Cloning of an isoform of integrin-linked kinase (ILK) that is upregulated in HT-144 melanoma cells following TGF-beta1 stimulation.
We have shown previously that integrin-linked kinase (ILK) is upregulated in human HT-144 melanoma cells following TGF-beta1 stimulation. Using mRNA from TGF-beta1 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, differs 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 fibrosarcoma cell lines, but not in normal human tissues. Moreover, stimulation of HT-144 cells with TGF-beta1, but not with EGF, PDGF-AB or insulin, induced a selective overexpression of ILK-2 mRNA as compared to ILK-1 mRNA. Bacterially-expressed GST/ILK-2 autophosphorylated and labeled myelin basic protein as well as a recombinant GST/beta3 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-beta1.
Janji
B
B
Laboratoire Franco-Luxembourgeois de Recherche Biomedicale (CRP-Sante/CNRS), University Center, L-1511 Luxembourg, Grand-Duchy of Luxembourg.
Melchior
C
C
Vallar
L
L
Kieffer
N
N
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002455:Cell Division; D018076:DNA, Complementary / Q000235:genetics; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D008545:Melanoma / Q000201:enzymology* / Q000473:pathology; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000737:chemistry* / Q000378:metabolism; D012150:Polymorphism, Restriction Fragment Length; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D014407:Tumor Cells, Cultured / Q000187:drug effects; D015854:Up-Regulation; D000099200:Scaffold Protein ILK
D018076:DNA, Complementary; D007527:Isoenzymes; D009363:Neoplasm Proteins; D017346:Protein Serine-Threonine Kinases; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta; D000099200:Scaffold Protein ILK