Conditional Knock-out of Integrin-linked Kinase Demonstrates an Essential Role in Protein Kinase B/Akt Activation

Protein kinase B (PKB/Akt) plays a pivotal role in signaling pathways downstream of phosphatidylinositol 3-kinase, regulating fundamental processes such as cell survival, cell proliferation, differentiation, and metabolism. PKB/Akt activation is regulated by phosphoinositide phospholipid-mediated plasma membrane anchoring and by phosphorylation on Thr-308 and Ser-473. Whereas the Thr-308 site is phosphorylated by PDK-1, the identity of the Ser-473 kinase has remained unclear and controversial. The integrin-linked kinase (ILK) is a potential regulator of phosphorylation of PKB/Akt on Ser-473. Utilizing double-stranded RNA interference (siRNA) as well as conditional knock-out of ILK using the Cre-Lox system, we now demonstrate that ILK is essential for the regulation of PKB/Akt activity. ILK knock-out had no effect on phosphorylation of PKB/Akt on Thr-308 but resulted in almost complete inhibition of phosphorylation on Ser-473 and significant inhibition of PKB/Akt activity, accompanied by significant stimulation of apoptosis. The inhibition of PKB/Akt Ser-473 phosphorylation was rescued by kinase-active ILK but not by a kinase-deficient mutant of ILK, suggesting a role for the kinase activity of ILK in the stimulation of PKB/Akt phosphorylation. ILK knock-out also resulted in the suppression of phosphorylation of GSK-3β on Ser-9 and cyclin D1 expression. These data establish ILK as an essential upstream regulator of PKB/Akt activation. Protein kinase B (PKB/Akt) plays a pivotal role in signaling pathways downstream of phosphatidylinositol 3-kinase, regulating fundamental processes such as cell survival, cell proliferation, differentiation, and metabolism. PKB/Akt activation is regulated by phosphoinositide phospholipid-mediated plasma membrane anchoring and by phosphorylation on Thr-308 and Ser-473. Whereas the Thr-308 site is phosphorylated by PDK-1, the identity of the Ser-473 kinase has remained unclear and controversial. The integrin-linked kinase (ILK) is a potential regulator of phosphorylation of PKB/Akt on Ser-473. Utilizing double-stranded RNA interference (siRNA) as well as conditional knock-out of ILK using the Cre-Lox system, we now demonstrate that ILK is essential for the regulation of PKB/Akt activity. ILK knock-out had no effect on phosphorylation of PKB/Akt on Thr-308 but resulted in almost complete inhibition of phosphorylation on Ser-473 and significant inhibition of PKB/Akt activity, accompanied by significant stimulation of apoptosis. The inhibition of PKB/Akt Ser-473 phosphorylation was rescued by kinase-active ILK but not by a kinase-deficient mutant of ILK, suggesting a role for the kinase activity of ILK in the stimulation of PKB/Akt phosphorylation. ILK knock-out also resulted in the suppression of phosphorylation of GSK-3β on Ser-9 and cyclin D1 expression. These data establish ILK as an essential upstream regulator of PKB/Akt activation. PKB/Akt 1The abbreviations used are: PKB/Akt, protein kinase B; CSF-1, colony-stimulating factor-1; GSK-3β, glycogen synthase kinase-3β; GFP, green fluorescent protein; PI 3-kinase, phosphatidylinositol 3-kinase; ILK, integrin linked kinase; PDK-1, PI 3-kinase-dependent kinase-1; siRNA, small interfering RNA; WT, wild-type; PTEN, protein with tensin homology; fl/fl, floxed/floxed. is activated in a PI 3-kinase-dependent manner and regulates cell survival, proliferation, differentiation, motility, and metabolism (1Nicholson K.M. Anderson N.G. Cell Signal. 2002; 14: 381-395Crossref PubMed Scopus (1388) Google Scholar). The activity of PKB/Akt is constitutively activated under situations of chronic activation of PI 3-kinase, for example, by the mutational inactivation of the tumor suppressor PTEN. PKB/Akt regulates apoptosis and cell cycle progression by promoting the phosphorylation of proapoptotic proteins such as Bad, Forkhead transcription factors, and the cell cycle inhibitor p27(kip1) (2Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5434) Google Scholar, 3Kops G.J. de Ruiter N.D. De Vries-Smits A.M. Powell D.R. Bos J.L. Burgering B.M. Nature. 