Identification of two novel human dynein light chain genes, DNLC2A and DNLC2B, and their expression changes in hepatocellular carcinoma tissues from 68 Chinese patients
Introduction
Cytoskeletal molecular motors are a group of proteins or protein complexes that are thought to be responsible for a wide variety of cellular functions. These motor proteins enable cells to move, contract, change shape, secrete, endocytosis, organize the cytoplasm, and conduct karyokinesis and cytokinesis (George, 2000). They fall into three superfamilies: myosins, kinesins and dyneins (George, 2000). Myosins are predominantly present in muscle cells as the only actin-based motors involved in plus-directed (from nuclear towards the membrane) movement of intracellular cargo along actin filaments. Dyneins and kinesins are the microtubule-dependent motors in eukaryotic cells. Kinesins are responsible for the plus-directed cargo trafficking. While the minus end-directed cargo movements are performed by dyneins and minus end-directed kinesins. Cytoplasmic Dynein contains of two or more heavy chains (HCs) of ∼530 kDa that form the globular heads and stems of the particles and are believed to be the sites of ATP hydrolysis and microtubule-binding (Bowman et al., 1999, King et al., 1996a). Associated to these dynein stems are intermediate chains (ICs) with multiple Trp–Asp amino acid repeats and several light chains (LCs). ICs act directly in the attachment of the dynein motor to its cargo (King, 2000). LCs is essential for dynein assembly. There is growing evidence suggesting that these accessory proteins are essential for dynein function and regulation (Bowman et al., 1999, King, 2000).
The Drosophila roadblock (robl) protein gene was identified from a genetic mutant that exhibited diverse defects in intracellular transport including a distal biased axonal transport defect, massive axonal loss and nerve degeneration. They also cause severe mitotic defect and female sterile (Bowman et al., 1999, King, 2000). Sequence analysis showed that the protein encoded by Drosophila robl is of 70% similarity to Chlamydomonas dynein light chain LC7 (Bowman et al., 1999). These homologues belong to a conserved new family of dynein-associated proteins whose molecular structure are different from other known dynein light chains, such as human DNLC1 and Drosophilas ddlc, etc. They function in axonal transport, flagellar motility, and mitosis though there is some variability in the number of robl/LC7-like proteins across species (Bowman et al., 1999). In the mammalian expressed sequence tag database, they fall into two classes. However, little is known about how and under what conditions these robl/LC7 proteins modulate dynein activity, and whether they have different expression in the actively growing and proliferating tumor cells.
Human hepatocellular carcinoma (HCC) is one of the commonest malignant tumors worldwide and a leading cause of death in many countries, especially in Asia and Africa (Bosch, 1997, Murray and Lopez, 1997). Accumulating evidence suggests that HCC may arise through accumulation of a number of genetic lesions besides some well defined risk factors, such as chronic viral hepatitis, alcohol, and metabolic disorders, in the contest of increased cellular turnover induced by chronic liver injury, regeneration and cirrhosis (Moradpour and Blum, 2000, Brechof et al., 2000). As generally observed in other tumors, the genetic lesions contributing to the development of HCC may include activation of cellular oncogenes, inactivation of tumor suppressor genes, over-expression of certain growth factors, and possibly telomerase activation and DNA mismatch repair defects (Bucndia, 2000, Chordhury et al., 1996). Besides these common causes for HCC carcinogenesis, some other proteins such as DNLC-1 were also reported to be over-expressed in nasopharyngeal carcinoma (Fung et al., 2000), and promote cell survival and inhibit apoptosis. LC8, another dynein light chain, also named PIN (protein inhibitor of neuronal nitric oxide synthase), can inhibit nerve growth factor withdrawal apoptosis in differentiated pc12 cells. However, these findings are, in most cases, dependent on precedent knowledge based on oncogene analysis in other types of cancer. Therefore, the wide variety of genetic differences between HCC and tumor-free liver remain to be elucidated. Here, we report cloning and characterization of two novel members of human DNLC family and describe the results of the expression changes of the two novel genes in hepatocellular carcinoma tissues from 68 Chinese patients.
Section snippets
Identification of the expressed sequence tag sequences
To find new members of the human DNLC, the nucleotide sequence of the Rattus norvegicus robl/LC7-like gene 1 (GenBank accession no. AF073839) was used to screen the human EST divisions of the GenBank database at NCBI. A number of ESTs (D80282, D59735, W17228, A120752, D61624, AA340594, etc.) were obtained and assembled into two EST-contig, termed RD1 and RD2.
