Cloning of an isoform of integrin-linked kinase (ILK) that is upregulated in HT-144 melanoma cells following TGF-beta 1 stimulation

We have shown previously that integrin-linked kinase (ILK) is upregulated i n human HT-144 melanoma cells following TGF-beta 1 stimulation, Using mRNA from TGF-beta 1 stimulated HT-144 cells and reverse transcriptase polymeras e chain reaction, we have isolated a cDNA encoding a protein highly homolog ous to ILK. Sequencing of the full-length 1359 base pair cDNA and polypepti de translation revealed that this protein, designated ILK-2, differs from t he known ILK (hereafter called ILK-1) by only four amino acids, while the c DNA sequence diverges by 102 nucleotides, thus excluding that ILK-2 is an a llelic variant of ILK-1, Expression of ILK2 mRNA was observed in metastatic human HT-144 melanoma and HT-1080 fibrosarcoma tell lines, but not in norm al human tissues, Moreover, stimulation of HT-144 cells with TGF-beta 1, bu t not with EGF, PDGF-AB or insulin, induced a selective overexpression of I LK-2 mRNA as compared to ILK-1 mRNA, Bacterially-expressed GST/ILK-2 autoph osphorylated and labeled myelin basic protein as well as a retombinant GST/ beta 3 integrin cytoplasmic tail peptide, Transfection of either ILK-2 or I LK-1 cDNA into the non-metastatic melanoma cell line SK-Mel-2, expressing e xclusively ILK-1, induced anchorage independent cell growth and cell prolif eration, 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 invas ive tumor cell lines but not in normal adult human tissues and whose expres sion is regulated by TGF-beta 1.