1999; 398: 630-634Crossref PubMed Scopus (952) Google Scholar, 4Liang J. Zubovitz J. Petrocelli T. Kotchetkov R. Connor M.K. Han K. Lee J.H. Ciarallo S. Catzavelos C. Beniston R. Franssen E. Slingerland J.M. Nat. Med. 2002; 8: 1153-1160Crossref PubMed Scopus (811) Google Scholar, 5Viglietto G. Motti M.L. Bruni P. Melillo R.M. D'Alessio A. Califano D. Vinci F. Chiappetta G. Tsichlis P. Bellacosa A. Fusco A. Santoro M. Nat. Med. 2002; 8: 1136-1144Crossref PubMed Scopus (606) Google Scholar). The full activation of PKB/Akt requires phosphorylation on Thr-308 and Ser-473 (6Kobayashi T. Cohen P. Biochem. J. 1999; 339: 319-328Crossref PubMed Scopus (529) Google Scholar). Whereas PDK-1 has been demonstrated to phosphorylate PKB/Akt on Thr-308 (7Alessi D.R. James S.R. Downes C.P. Holmes A.B. Gaffney P.R. Reese C.B. Cohen P. Curr. Biol. 1997; 7: 261-269Abstract Full Text Full Text PDF PubMed Google Scholar), the kinase responsible for phosphorylation at the hydrophobic Ser-473 site (PDK-2) has not been identified despite extensive efforts. PDK-1 was initially proposed as also being responsible for the stimulation of phosphorylation of Ser-473 (8Balendran A. Casamayor A. Deak M. Paterson A. Gaffney P. Currie R. Downes C.P. Alessi D.R. Curr. Biol. 1999; 9: 393-404Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar). However, this site is inducibly phosphorylated in PDK-1 knock-out cells, pointing to the existence of a distinct Ser-473 kinase (9Williams M.R. Arthur J.S. Balendran A. van der Kaay J. Poli V. Cohen P. Alessi D.R. Curr. Biol. 2000; 10: 439-448Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar). Autophosphorylation is another proposed mechanism for the phosphorylation on Ser-473 (10Toker A. Newton A.C. J. Biol. Chem. 2000; 275: 8271-8274Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar). The integrin-linked kinase (ILK), an integrin- and growth factor-regulated PI 3-kinase-dependent kinase (11Delcommenne M. Tan C. Gray V. Ruel L. Woodgett J. Dedhar S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11211-11216Crossref PubMed Scopus (947) Google Scholar, 12Wu C. Dedhar S. J. Cell Biol. 2001; 155: 505-510Crossref PubMed Scopus (350) Google Scholar), has also been shown to promote the phosphorylation of PKB/Akt on Ser-473 but not on Thr-308 (1Nicholson K.M. Anderson N.G. Cell Signal. 2002; 14: 381-395Crossref PubMed Scopus (1388) Google Scholar, 11Delcommenne M. Tan C. Gray V. Ruel L. Woodgett J. Dedhar S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11211-11216Crossref PubMed Scopus (947) Google Scholar, 12Wu C. Dedhar S. J. Cell Biol. 2001; 155: 505-510Crossref PubMed Scopus (350) Google Scholar, 13Lynch D.K. Ellis C.A. Edwards P.A. Hiles I.D. Oncogene. 1999; 18: 8024-8032Crossref PubMed Scopus (183) Google Scholar, 14Persad S. Attwell S. Gray V. Mawji N. Deng J.T. Leung D. Yan J. Sanghera J. Walsh M.P. Dedhar S. J. Biol. Chem. 2001; 276: 27462-27469Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar, 15Cruet-Hennequart S. Maubant S. Luis J. Gauduchon P. Staedel C. Dedhar S. Oncogene. 2003; 22: 1688-1702Crossref PubMed Scopus (128) Google Scholar). Growth factor- and extracellular matrix-induced PKB/Akt Ser-473 phosphorylation is inhibited by kinase-deficient, dominant-negative ILK (1Nicholson K.M. Anderson N.G. Cell Signal. 2002; 14: 381-395Crossref PubMed Scopus (1388) Google Scholar, 13Lynch D.K. Ellis C.A. Edwards P.A. Hiles I.D. Oncogene. 1999; 18: 8024-8032Crossref PubMed Scopus (183) Google Scholar, 14Persad S. Attwell S. Gray V. Mawji N. Deng J.T. Leung D. Yan J. Sanghera J. Walsh M.P. Dedhar S. J. Biol. Chem. 2001; 276: 27462-27469Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar), as well as by small molecule ILK kinase inhibitors (14Persad S. Attwell S. Gray V. Mawji N. Deng J.T. Leung D. Yan J. Sanghera J. Walsh M.P. Dedhar S. J. Biol. Chem. 2001; 276: 27462-27469Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar, 16Persad S. Attwell S. Gray V. Delcommenne M. Troussard A. Sanghera J. Dedhar S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3207-3212Crossref PubMed