Role of integrin-linked kinase in nerve growth factor-stimulated neurite outgrowth - PubMed (original) (raw)

ILK inhibition reduces AKT and GSK-3 phosphorylation in PC12 cells after NGF receptor stimulation.A, NGF increases ILK activity in a concentration-dependent manner. PC12 cells, plated onto laminin, were exposed to increasing concentrations of NGF for 30 min. After immunoprecipitation, ILK activity was measured by AKT substrate radiolabeling. Top, Average CPM of AKT substrate radiolabeling; n = 3 ± SEM.Bottom, Representative in vitro kinase assay. B, NGF increases ILK phosphorylation of AKT Ser-473. ILK kinase activity was measured in PC12 cells plated onto collagen I after a 10 min exposure to NGF (100 ng/ml). ILK activity was measured using a His-tagged fusion protein corresponding to human AKT as an exogenous substrate. Phosphorylation of the substrate by ILK is detected using a phosphospecific anti-AKT Ser-473 antibody. The antibody control (lane 3) corresponds to lysate immunoprecipitated with an irrelevant antibody (anti-flag monoclonal antibody; Upstate Biotechnology). C, KP-392 inhibits NGF stimulation of AKT and GSK phosphorylation in a dose-dependent manner. Western blots of lysates from PC12 cells grown on collagen I and treated with NGF (50 ng/ml) for 4 hr in the presence of vehicle control or increasing concentrations of KP-392. Representative Western blots of cell lysates, run in parallel and probed with antibodies for phospho-AKT, phospho-GSK-3, or phospho-p38 MAPK; n = 3–5. These same blots were subsequently stripped and reprobed for AKT, GSK-3, or p38 MAPK as a control for protein expression levels. D, KP-392 does not inhibit phosphorylation of PI-3 kinase or TrkA. Left, Representative Western blot of PC12 cell lysates from PC12 cells treated for 4 hr with NGF in the presence or absence of KP-392 (100 μ

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) and probed with a phosphospecific antibody for TrkA (Tyr490). Right, Levels of the p85 regulatory subunit of PI-3 kinase were analyzed in anti-phosphotyrosine immunoprecipitates by Western blotting. E, Long-term effects of KP-392 (100 μ

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) on cell signaling in PC12 cells grown on collagen I (Col I), collagen IV (Col IV), or laminin (L) and incubated in the presence or absence of NGF (50 ng/ml) for 48 hr. Representative Western blots of cell lysates, run in parallel and probed with antibodies for phospho-AKT, phospho-GSK-3, or phospho-ERK; n = 3. After membrane stripping, Western blots were subsequently reprobed for AKT, GSK-3, and ERK.F, Overexpression of ILK-DN decreases NGF-induced stimulation of AKT and GSK-3 phosphorylation. PC12 cells were transfected with ILK-DN:V5 or EmptyV5. Twenty-four hours after transfection, cells were replated onto collagen I and were serum-starved overnight. Representative Western blots of phospho-AKT and phospho-GSK-3 after a 15 min exposure to NGF (50 ng/ml); n = 3. These same Western blots were subsequently stripped and reprobed with an anti-V5 or anti-GSK-3 antibody, respectively.