title: Integrin-linked kinase is a functional Mn2+-dependent protein kinase that regulates glycogen synthase kinase-3β (GSK-3beta) phosphorylation.
abstract: BACKGROUND
  Integrin-linked kinase (ILK) is a highly evolutionarily conserved, multi-domain signaling protein that localizes to focal adhesions, myofilaments and centrosomes where it forms distinct multi-protein complexes to regulate cell adhesion, cell contraction, actin cytoskeletal organization and mitotic spindle assembly. Numerous studies have demonstrated that ILK can regulate the phosphorylation of various protein and peptide substrates in vitro, as well as the phosphorylation of potential substrates and various signaling pathways in cultured cell systems. Nevertheless, the ability of ILK to function as a protein kinase has been questioned because of its atypical kinase domain.
  
  
  RESULTS
  Here, we have expressed full-length recombinant ILK, purified it to >94% homogeneity, and characterized its kinase activity. Recombinant ILK readily phosphorylates glycogen synthase kinase-3 (GSK-3) peptide and the 20-kDa regulatory light chains of myosin (LC(20)). Phosphorylation kinetics are similar to those of other active kinases, and mutation of the ATP-binding lysine (K220 within subdomain 2) causes marked reduction in enzymatic activity. We show that ILK is a Mn-dependent kinase (the K(m) for MnATP is approximately 150-fold less than that for MgATP).
  
  
  CONCLUSIONS
  Taken together, our data demonstrate that ILK is a bona fide protein kinase with enzyme kinetic properties similar to other active protein kinases.
authors.affiliation: Department of Integrative Oncology, BC Cancer Research Centre, Vancouver, British Columbia, Canada.
authors.forename: Mykola
authors.initials: M
authors.lastname: Maydan
authors.forename: Paul C
authors.initials: PC
authors.lastname: McDonald
authors.forename: Jasbinder
authors.initials: J
authors.lastname: Sanghera
authors.forename: Jun
authors.initials: J
authors.lastname: Yan
authors.forename: Charalampos
authors.initials: C
authors.lastname: Rallis
authors.forename: Sheena
authors.initials: S
authors.lastname: Pinchin
authors.forename: Gregory E
authors.initials: GE
authors.lastname: Hannigan
authors.forename: Leonard J
authors.initials: LJ
authors.lastname: Foster
authors.forename: David
authors.initials: D
authors.lastname: Ish-Horowicz
authors.forename: Michael P
authors.initials: MP
authors.lastname: Walsh
authors.forename: Shoukat
authors.initials: S
authors.lastname: Dedhar
mesh_terms: D000185:Actinin / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D006801:Humans; D007700:Kinetics; D008239:Lysine; D008345:Manganese / Q000378:metabolism*; D008840:Microfilament Proteins; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D000099200:Scaffold Protein ILK; D000096985:Calponins
chemical_list: D000185:Actinin; D000255:Adenosine Triphosphate; D004791:Enzyme Inhibitors; D038362:Glycogen Synthase Kinase 3; D000071679:Glycogen Synthase Kinase 3 beta; D008239:Lysine; D008345:Manganese; D008840:Microfilament Proteins; D010455:Peptides; D017346:Protein Serine-Threonine Kinases; D011994:Recombinant Proteins; D000099200:Scaffold Protein ILK; C486590:PARVA protein, human; D000096985:Calponins; C000605506:GSK3B protein, human
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