Scopus (379) Google Scholar). Inhibition of ILK activity also results in inhibition of constitutively activated PKB/Akt in PTEN-null cancer cells (16Persad S. Attwell S. Gray V. Delcommenne M. Troussard A. Sanghera J. Dedhar S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3207-3212Crossref PubMed Scopus (379) Google Scholar). In addition, ILK was identified as a PKB/Akt Ser-473 kinase by in-gel kinase analysis and protein purification (14Persad S. Attwell S. Gray V. Mawji N. Deng J.T. Leung D. Yan J. Sanghera J. Walsh M.P. Dedhar S. J. Biol. Chem. 2001; 276: 27462-27469Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar). These data support a role of ILK in the regulation of phosphorylation of PKB/Akt on Ser-473. However, recently, a lipid-raft associated PKB Ser-473 kinase activity distinct from PDK-1, PKB, or ILK was detected (17Hill M.M. Feng J. Hemmings B.A. Curr. Biol. 2002; 12: 1251-1255Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar), although its identity remains unknown. This finding and the reports showing that the PI 3-kinase pathway and PKB/Akt activation do not appear to be affected in Caenorhabditis elegans and Drosophila ILK mutants have prompted a debate over the physiological role of ILK in PKB/Akt activation (23Zervas C.G. Gregory S.L. Brown N.H. J. Cell Biol. 2001; 152: 1007-1018Crossref PubMed Scopus (238) Google Scholar, 24Mackinnon A.C. Qadota H. Norman K.R. Moerman D.G. Williams B.D. Curr. Biol. 2002; 12: 787-797Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar). To definitively address the question of whether ILK is essential for the promotion of phosphorylation of PKB/Akt on Ser-473, we have taken two genetic approaches to down-regulate or eliminate the expression of ILK in mammalian cells and then to assess whether phosphorylation of PKB/Akt on Ser-473 is specifically affected. We find that ILK expression is down-regulated in HEK-293 human kidney epithelial cells by double-stranded RNA interference (siRNA) and in immortalized mouse macrophages by conditional deletion of the ILK gene using the Cre-Lox system. ILK knock-out results in the inhibition of PKB/Akt activity and specific suppression of phosphorylation of PKB/Akt on Ser-473, without any effect on PKB/Akt expression or phosphorylation on Thr-308. Furthermore, the inhibition of PKB/Akt Ser-473 phosphorylation is rescued by transfection of a kinase-active ILK, but not by a kinase-inactive ILK. ILK knock-out also results in the inhibition of phosphorylation of GSK-3β and expression of cyclin D1, two known targets of PKB/Akt and ILK. Cells and Plasmids—Human embryonic kidney HEK-293 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% donor calf serum (Invitrogen). Bone marrow cells were isolated from femurs and plated in tissue culture plates for 2 h at 37 °C in 100 ng/ml CSF-1 in 10% fetal calf serum/Iscove's modified Dulbecco's media supplemented with 30% conditioned media from cells producing the pZip-Tex virus (18Timms J.F. Carlberg K. Gu H. Chen H. Kamatkar S. Nadler M.J. Rohrschneider L.R. Neel B.G. Mol. Cell. Biol. 1998; 18: 3838-3850Crossref PubMed Scopus (174) Google Scholar). The non-adherent cells were then removed and placed in fresh tissue culture plates and allowed to differentiate into macrophages. After several passages over weeks, 100% of the cells were Mac-1-positive. Transfection of the following plasmids were carried out in macrophages using LipofectAMINE 2000: pcDNA-3-ILK-WT, pcDNA-3-ILK-S343A, pcDNA-3-ILK-S343D (14Persad S. Attwell S. Gray V. Mawji N. Deng J.T. Leung D. Yan J. Sanghera J. Walsh M.P. Dedhar S. J. Biol. Chem. 2001; 276: 27462-27469Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar), and pEGFP. siRNA—siRNA duplexes were synthesized by Xeragon, Inc., Germantown, MD. Twenty-one-base sequences of the human ILK gene (GenBank™ accession number gi 3150001) specifically targeting the kinase domain (ILK-FSF) and the pH domain (ILK-H) were chosen. A control-NS, non-silencing siRNA (from Xeragon's data base, 16-base overlap with Thermotoga maritimia), a control-HC, inverted ILK-FSF siRNA, and a siRNA targeting calreticulin (GenBank™ accession number gi 16151096) were also designed. Each sequence was blasted to assess specificity. Transient transfections of HEK-293 cells were carried out by using 6 μl of Lipofectin reagent (Invitrogen), according to the manufacturer's guidelines. Excision of the Floxed ILK Gene—Immortalized ILK fl/fl macrophages were infected with CRE recombinase adenoviruses. Cells were plated in a 6-well plate and used when they were 70% confluent. Cells were incubated with 0, 1, or 3 μl of AdCRE (5.29 × 1012 viral particles/ml) in 500 μl of Dulbecco's modified Eagle's medium for 1 h at 37 °C. The volume was then increased to 2 ml, and the cells were incubated for an additional 3 h before replacement by complete media. By PCR, we assessed the presence of CRE recombinase (forward primer, 5′-CATTTCTGGGGATTGCTTATAACAC-3′; reverse, 5′-TATTGAAACTCCAGCGCGGGCC-3′), the presence of the floxed ILK gene (forward, 5′-AAGGTGCTGAAGGTTCGAGA-3′; reverse, 5′-CAAGGAATAAGGTGAGCTTCAGAA-3′), and the presence of excision products (forward, 5′-CCAGGTGGCAGAGGTAAGTA-3′; reverse, 5′-CAAGGAATAAGGTGAGCTTCAGAA-3′). After infection, cells were maintained in 10% fetal bovine serum and 100 ng/ml CSF-1. ILK protein expression was then evaluated by Western blotting as described below. For the rescue experiments, macrophages were co-transfected with 1 μg of the indicated plasmids and 0.5 μg of pEGFP, prior to infection with AdCre. Transfection efficiencies were determined by counting GFP-positive cells. The transfection efficiencies varied from ∼20 to 40%. Because of this variability and the different amounts of GFP detected by Western blotting, we performed densitometric analyses and then normalized the phosphorylation status of PKB/Akt on Ser-473 relative to GFP expression. Ratios are shown under “Results.” Immunoblots—Cells, grown in the described were in pH 1 inhibitor 1 1 and Protein were then by and were with of the following Inc., D1 or was carried out with the and an analyses were performed using from kinase were performed following the manufacturer's μg of protein were using an and were incubated with a protein as a of of GSK-3β, as a of kinase activity, was then detected by Western were with and according to the manufacturer's cells apoptosis were then detected by To whether ILK is essential for the regulation of PKB/Akt phosphorylation on Ser-473, we have taken two approaches to the expression of ILK and then assess PKB/Akt Ser-473 phosphorylation. The was the of double-stranded RNA (siRNA) to ILK expression. We identified and synthesized two specific ILK as well as shown in the ILK siRNA but not a RNA in a the expression of ILK into HEK-293 cells. We determined the of the ILK a non-silencing siRNA and a siRNA a sequence from to down-regulate the expression of ILK. shown in transfection of of the but not the non-silencing siRNA or the calreticulin siRNA resulted specifically in of ILK expression in HEK-293 cells as determined by Western The expression of calreticulin protein was down-regulated by the calreticulin siRNA, but this siRNA had no effect on ILK expression. Transfection of the siRNA, ILK was the siRNA that also phosphorylation of PKB/Akt on Ser-473 To the of the effect of ILK expression on PKB/Akt the of PKB/Akt phosphorylation of Thr-308 to phosphorylation of was determined by Western blotting using shown in expression of and PKB/Akt, as well as the phosphorylation of PKB/Akt on Thr-308 were ILK the phosphorylation of PKB/Akt on Ser-473 was and specifically with ILK This effect was with two different of ILK by siRNA on downstream ILK of the indicated siRNA in HEK-293 cells, the phosphorylation status of PKB/Akt on Ser-473 and Thr-308 was ILK expression by siRNA phosphorylation on Ser-473, but Thr-308 phosphorylation and PKB/Akt protein expression were not affected. indicated are the from densitometric shown are of in the as suppression of ILK expression by siRNA inhibited phosphorylation of GSK-3β on but not GSK-3β protein expression. D1 expression was also indicated are the from densitometric shown are of ILK has been shown to the phosphorylation of Ser-9 of GSK-3β, as well as expression of cyclin D1 M. J. C. M. K. R. M.P. M. J. A. Troussard Dedhar S. