Elsevier

Gene

Volume 273, Issue 1, 25 July 2001, Pages 29-39
Gene

Differential expression and subcellular distribution of the mouse metastasis-associated proteins Mta1 and Mta3

https://doi.org/10.1016/S0378-1119(01)00563-7Get rights and content

Abstract

The human metastasis-associated gene (MTA1) is overexpressed in cell lines and tissues representing metastatic tumors. Here we report cloning of the mouse Mta1 as well as a novel structurally related mouse gene, Mta3. The mouse Mta1 protein shares 94 and 59% homology to the human MTA1 and mouse Mta3 proteins, respectively. Northern blotting analysis using an Mta1 cDNA probe revealed a prevalent 3 kb hybridization signal in all mouse tissues except the skeletal muscle while a smaller ∼1.0 kb mRNA product was also detected in the heart. Mta3 transcripts (∼2 kb) were detected in most tissues with an additional ∼6.2 kb signal detected in the brain. In vitro transcription/translation of the full-length Mta1 and Mta3 cDNAs generated products of the expected molecular masses, i.e. 80 and 60 kDa, respectively. To assess subcellular localization, green fluorescence protein (GFP)-tagged expression constructs of Mta1 and Mta3 and various deletion constructs of GFP-Mta1 were transiently expressed in Balb/MK keratinocytes. GFP-Mta1 was found exclusively in the nucleus while GFP-Mta3 was present in both the nucleus and cytoplasm. Compared to Mta3, the carboxy terminal end of Mta1 contains an additional nuclear localization signal (NLS) and a proline-rich Src homology 3 (SH3) ligand. The results of transient expression experiments of various Mta1 fragments containing these domains in different combinations indicated that nuclear localization of Mta1 depended on the presence of at least one NLS and one SH3 binding site. These SH3 ligands appeared to be functional as they facilitated interaction with the adaptor protein, Grb2, and the Src-family tyrosine kinase, Fyn.

Introduction

The metastasis associated gene 1 (mta1) was originally identified by differential cDNA screening using rat adenocarcinoma cell lines with high and low metastatic potential (Pencil et al., 1993, Toh et al., 1994, Toh et al., 1995). Rat mta1 is overexpressed four-fold in highly metastatic (MTLn3) as compared to non-metastatic (MTC.4) cells (Toh et al., 1994). Similarly, human MTA1 is expressed at high levels in highly metastatic breast, esophageal, colorectal and gastric carcinomas as compared to less aggressive lesions or normal tissues (Toh et al., 1994, Toh et al., 1997, Toh et al., 1999). In culture, highly proliferative breast adenocarcinoma cells overexpress MTA1 compared to normal human breast epithelial cells and growth is inhibited by treatment with MTA1 antisense phosphorothioate oligonucleotides (Nawa et al., 2000). It is unclear whether and how MTA1 contributes to the metastatic phenotype of tumor cells. However, recent evidence has implicated MTA1 in the regulation of gene expression by covalent modification of histone proteins. In support of this notion, MTA1 and its homolog, MTA2, are components of and functionally contribute to the nucleosome-remodeling histone-deacetylase (NuRD) complex (Toh et al., 2000, Xue et al., 1998, Zhang et al., 1998, Zhang et al., 1999). The NuRD complex has both histone deacetylase and nucleosome remodeling activities (Tong et al., 1998, Xue et al., 1998, Zhang et al., 1998). The core complex is composed of histone deacetylases (HDAC1 and HDAC2) and the histone binding proteins RbAp48 and RbAp46. MTA2 is recruited to the core complex by the methyl-CpG-binding-domain-containing protein (MDB3) and serves to enhance the complex's histone deacetylase activity (Zhang et al., 1999). Histone deacetylation by the NuRD complex (Herman et al., 1999, Paterno et al., 1997) has been linked to transcriptional repression, cellular proliferation and cancer (Kadosh and Struhl, 1998, Kuo and Allis, 1998, Archer and Hodin, 1999). However, the contributory role of members of the MTA family and NuRD complex to the metastatic potential of tumor cells remains to be defined.

MTA1 is representative of a family of genes which is highly conserved through evolution and includes the metastasis associated 1-like protein (MTA1-L1) (Futamura et al., 1999, Zhang et al., 1999), and MTA2 (Zhang et al., 1999). MTA1-L1 has been cloned from human (Futamura et al., 1999), mouse (Zhang et al., 1999), Drosophila melanogaster (GenBank Accession number: AF170345), and Xenopus laevis (GenBank Accession number: AF170344). In addition, in pancreatic acinar cells a derivative of the rat mta1 gene, ZG29, has been identified (Kleene et al., 1999). ZG29 encodes an N-terminally truncated form of mta1 due to alternative transcription initiation of the rat mta1 gene and is highly expressed in hormonally stimulated acinar cells.

Here we describe cloning and initial characterization of mouse Mta1 and a novel Mta family member, Mta3. We compared tissue expression patterns and subcellular localizations of Mta1 and Mta3 transcripts and proteins, respectively. We provide evidence for the presence of both Mta1 and Mta3 in cell nuclei consistent with roles in histone modification or transcriptional regulation. Furthermore, we show enhanced nuclear retention of Mta1, compared to Mta3, possibly imparted by the carboxy terminal sequences of Mta1 containing an additional nuclear localization signal (NLS) and a proline-rich SH3 binding site. Finally, we demonstrate that the putative SH3 ligand sites on Mta1 and Mta3 enable binding to SH3 domain-containing proteins such as Fyn and Grb2.

Section snippets

Cloning of mouse Mta1 and Mta3

The following primers, forward 5′-ACA TGG CCG CCA ACA TGT AC-3′ (position −2–18) and reverse 5′-CTG CCA GCA GGG TCT TCT GC-3′ (position 468–487), were used to PCR amplify a 489 bp fragment of human cDNA (Clontech, Palo Alto, CA) encoding for human MTA1. The PCR conditions were as follows: 94°C for 2 min, followed by 35 cycles of 94°C for 30 s, 58°C for 1 min, and 72°C for 1 min. This DNA product was used to probe a mouse keratinocyte cDNA Uni-Zap XR library (Stratagene, La Jolla, CA) according

Identification and cloning of mouse Mta1 and Mta3

A 489 bp fragment of human cDNA encoding for human MTA1 was generated by RT-PCR and used to screen a mouse keratinocyte cDNA Uni-Zap XR library. We obtained one clone containing the complete cDNA for Mta1 of 2.8 kb. The cDNA includes a 2.1 kb open reading frame encoding a protein of 698 amino acids (Fig. 1A). The putative translation initiation codon (ATG) lies downstream of a GC-rich region (96% within 102 bp). The open reading frame is followed by a translation termination codon (TAG) and a

Discussion

Here we report two new members of the mouse Mta family, Mta1 and Mta3. They share sequence homology with rat mta1 and the human MTA1 and MTA1-L1, genes that are expressed at high levels in cell lines and tissues representing highly metastatic tumors. Mouse Mta1 and Mta3 contain different functional domains associated with distinct subcellular expression patterns.

Mta3 was expressed in all tissues examined except skeletal muscle. A similar expression pattern has been reported previously for human

Acknowledgements

We would like to thank Dayna Levin, Sonul Mehta and Rachel Altman for their excellent technical help. We thank Drs Gabriele Richard and Joy Mulholland for critically reading this manuscript. We also thank Drs Paul Stein and John Seykora (Department of Dermatology, University of Pennsylvania, Philadelphia, PA) for the Fyn-GST proteins and Dr Jeffrey Benovic (Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, PA) for the Nck- and Grb2-GST fusion proteins. This work was supported

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