role and the potential mechanisms in the Oral Squamous Cell Carcinoma (OSCC) remain unclear. We examined the expression of Kank1 in OSCC tissues and explored its clinical significance. In addition, we investigated the effects of Kank1 on the biological behavior of OSCC cells and their specific molecular mechanisms. We found that Kank1 was poorly expressed in OSCC tissues and it is correlated with the OSCC stage and the patient's poor prognosis. By overexpression of Kank1, we found that the proliferation ability of the OSCC cells decreased both in vitro and in vivo, the proportion of apoptotic cells increased, and the mitochondrial transmembrane potential decreased. In terms of the molecular mechanism, we confirmed that Kank1 could inhibit the occurrence of OSCC by regulating Yap to inhibit the proliferation and promote apoptosis of the OSCC cells. Moreover, it was found that the overexpression of YAP reversed those effects caused by Kank1 overexpression on the OSCC cells. In conclusion, the research indicated that Kank1 might play an anti-oncogenic role in OSCC and it could be considered to be a target for the diagnosis and the treatment of OSCC.     

Kank Is an EB1 Interacting Protein that Localises to Muscle-Tendon Attachment Sites in Drosophila

Little is known about how microtubules are regulated in different cell types during development. EB1 plays a central role in the regulation of microtubule plus ends. It directly binds to microtubule plus ends and recruits proteins which regulate microtubule dynamics and behaviour. We report the identification of Kank, the sole Drosophila orthologue of human Kank proteins, as an EB1 interactor that predominantly localises to embryonic attachment sites between muscle and tendon cells. Human Kank1 was identified as a tumour suppressor and has documented roles in actin regulation and cell polarity in cultured mammalian cells. We found that Drosophila Kank binds EB1 directly and this interaction is essential for Kank localisation to microtubule plus ends in cultured cells. Kank protein is expressed throughout fly development and increases during embryogenesis. In late embryos, it accumulates to sites of attachment between muscle and epidermal cells. A kank deletion mutant was generated. We found that the mutant is viable and fertile without noticeable defects. Further analysis showed that Kank is dispensable for muscle function in larvae. This is in sharp contrast to C. elegans in which the Kank orthologue VAB-19 is required for development by stabilising attachment structures between muscle and epidermal cells.     

Kank regulates RhoA-dependent formation of actin stress fibers and cell migration via 14-3-3 in PI3K-Akt signaling

Phosphoinositide-3 kinase (PI3K)/Akt signaling is activated by growth factors such as insulin and epidermal growth factor (EGF) and regulates several functions such as cell cycling, apoptosis, cell growth, and cell migration. Here, we find that Kank is an Akt substrate located downstream of PI3K and a 14-3-3-binding protein. The interaction between Kank and 14-3-3 is regulated by insulin and EGF and is mediated through phosphorylation of Kank by Akt. In NIH3T3 cells expressing Kank, the amount of actin stress fibers is reduced, and the coexpression of 14-3-3 disrupted this effect. Kank also inhibits insulin-induced cell migration via 14-3-3 binding. Furthermore, Kank inhibits insulin and active Akt-dependent activation of RhoA through binding to 14-3-3. Based on these findings, we hypothesize that Kank negatively regulates the formation of actin stress fibers and cell migration through the inhibition of RhoA activity, which is controlled by binding of Kank to 14-3-3 in PI3K-Akt signaling.     