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar, S. Troussard Dedhar S. J. Cell Biol. 2001; PubMed Scopus Google Scholar). In addition, activated PKB/Akt also GSK-3β on Ser-9 de K.M. de Vries-Smits A.M. van J. Burgering B.M. J. Biol. Chem. 1998; Full Text Full Text PDF Scopus Google Scholar). We to whether the of ILK also inhibited the phosphorylation and expression of GSK-3β and cyclin D1, shown in the phosphorylation of GSK-3β is also inhibited the of ILK the of GSK-3β protein remained In addition, the expression of cyclin D1 are also by the ILK with the the at phosphorylation of PKB/Akt and GSK-3β were was a effect on cell growth but no significant effect on cell as the cells remained and not Inhibition of ILK activity has been shown to apoptosis and cell cycle C. Dedhar S. J. Cell Biol. 2001; 155: 505-510Crossref PubMed Scopus (350) Google Scholar, 13Lynch D.K. Ellis C.A. Edwards P.A. Hiles I.D. Oncogene. 1999; 18: 8024-8032Crossref PubMed Scopus (183) Google Scholar, 15Cruet-Hennequart S. Maubant S. Luis J. Gauduchon P. Staedel C. Dedhar S. Oncogene. 2003; 22: 1688-1702Crossref PubMed Scopus (128) Google Scholar, 16Persad S. Attwell S. Gray V. Delcommenne M. Troussard A. Sanghera J. Dedhar S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3207-3212Crossref PubMed Scopus (379) Google Scholar). data genetic support for that inhibition of ILK activity by dominant-negative ILK or small molecule ILK inhibitors PKB/Akt Ser-473 phosphorylation. In to siRNA, we have used another for out expression of ILK in mammalian cells. We have in to out ILK expression in a manner by with under a The of is described in J. A. S. G. S. and R. To ILK expression in cells from the we isolated and immortalized macrophages from as described under The cells were then infected with V. A. 2001; PubMed Scopus Google to the ILK gene and eliminate its expression. the expression of the as well as the excision of the site from the floxed ILK gene The expression of ILK protein was inhibited in a manner of the shown in phosphorylation of PKB/Akt on Ser-473 was inhibited with the of ILK expression in cells grown in the presence of 10% fetal bovine to activation of of PKB/Akt on Thr-308 was as were the expression of PKB/Akt and phosphorylation of PKB/Akt on Ser-473 was detected in the of serum and CSF-1 in cells not infected with not ILK expression was also under not shown in ILK also resulted in the inhibition of phosphorylation of GSK-3β on Ser-9 as well as cyclin D1 GSK-3β protein remained A analysis in epithelial cells from the also resulted in inhibition of PKB/Akt Ser-473 phosphorylation ILK deletion by CRE N. and S. Furthermore, as shown in phosphorylation of PKB/Akt on Ser-473 and PKB/Akt kinase activity were inhibited of ILK expression. These data support results from the siRNA and demonstrate that ILK expression is essential for the specific phosphorylation of PKB/Akt on Ser-473 and for the regulation of PKB/Akt activity. The macrophages in ILK expression was were to with the cells. demonstrated that cells were apoptosis as determined by as be of the inhibition of PKB/Akt activity. The role of the kinase activity of ILK in its has been on data showing that ILK kinase activity is not for in C. elegans and Drosophila (23Zervas C.G. Gregory S.L. Brown N.H. J. Cell Biol. 2001; 152: 1007-1018Crossref PubMed Scopus (238) Google Scholar, 24Mackinnon A.C. Qadota H. Norman K.R. Moerman D.G. Williams B.D. Curr. Biol. 2002; 12: 787-797Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar). In addition, has been that ILK PKB/Akt phosphorylation D.K. Ellis C.A. Edwards P.A. Hiles I.D. Oncogene. 