ILKAP regulates ILK signaling and inhibits anchorage-independent growth

Abstract

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 GSK3b. Silencing of endogenous ILKAP by short interfering RNA (siRNA) stimulated GSK3b 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 GSK3b without affecting S473 phosphorylation of PKB. The ILKAP-mediated inhibition of S9 phosphorylation was rescued by overexpression of ILK, but not by a dominantnegative ILK mutant. The expression level of cyclin D1, a target of ILK-GSK3b 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-GSK3b signaling axis, and suggest that ILKAP activity plays an important role in inhibiting oncogenic transformation.

Inhibition of IGF-I receptor in anchorage-independence attenuates GSK-3β constitutive phosphorylation and compromises growth and survival of medulloblastoma cell lines

“…This interaction involves cell attachment-induced recruitment of integrin-linked kinase (ILK) to the integrin complex, ILK-mediated phosphorylation of Akt, and Akt-mediated phosphorylation of GSK3b (Yau et al, 2005). It has been also proposed that ILK can phosphorylate GSK3b directly without the contribution of Akt, which could additionally enhance GSK3b inactivation and cell survival following interaction with ECM (Cordes and van Beuningen, 2003;Kumar et al, 2004).…”

An Essential Role for the Integrin-Linked Kinase–Glycogen Synthase Kinase-3β Pathway during Dendrite Initiation and Growth

“…Cultures were then washed free of the adenovirus and grown for 3 d in 10 ng/ml NGF or 10 ng/ml NGF plus 50 mM KCl. Recombinant adenoviruses used were dominant-negative ILK (dnILK) and wild-type ILK (wtILK), both expressing EGFP bicistronically (Kumar et al, 2004). An EGFP-expressing recombinant adenovirus (Pozniak et al, 2000) was used as a control.…”

“…Ini-tially, we transduced sympathetic neurons with a previously described dnILK (Kumar et al, 2004) using a replication-defective adenovirus that also expressed EGFP. As a control, we used an adenovirus expressing EGFP only in the same adenoviral backbone.…”

Contributions of the Raf/MEK/ERK, PI3K/PTEN/Akt/mTOR and Jak/STAT pathways to leukemia

“…A second kinase phosphorylates Akt on an S regulatory residue (S473/474/472). [63][64][65][66][67] The identity of the second kinase (often referred to as PDK2) that phosphorylates Akt has remained elusive. Integrin linked kinase (ILK), PDK1, Rictor-mTOR complex (see below) and Akt autophosphorylation have all been suggested to be responsible for phosphorylation of Akt at the second S phosphorylation site.…”