HIC-5: A Mobile Molecular Scaffold Regulating the Anchorage Dependence of Cell Growth

Hindawi Publishing Corporation International Journal of Cell Biology Volume 2012, Article ID 426138, 8 pages doi:10.1155/2012/426138 Review Article HIC-5: A Mobile Molecular Scaffold Regulating the Anchorage Dependence of Cell Growth Motoko Shibanuma, Kazunori Mori, and Kiyoshi Nose Department of Cancer Cell Biology, Showa University School of Pharmacy, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan Correspondence should be addressed to Motoko Shibanuma, Received 31 July 2011; Accepted 2 September 2011 Academic Editor: Motoharu Seiki Copyright © 2012 Motoko Shibanuma et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. HIC-5 is a multidomain LIM protein homologous to paxillin that serves as a molecular scaffold at focal adhesions and in the nucleus. It forms mobile molecular units with LIM-only proteins, PINCH, and CRP2 and translocates in and out of the nucleus via a nuclear export signal (NES). Of note, NES of HIC-5 is distinctive in its sensitivity to the cellular redox state. Recently, the mobile units of HIC-5 have been suggested to be involved in the regulation of the anchorage dependence of cell growth. On loss of adhesion, an increase in reactive oxygen species in the cells modifies NES and stops shuttling, which leads to cell-cycle control. More specifically, the system circumvents nuclear localization of cyclin D1 and transactivates p21Cip1 in detached cells, thereby avoiding anchorage-independent cell growth. Thus, the HIC-5-LIM only protein complex has emerged as a fail-safe system for regulating the anchorage dependence of cell growth. 1. Introduction Hydrogen peroxide-inducible clone 5 or Hic-5 is a gene we isolated by subtractive hybridization in 1994 as a cDNA clone induced by transforming growth factor β (TGF-β) or hydrogen peroxide [1]. At that time, we studied TGF-β signalling and pursued the possibility that reactive oxygen species (ROS) function was an intracellular TGF-β signal. After isolating the gene, we conducted a number of studies of Hic-5 at a molecular as well as cellular level. Its amino acid sequence revealed that HIC-5 is a homologue of paxillin, which is a multidomain LIM (Lin-11, Isl-1, and Mec-3) protein that is localized at focal adhesions and was originally identified as a substrate of the v-src oncogene [2] (Figure 1). Together with its family members (Leupaxin specifically expressed in lymphocytes, PaxB, an orthologue of paxillin in slime mold, and HIC-5), paxillin has now been established as a molecular adaptor that transduces signals in response to changes in the adhesion environment of cells. A famous example of a molecular adaptor is the Grb2-SOS system that transduces signals from growth factor receptors to RAS. Paxillin transduces signals from extracellular matrix receptors, integrins, to intracellular downstream molecules such as MAP kinase. Of these family members, HIC-5 is most homologous to paxillin, and thus, analyses of HIC-5 have been conducted in reference to and in comparison with paxillin. For example, the intracellular localization of HIC-5 is, like paxillin, mainly confined to so-called focal adhesion sites where cells adhere to the extracellular matrix via integrins. In terms of expression in tissues and cell types, paxillin is relatively ubiquitously expressed, whereas expression of HIC-5 is prominent in the smooth muscle layer of tissues such as the large intestine and uterus [3]. Furthermore, expression of HIC-5 is relatively high in the lung and spleen [1]. In cell culture systems, HIC-5 expression is detectable in most cell lines with varying degrees of expression. High expression of HIC-5 is detected in mesenchymal cell lines including fibroblastic and osteoblastic cell lines; however, it is generally low in epithelial cell lines. In a knockout mouse model, HIC-5 was suggested to be inessential for the development and maintenance of homeostasis of the animal, and no remarkable functional abnormality was found under standard rearing 2 International Journal of Cell Biology 31 118 PY PY 188/190 PY Paxillin LD1 LD2 LD3 LD4 LD5 LIM1 LIM2 LIM3 LIM4 LIM1 LIM2 LIM3 LIM4 Src Crk 60 PY Hic-5 LD1 LD2 LD3 LD4 Vinculin FAK GIT PTP-PEST Csk HSP27 PKL PYK2 Leupaxin LD1 LD2 LD3 LD4 LIM1 LIM2 LIM3 LIM4 LD3 LIM1 LIM2 LIM3 LIM4 PaxB LD1 LD2 LD4 Figure 1: The paxillin/focal adhesion-associated adaptor protein family; domain structure and binding factors. The paxillin family includes HIC-5, Leupaxin, which is preferentially expressed in hematopoietic cells, and PaxB, an orthologue of paxillin in the slime mould Dictyostellium discoidium. The family members share many of their structural characteristics and binding factors. They have four to five leucine-rich motifs (LD repeats) in the N-terminal half and four cysteine-rich regions composed of two zinc fingers (LIM domains) in the C-terminal half. These domains mediate the protein-protein interactions that allow paxillin to bind a variety of structural and signalling molecules, such as the structural protein vinculin, the SH2-SH3 adaptor protein Crk, Src, focal adhesion kinase (FAK), PTK2B protein tyrosine kinase 2 beta (PYK2), a negative regulator of Src, the Csk nonreceptor tyrosine kinase, the G protein-coupled receptor kinase interactor Arf GAP1 (GIT-1), paxillin-kinase linker (PKL), protein tyrosine phosphatase-PEST (PTP-PEST), and heat shock protein 27 (HSP27). The LIM domains also mediate the localization of Hic-5 at the nucleus and at focal adhesions. HIC-5 has the same binding partners, except for Crk and Src, as paxillin. conditions [4]. In contrast, the paxillin knockout mouse is reportedly embryonic lethal [5]. Similar to fibronectin, it exhibits abnormal development of extraembryonic tissues and heart and body segmentation, resulting in death at 9.5 foetal days. The embryonic lethality of the paxillin knockout mouse means that HIC-5 cannot substitute the functions of paxillin, at least those associated with development. These results together with the abovementioned differences in expression patterns indicate that it is most likely that paxillin and HIC-5 have different functions in mammals. 2. Structure of HIC-5 and Interacting Factors The genomic structure of Hic-5 features a long intron between the N-terminal and C-terminal domains, a sign that Hic-5 evolved from the fusion of two different genes [6]. Accordingly, the protein structure can also be broadly divided from the centre into N-terminal and C-terminal regions. The N-terminal region comprises four domains, the LD domains, which are rich in Leu and Asp; LD1 is deleted in one isoform. The C-terminal region comprises four LIM domains having two zinc fingers (Figure 1). These features are almost identical to those of paxillin, with minor differences in the number of LD domains in the Nterminal (...truncated)