ILKAP regulates ILK signaling and inhibits anchorage-independent growth.
ILKAP is a protein phosphatase 2C that selectively associates with integrin linked kinase, ILK, to modulate cell adhesion and growth factor signaling. We investigated the role of endogenous cellular ILKAP in antagonizing ILK signaling of two key targets, PKB and GSK3beta. Silencing of endogenous ILKAP by short interfering RNA (siRNA) stimulated GSK3beta phosphorylation at S9, with no effect on PKB S473 phosphorylation. In LNCaP prostate carcinoma cells, transient or stable expression of ILKAP suppressed ILK immune complex kinase activity, demonstrating an interaction between ILKAP and ILK. Consistent with the silencing data, ILKAP inhibition of ILK selectively inhibited S9 phosphorylation of GSK3beta without affecting S473 phosphorylation of PKB. The ILKAP-mediated inhibition of S9 phosphorylation was rescued by overexpression of ILK, but not by a dominant-negative ILK mutant. The expression level of cyclin D1, a target of ILK-GSK3beta signaling, was inversely correlated with ILKAP protein levels, suggesting that antagonism of ILK modulates cell cycle progression. ILKAP expression increased the proportion of LNCaP cells in G1, relative to vector control cells, and siRNA suppression of ILKAP increased entry of cells into the S phase, consistent with ILK antagonism. Anchorage-independent growth of LNCaP cells was inhibited by ILKAP, suggesting a critical role in the suppression of cellular transformation. Taken together, our results indicate that endogenous ILKAP activity inhibits the ILK-GSK3beta signaling axis, and suggest that ILKAP activity plays an important role in inhibiting oncogenic transformation.Oncogene (2004) 23, 3454-3461. doi:10.1038/sj.onc.1207473 Published online 1 March 2004
Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada.
Ashu S
AS
Kumar
Izabela
I
Naruszewicz
Ping
P
Wang
Chungyee
C
Leung-Hagesteijn
Gregory E
GE
Hannigan
D002453:Cell Cycle; D002455:Cell Division; D002471:Cell Transformation, Neoplastic; D002478:Cells, Cultured; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D006801:Humans; D010749:Phosphoprotein Phosphatases / Q000502:physiology*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D034741:RNA, Small Interfering / Q000494:pharmacology; D015398:Signal Transduction; D000099200:Scaffold Protein ILK
D038362:Glycogen Synthase Kinase 3; D000071679:Glycogen Synthase Kinase 3 beta; D010749:Phosphoprotein Phosphatases; D017346:Protein Serine-Threonine Kinases; D034741:RNA, Small Interfering; D000099200:Scaffold Protein ILK; C000605506:GSK3B protein, human; C426826:ILKAP protein, human