Mutations in the Paxillin-binding Site of Integrin-linked Kinase (ILK) Destabilize the Pseudokinase Domain and Cause Embryonic Lethality in Mice

• Published in: The Journal of biological chemistry Vol. 288; no. 26; pp. 18863 - 18871
• Main Authors: Moik, Daniel, Böttcher, Anika, Makhina, Tatiana, Grashoff, Carsten, Bulus, Nada, Zent, Roy, Fässler, Reinhard
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 28.06.2013; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Motifs; Animals; Binding Sites; Cell Adhesion; Cell Biology; Cell Migration; Cell Movement; Development; Embryo, Mammalian - embryology; Embryonic Development; Flow Cytometry; Focal Adhesions - metabolism; Gene Expression Regulation, Developmental; Genes, Lethal; Integrin-linked Kinase; Mice; Microfilament Proteins - metabolism; Mutation; Parvin; Paxillin; Paxillin - metabolism; PINCH; Protein Binding; Protein Conformation; Protein Stability; Protein Structure, Tertiary; Protein-Serine-Threonine Kinases - genetics; Protein-Serine-Threonine Kinases - metabolism; Time Factors; Vasculogenesis; Cell Migration; PINCH; Integrin-linked Kinase; Development; Parvin; Protein Stability; Vasculogenesis; Embryonic Development; Cell Adhesion; Paxillin
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M113.470476

Integrin-linked kinase (ILK) localizes to focal adhesions (FAs) where it regulates cell spreading, migration, and growth factor receptor signaling. Previous reports showed that overexpressed ILK in which Val386 and Thr387 were substituted with glycine residues (ILK-VT/GG) could neither interact with paxillin nor localize to FA in cells expressing endogenous wild-type ILK, implying that paxillin binding to ILK is required for its localization to FAs. Here, we show that introducing this mutation into the germ line of mice (ILK-VT/GG) caused vasculogenesis defects, resulting in a general developmental delay and death at around embryonic day 12.5. Fibroblasts isolated from ILK-VT/GG mice contained mutant ILK in FAs, showed normal adhesion to and spreading on extracellular matrix substrates but displayed impaired migration. Biochemical analysis revealed that VT/GG substitutions decreased ILK protein stability leading to decreased ILK levels and reduced binding to paxillin and α-parvin. Because paxillin depletion did not affect ILK localization to FAs, the embryonic lethality and the in vitro migration defects are likely due to the reduced levels of ILK-VT/GG and diminished binding to parvins. Background: Integrin-linked kinase is believed to be recruited to focal adhesions (FAs) by binding paxillin via a conserved motif in the pseudokinase domain. Results: The paxillin-binding motif is not required for FA localization but for ILK stability, parvin and paxillin binding, and mouse development. Conclusion: ILK does not require paxillin for FA localization. Significance: Reducing ILK stability perturbs cell migration and development.