Assembly of the PINCH-ILK-CH-ILKBP complex precedes and is essential for localization of each component to cell-matrix adhesion sites.
PINCH, integrin-linked kinase (ILK) and calponin homology-containing ILK-binding protein (CH-ILKBP) form a ternary complex that plays crucial roles at cell-extracellular matrix adhesion sites. To understand the mechanism underlying the complex formation and recruitment to cell-adhesion sites we have undertaken a combined structural, mutational and cell biological analysis. Three-dimensional structure-based point mutations identified specific PINCH and ILK sites that mediate the complex formation. Analyses of the binding defective point mutants revealed that the assembly of the PINCH-ILK-CH-ILKBP complex is essential for their localization to cell-extracellular matrix adhesion sites. The formation of the PINCH-ILK-CH-ILKBP complex precedes integrin-mediated cell adhesion and spreading. Furthermore, inhibition of protein kinase C, but not that of actin polymerization, inhibited the PINCH-ILK-CH-ILKBP complex formation, suggesting that the PINCH-ILK-CH-ILKBP complex likely serves as a downstream effector of protein kinase C in the cellular control of focal adhesion assembly. Finally, we provide evidence that the formation of the PINCH-ILK-CH-ILKBP complex, while necessary, is not sufficient for ILK localization to cell-extracellular matrix adhesion sites. These results provide new insights into the molecular mechanism underlying the assembly and regulation of cell-matrix adhesion structures.
Zhang
Yongjun
Y
Department of Pathology, University of Pittsburgh, Pittsburgh, PA 15261, USA.
Chen
Ka
K
Tu
Yizeng
Y
Velyvis
Algirdas
A
Yang
Yanwu
Y
Qin
Jun
J
Wu
Chuanyue
C
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D002448:Cell Adhesion / Q000502:physiology*; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D005455:Fluorescent Antibody Technique; D006801:Humans; D060588:LIM Domain Proteins; D008565:Membrane Proteins; D051379:Mice; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism / Q000502:physiology*; D000099200:Scaffold Protein ILK
D048868:Adaptor Proteins, Signal Transducing; D004268:DNA-Binding Proteins; D060588:LIM Domain Proteins; D008565:Membrane Proteins; D017346:Protein Serine-Threonine Kinases; D000099200:Scaffold Protein ILK; C088088:LIMS1 protein, human; C490232:Lims1 protein, mouse