1999; 18: 8024-8032Crossref PubMed Scopus (183) Google Scholar). We carried out rescue to assess the role of the ILK kinase activity in the regulation of PKB/Akt Ser-473 phosphorylation. We co-transfected the cells from the with expression ILK or kinase-inactive mutant (14Persad S. Attwell S. Gray V. Mawji N. Deng J.T. Leung D. Yan J. Sanghera J. Walsh M.P. Dedhar S. J. Biol. Chem. 2001; 276: 27462-27469Abstract Full Text Full Text PDF PubMed Scopus (416) Google and a GFP and then infected the cells with AdCre. We have demonstrated that the mutant of ILK not have activity as determined by in kinase of proteins (14Persad S. Attwell S. Gray V. Mawji N. Deng J.T. Leung D. Yan J. Sanghera J. Walsh M.P. Dedhar S. J. Biol. Chem. 2001; 276: 27462-27469Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar). shown in analysis of the phosphorylation of PKB/Akt Ser-473 relative to the of GFP expression that was at the inhibition of phosphorylation of PKB/Akt the kinase-inactive Because the transfection efficiencies for mutant varied we the of phosphorylation to GFP for transfection from The data that rescued Ser-473 phosphorylation by an of with the This is significant In we also the effect of the kinase-active mutant (14Persad S. Attwell S. Gray V. Mawji N. Deng J.T. Leung D. Yan J. Sanghera J. Walsh M.P. Dedhar S. J. Biol. Chem. 2001; 276: 27462-27469Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar). kinase-active and rescued phosphorylation of PKB/Akt on Ser-473. the kinase-inactive mutant was at the phosphorylation. After densitometric analyses that phosphorylation of Ser-473 PKB/Akt rescue by or was and with rescue by These results demonstrate that the regulation of the phosphorylation of PKB/Akt on Ser-473 by ILK is on its kinase activity and that ILK phosphorylate this site These data are in with data showing that ILK PKB/Akt in an in-gel kinase (14Persad S. Attwell S. Gray V. Mawji N. Deng J.T. Leung D. Yan J. Sanghera J. Walsh M.P. Dedhar S. J. Biol. Chem. 2001; 276: 27462-27469Abstract Full Text Full Text PDF PubMed Scopus (416) Google and that specific small molecule inhibitors of ILK kinase activity PKB/Akt phosphorylation on Ser-473 (14Persad S. Attwell S. Gray V. Mawji N. Deng J.T. Leung D. Yan J. Sanghera J. Walsh M.P. Dedhar S. J. Biol. Chem. 2001; 276: 27462-27469Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar, 16Persad S. Attwell S. Gray V. Delcommenne M. Troussard A. Sanghera J. Dedhar S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3207-3212Crossref PubMed Scopus (379) Google Scholar). be that although the kinase domain of ILK from kinase ILK has been demonstrated to as a kinase in the of phosphorylation of PKB/Akt, such as and and J.T. C. Walsh M.P. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, J.T. C. M. Walsh M.P. Biochem. J. 2002; PubMed Scopus Google Scholar, A. Deng J.T. Walsh M.P. F. E. Biochem. J. 2002; PubMed Google Scholar). on the extensive of inhibition of phosphorylation of Ser-473 of ILK expression in the cell ILK as a PKB/Akt Ser-473 kinase or is a regulator of the PKB/Akt Ser-473 kinase activity identified in a plasma membrane lipid-raft (17Hill M.M. Feng J. Hemmings B.A. Curr. Biol. 2002; 12: 1251-1255Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). an of results be that of ILK is a phosphorylation site for the of the phosphorylation of has not been demonstrated we have shown that ILK and PKB and that of with results in the inhibition of with PKB/Akt, with an the (14Persad S. Attwell S. Gray V. Mawji N. Deng J.T. Leung D. Yan J. Sanghera J. Walsh M.P. Dedhar S. J. Biol. Chem. 2001; 276: 27462-27469Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar). and the mutant rescue PKB/Akt Ser-473 phosphorylation they promote and phosphorylation by ILK or by another the mutant do A a role of ILK in the regulation of suppression of apoptosis. has been demonstrated N. Tan L. Mol. Cell. 2002; 10: Full Text Full Text PDF PubMed Scopus Google that PKB/Akt activation the specific of ILK and PDK-1, a mechanism for the of PKB/Akt data demonstrate an essential role for ILK in the regulation of phosphorylation of PKB/Akt on Ser-473 and GSK-3β on as well as in the regulation of expression of cyclin In support for the knock-out of ILK in by the with also results in the inhibition of PKB/Akt Ser-473 phosphorylation and suppression of expression of cyclin D1 in in the of The data in this establish ILK as an and essential physiological regulator of the phosphorylation of PKB/Akt on Ser-473 and PKB/Akt activation.

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