A major mutation of KIF21A associated with congenital fibrosis of the extraocular muscles type 1 (CFEOM1) enhances translocation of Kank1 to the membrane

Congenital fibrosis of the extraocular muscles type 1 (CFEOM1) is associated with heterozygous mutations in the KIF21A gene, including a major (R954W) and a minor (M947T) mutation. Kank1, which regulates actin polymerization, cell migration and neurite outgrowth, interacted with the third and fourth coiled-coil domains of KIF21A protein at its ankyrin-repeat domain. While both KIF21A^R954W and KIF21A^M947T enhanced the formation of a heterodimer with the wild type, KIF21A^WT, these mutants also enhanced the interaction with Kank1. Knockdown of KIF21A resulted in Kank1 predominantly occurring in the cytosolic fraction, while KIF21A^WT slightly enhanced the translocation of Kank1 to the membrane fraction. Moreover, KIF21A^R954W significantly enhanced the translocation of Kank1 to the membrane fraction. These results suggest that KIF21A regulates the distribution of Kank1 and that KIF21A mutations associated with CFEOM1 enhanced the accumulation of Kank1 in the membrane fraction. This might cause an abrogation of neuronal development in cases of CFEOM1 through over-regulation of actin polymerization by Kank1.     

NBP, a zebrafish homolog of human Kank3, is a novel Numb interactor essential for epidermal integrity and neurulation

Numb is an adaptor protein implicated in diverse basic cellular processes. Using the yeast-two hybrid system we isolated a novel Numb interactor in zebrafish called NBP which is an ortholog of human renal tumor suppressor Kank. NBP interacts with the PTB domain of Numb through a region well conserved among vertebrate Kanks containing the NGGY sequence. Similar NBP and Numb morphant phenotype such as impaired convergence and extension movements during gastrulation, neurulation and epidermis defects and enhanced phenotypic aberrations in double morphants suggest that the genes interact genetically. We demonstrate that the expression of NBP undergoes quantitative and qualitative changes during embryogenesis and that the protein accumulates at the cell periphery to sites of cell-cell contact during gastrulation and later in development it concentrates at the basal poles of differentiated cells. These findings imply a possible role of NBP in establishing and maintaining cell adhesion and tissue integrity.     

Pleiotrophin stimulates fibroblasts and endothelial and epithelial cells and is expressed in human cancer.

Previously we reported the purification of the heparin-binding growth factor pleiotrophin (PTN) from supernatants of the human breast cancer cell line MDA-MB-231. To investigate further the biological activities of PTN and its potential role in cancer, we cloned a PTN cDNA and expressed the gene in a human kidney and in a human adrenal carcinoma cell line (SW-13). The supernatants harvested from cells transfected with PTN contained a heparin-binding specific protein of an apparent molecular mass of 18 kDa. These supernatants stimulated the proliferation of endothelial cells as well as the anchorage-independent growth of SW-13 cells and of normal rat kidney fibroblasts. Furthermore, SW-13 cells transfected with PTN acquired autonomous growth in soft agar and were tumorigenic in athymic nude mice. In contrast to these results with PTN from human cells, PTN obtained from insect cells (Sf9) using recombinant baculovirus as a vector was biologically inactive. We detected high levels of PTN mRNA in 16 of 27 primary human breast cancer samples (62%) as well as in 8 of 8 carcinogen-induced rat mammary tumors. Furthermore, 9 of 34 human tumor cell lines of different origin showed detectable PTN mRNA. We conclude that PTN may function as a tumor growth and angiogenesis factor in addition to its role during embryonic development.     

Variation of human mucin gene expression in gastric cancer cell lines and gastric mucous cell primary cultures

