Cloning of an isoform of integrin-linked kinase (ILK) that is upregulated in HT-144 melanoma cells following TGF-β1 stimulation

Abstract

We have shown previously that integrin-linked kinase (ILK) is upregulated in human HT-144 melanoma cells following TGF-b1 stimulation. Using mRNA from TGFb1 stimulated HT-144 cells and reverse transcriptase polymerase chain reaction, we have isolated a cDNA encoding a protein highly homologous to ILK. Sequencing of the full-length 1359 base pair cDNA and polypeptide translation revealed that this protein, designated ILK-2, di ers from the known ILK (hereafter called ILK-1) by only four amino acids, while the cDNA sequence diverges by 102 nucleotides, thus excluding that ILK-2 is an allelic variant of ILK-1. Expression of ILK-2 mRNA was observed in metastatic human HT-144 melanoma and HT-1080 ®brosarcoma cell lines, but not in normal human tissues. Moreover, stimulation of HT-144 cells with TGF-b1, but not with EGF, PDGF-AB or insulin, induced a selective overexpression of ILK-2 mRNA as compared to ILK-1 mRNA. Bacteriallyexpressed GST/ILK-2 autophosphorylated and labeled myelin basic protein as well as a recombinant GST/b3 integrin cytoplasmic tail peptide. Transfection of either ILK-2 or ILK-1 cDNA into the non-metastatic melanoma cell line SK-Mel-2, expressing exclusively ILK-1, induced anchorage independent cell growth and cell proliferation, as demonstrated by growth in soft agar. Our data provide evidence that ILK-2 is a new isoform of ILK-1 that is expressed in some highly invasive tumor cell lines but not in normal adult human tissues and whose expression is regulated by TGF-b1.

Integrin-linked Kinase (ILK) Binding to Paxillin LD1 Motif Regulates ILK Localization to Focal Adhesions

“…A monoclonal antibody raised against a sequence in the carboxyl terminus of ILK that is 100% conserved between human ILK-1 and rat ILK, recognized a single 50-kDa band on Western blots of all tissues and cells examined. Thus, whereas it has been suggested that the expression of different ILK isoforms may be developmentally and spatially restricted (23,27), the reason for the discrepancy in the reported size for ILK is unclear at this time.…”

“…ILK activity has been implicated in growth factor and Wnt signaling pathways (14,17,18), as well as in integrin-mediated cellular processes including regulation of cell adhesion (14), fibronectin matrix assembly (19), and cell adhesion-dependent cell cycle progression (20 -22). A second human ILK isoform (ILK-2) that is selectively up-regulated in metastatic melanoma cells has recently been described (23).…”

Integrin-Linked Kinase, a Hypoxia-Responsive Molecule, Controls Postnatal Vasculogenesis by Recruitment of Endothelial Progenitor Cells to Ischemic Tissue

“…24 However, neither blockade of TGF-␤ with neutralizing antibody nor blockade of intracellular TGF-␤ signaling with Smad7 transfection could block the upregulation of ILK in hypoxic ECs (data not shown). In a previous report by Melchior et al, 25,26 there were 2 isoforms of ILK, namely, ILK1 and ILK2, that could only be discriminated by BamHI cDNA restriction. ILK1, which has no TGF-␤ responsive element in its promoter site and is not restricted by BamHI, was found to have typical characteristics of a housekeeping gene.…”

Tumor expression of Integrin-linked kinase (ILK) correlates with the expression of the E-cadherin repressor Snail: an immunohistochemical study in ductal pancreatic adenocarcinoma

“…Overexpression of ILK promotes oncogenic transformation [14,15] and promotes tumor invasion partially due to the inhibition of E-cadherin [16,17]. The transcription factor Snail has been shown to directly regulate expression of E-cadherin via the ILK pathway, as overexpression of ILK causes a stimulation of expression of Snail [13].…”