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-3beta on Ser-9 and cyclin D1 expression. These data establish ILK as an essential upstream regulator of PKB/Akt activation.
Troussard
Armelle A
AA
British Columbia Cancer Agency, University of British Columbia, Jack Bell Research Centre, Vancouver, British Columbia V6H 3Z6, Canada.
Mawji
Nasrin M
NM
Ong
Christopher
C
Mui
Alice
A
St -Arnaud
René
R
Dedhar
Shoukat
S
D001483:Base Sequence; D001854:Bone Marrow Cells / Q000201:enzymology; D002460:Cell Line; D017931:DNA Primers; D004789:Enzyme Activation; D017353:Gene Deletion; D006801:Humans; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D016133:Polymerase Chain Reaction; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D034741:RNA, Small Interfering / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D000099200:Scaffold Protein ILK
D017931:DNA Primers; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D000099200:Scaffold Protein ILK; C494918:AKT1 protein, human