Human gastric mucous cells - gastric cancer cell lines mucin gene expression - TNFalpha - RT-PCR immunocytochemistry Little is known on the expression pattern of mucin genes in human gastric cancer cell lines in relation to mucin expression in normal gastric epithelial cells. Thus, the aim of this study was to compare gastric cancer cell lines and non-transformed epithelial cells in their expression of the different mucin genes, in order to use these cells as models for physiological MUC expression in human stomach. Human gastric mucous cell primary cultures which were obtained from surgical specimen by collagenase/pronase treatment and a panel of six human gastric cancer cells were screened for mRNA expression of the mucin genes MUC1, MUC2, MUC5AC, MUC5B, and MUC6. Mucin gene expression was analyzed by semi-quantitative RT-PCR, and by Western blotting and immunocytochemistry. Primary cultured human gastric mucous cells retained the stomach-specific pattern of mRNA expression found in gastric mucosal biopsies (MUC1, MUC5AC, MUC6), whereas any gastric cancer cell line exhibited an aberrant mucin gene expression. Mucin gene expression showed large variations in levels and patterns from cell line to cell line, but MUC2 was aberrantly expressed in all cancer cells. Immunocytochemistry confirmed aberrant MUC2 protein expression in cancer cells. The expression of the secretory mucin genes MUC2 and MUC5AC varied in relation to the length of cultivation of the cancer cell lines. Treatment of the gastric cancer cells with TNFalpha resulted in an enhanced mRNA expression of MUC1, MUC2, and MUC5AC (2-fold increase within 3 hours; p <0.05). In contrast, immunocytochemistry disclosed a decrease in MUC2 and MUC5AC staining intensity. Our results indicate that primary cultured human gastric mucous cells provide a physiological in vitro system for investigations of gastric mucin gene regulation. In gastric cancer cells marked changes in the mucin gene expression pattern are found with coexpression of non-gastric type mucins. Gastric mucin gene expression may be regulated by proinflammatory cytokines which could have implications in gastritis.     

Cloning of the human keratin 18 gene and its expression in nonepithelial mouse cells.

Human keratin 18 (K18) and the homologous mouse protein, Endo B, are intermediate filament subunits of the type I keratin class. Both are expressed in many simple epithelial cell types including trophoblasts, the first differentiated cell type to appear during mouse embryogenesis. The K18 gene was identified and cloned from among the 15 to 20 similar sequences identified within the human genome. The identity of the cloned gene was confirmed by comparing the sequence of the first two exons to the K18 cDNA sequence and transfecting the gene into various murine cell lines and verifying the encoded protein as K18 by immunoprecipitation and partial peptide mapping. The transfected K18 gene was expressed in mouse HR9 parietal endodermal cells and mouse fibroblasts even though the fibroblasts fail to express endogenous Endo B. S1 nuclease protection analysis indicated that mRNA synthesized from the transfected K18 gene is initiated at the same position as authentic K18 mRNA found in both BeWo trophoblastoma cells and HeLa cells. Pulse-chase experiments indicated that the human K18 protein is stable in murine parietal endodermal cells (HR9) which express EndoA, a complementary mouse type II keratin. Surprisingly, however, K18 was degraded when synthesized in cells which lack a type II keratin. This turnover of K18 may be an important mechanism by which epithelial cells maintain equal molar amounts of both type I and II keratins. In addition, the levels of the endogenous type I Endo B in parietal endodermal cells were compensatingly down regulated in the presence of the K18 protein, while the levels of the endogenous type II Endo A were not affected in any of the transfected cell lines.     

Human leukocyte formin: a novel protein expressed in lymphoid malignancies and associated with Akt

The very large family of Formin proteins is involved in processes such as morphogenesis, embryonic differentiation, cell polarity, and cytokinesis. A novel human gene from the Formin family, denominated human leukocyte formin gene, was cloned. The cDNA of the gene was determined to be 3959bp long with an open reading frame of 3302bp and computational analysis located this gene on chromosome 17, suggesting that it is composed of 27 exons. Northern blot analysis revealed a restricted expression of mRNA in the thymus, spleen, and peripheral blood leukocytes in normal human tissues. Western blot analysis demonstrated that the protein encoded by this gene is overexpressed in lymphoid malignancies; cancer cell lines and peripheral blood leukocyte from chronic lymphocytic leukemia (CLL) patients. Furthermore, the human leukocyte formin protein was observed to associate with Akt, a critical survival regulator in many different cell types.     