Cloning and sequencing
To obtain full-length cDNA sequence of RD1 and RD2, we designed two pairs of specific primers, RD1-A (5′-GGACTCGCTAAGTGTTCGCTACG-3′),
Cloning of the DNLC2A and DNLC2B cDNA
Two contigs were identified by screening the human EST database and were verified by amplifying and sequencing the specific PCR fragments with primers RDA1, A2 and RDB1, B2. The full length of DNLC2A cDNA is 1028 bp (Fig. 1A) (GenBank accession no. AF132750) and of DNLC2B cDNA is 506 bp (Fig. 1B) (GenBank accession no. AF125108), which contain the whole open reading frame. The putative proteins of DNLC2A and DNLC2B both consist of 96 amino acid residues, with a predicted molecular weight of
Discussion
Dynein is a large protein complex composed of a number of subunits. Dynein heavy chains contain ATPase and motor activities, and intermediate chains are involved in cargo binding, regulated by a group of light chains (King, 2000). However, the functions of LCs remain to be discovered. Until now, eight dynein LCs have been identified, among which LC8 is the most well characterized. LC8 was firstly identified as an integral component of the Chlamydomonas outer dynein arm and is required for outer
Acknowledgements
This work was supported by the national 973 program (973-010203), 863 Project (863-Z020401) of China. We also give thanks to Dr. Jian Huang, the director of Qidong Liver Cancer Institute, Jiangsu province, China and Dr. Changyuan Wei, of the liver cancer department of Guangxi Tumor hospital, Guangxi province, China, for supplying fresh surgical specimens of liver cancer.
References (22)
- et al.
Differential gene expression in nasopharyngeal carcinoma cells
Life Sci.
(2000) - et al.
Cytoplasmic dynein and dynactin in cell and intracellular transport
Curr. Opin. Cell Biol.
(1999) The dynein microtubule motor
Biochim. Biophys. Acta
(2000)- et al.
Brain cytoplasmic and flagellar outer arm dyneins share a highly conserved Mr 8000 light chain
J. Biol. Chem.
(1996) - et al.
The mouse t-complex-encoded protein Tctex-1 is a light chain of brain cytoplasmic dynein
J. Biol. Chem.
(1996) - et al.
Dynein light chains of the roadblock/LC7 group belong to an ancient protein superfamily implicated in NTPase regulation
Curr. Biol.
(2000) - et al.
Mortality by cause for eight regions of the world: global burden of disease study
Lancet
(1997) - et al.
Light-induced down-regulation of the rat class 1 dynein-associated protein robl/LC7-like gene in visual cortex
J. Biol. Chem.
(2000) Global epidemiology of hepatocellular carcinoma
- et al.
Drosophila roadblock and Chlamydomonas LC7: A conserved family of dynein-associated proteins involved in axonal transport, flagellar motility, and mitosis
J. Cell Biol.
(1999)
Molecular bases for the development of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC)
Cancer Biol.
Cited by (56)
DYNLRB1 is essential for dynein mediated transport and neuronal survival
2020, Neurobiology of DiseaseCitation Excerpt :There are three families of dynein light chains, LC7/road-block, LC8 and Tctx1/rp3, the latter playing a role in the direct binding of cargoes to the dynein motor complex (Olenick and Holzbaur, 2019; Reck-Peterson et al., 2018; Tai et al., 1999). Dynein Light Chain Roadblock-Type 1 (DYNLRB1), also known as Km23, and DNLC2A (Jiang et al., 2001; Tang, 2002), was first identified in Drosophila during a genetic screen for axonal transport mutants (Bowman et al., 1999). Roadblock mutants exhibited defects in intracellular transport, including intra-axonal accumulation of synaptic cargoes, severe axonal degeneration and aberrant chromosome segregation (Bowman et al., 1999).
Severe skeletal abnormalities caused by defects in retrograde intraflagellar transport dyneins
2018, Dyneins: Dynein Mechanics, Dysfunction, and Disease: Second EditionStructural analysis of dynein intermediate and light chains
2018, Dyneins: Dynein Mechanics, Dysfunction, and Disease: Second EditionCytoplasmic dynein function defined by subunit composition
2017, Dyneins: The Biology of Dynein Motors: Second EditionRequirement for protein kinase A in the phosphorylation of the TGFΒ receptor-interacting protein km23-1 as a component of TGFΒ downstream effects
2013, Experimental Cell ResearchCitation Excerpt :We have previously identified km23-1 as a TGFβ receptor-interacting protein in a novel screen [14]. km23-1, also termed km23 [14–16], Robl1 [17], DNLC2A [18], mLC7-1 [14], DYNLRB1 [19] has been shown to interact with Rab6 [20], the human reduced folate carrier [21], dynein intermediate chain (DIC) [14,22], and LIN-5 and GPR-1/2 in Caenorhabditis elegans [23]. We have shown that km23-1 undergoes rapid phosphorylation on serine residues after TβR activation, in keeping with the kinase specificity of the TβRs [14].
Role of km23-1 in RhoA/actin-based cell migration
2012, Biochemical and Biophysical Research CommunicationsCitation Excerpt :As a result, there has been considerable interest in the possibility that specific proteins in the signal transduction pathways mediating these cytoskeletal events could be potential targets for cancer therapy [9]. km23-1 (also referred as km23 [10–12], Robl1 [13], DNLC2A [14], mLC7-1 [15], DYNLRB1 [16]) was originally identified as a TGFß receptor-interacting protein that is also a dynein light chain [15]. More recently, we have shown that Ras and km23-1 form a TGFß-regulated complex in vivo [17].