Different gamma-glutamyl transpeptidase mRNAs are expressed in human liver and kidney

In human, the two subunits of gamma-glutamyl transpeptidase (GGT) arise from a common precursor encoded by a multigene family. Until now, a single specific coding sequence for this precursor (type I) has been identified in human placenta and liver. In the present study, we have isolated from a human kidney cDNA library, a GGT specific clone (0.8 Kb). The sequence of which (type II) i) covers the carboxy terminal part of the GGT precursor, ii) exhibits 22 point mutations and a 30 bp deletion as compared to the type I GGT sequence. The sequencing of a human genomic clone reveals that this type II GGT mRNA is encoded by a different gene than the type I GGT mRNA. Both type I and type II GGT mRNAs are expressed in human liver, while almost exclusively type II GGT mRNA is detected in human kidney.     

Evolution, expression, and chromosomal location of a novel receptor tyrosine kinase gene, eph.

Partial sequence analysis of the genomic eph locus revealed that the splicing points of kinase domain-encoding exons were completely distinct from those of the other protein tyrosine kinase members reported, suggesting that this is the earliest evolutionary split within this family. In Northern (RNA) blot analysis, the eph gene was expressed in liver, lung, kidney, and testis of rat, and screening of 25 human cancers of various cell types showed preferential expression in cells of epithelial origin. Overexpression of eph mRNA was found in a hepatoma and a lung cancer without gene amplification. Comparison of cDNA sequences derived from a normal liver and a hepatoma that overproduces eph mRNA demonstrated that two of them were completely identical throughout the transmembrane to the carboxy-terminal portions. Southern blot analysis of DNAs from human-mouse hybrid clones with an eph probe showed that this gene was present on human chromosome 7.     

Tissue specific expression and chromosomal mapping of a human UDP-N-acetylglucosamine: alpha1,3-d-mannoside beta1, 4-N-acetylglucosaminyltransferase

A human cDNA for UDP- N -acetylglucosamine:alpha1,3-d-mannoside beta1,4- N- acetylglucosaminyltransferase (GnT-IV) was isolated from a liver cDNA library using a probe based on a partial cDNA sequence of the bovine GnT-IV. The cDNA encoded a complete sequence of a type II membrane protein of 535 amino acids which is 96% identical to the bovine GnT-IV. Transient expression of the human cDNA in COS7 cells increased total cellular GnT-IV activity 25-fold, demonstrating that this cDNA encodes a functional human GnT-IV. Northern blot analysis of normal tissues indicated that at least five different sizes of mRNA (9.7, 7.6, 5.1, 3.8, and 2.4 kb) forGnT-IV are expressed in vivo. Furthermore, these mRNAs are expressed at different levels between tissues. Large amounts of mRNA were detected in tissues harboring T lineage cells. Also, the promyelocytic leukemia cell line HL-60 and the lymphoblastic leukemia cell line MOLT-4 revealed abundant mRNA. Lastly, the gene was mapped at the locus on human chromosome 2, band q12 by fluorescent in situ hybridization.     

Upregulation of the netrin receptor (DCC) gene during activation of b lymphocytes and modulation by interleukins

The DCC (deleted in colon cancer) gene has a brain restricted high expression pattern. It encodes a transmembrane protein of the immunoglobulin superfamily identified as the netrin-1 receptor. It might be a member of the so called "brain-lymphoid" molecules, which control key cell surface events. To test this hypothesis we have assessed the DCC mRNA level in human normal and malignant myeloid and lymphoid cells. A high mRNA content has been observed only in mature B cells at the secreting or presecreting stage. Expression of DCC was also assessed in the anti-CD40 model of immunopoiesis. Activation of purified tonsillar B cells by anti-CD 40 antibody strongly increased the DCC mRNA level and this effect was dramatically enhanced by the association of IL-2 + IL-10, which is a potent and selective in vitro inducer of the B cell memory phenotype. In contrast no effect has been detected after activation of T cells by anti-CD3. These data suggest that the DCC encoded netrin receptor is involved in B cell immunopoiesis.