The Role of Conserved Amino Acid Motifs within the Integrin β3 Cytoplasmic Domain in Triggering Focal Adhesion Kinase Phosphorylation

Abstract

Integrin-mediated adhesion of cells to extracellular matrix proteins triggers a variety of intracellular signaling pathways including a cascade of tyrosine phosphorylations. In many cell types, the cytoplasmic focal adhesion tyrosine kinase, FAK, appears to be the initial protein that becomes tyrosine-phosphorylated in response to adhesion; however, the molecular mechanisms regulating integrin-triggered FAK phosphorylation are not understood. Previous studies have shown that the integrin ␤ 1 , ␤ 3 , and ␤ 5 subunit cytoplasmic domains all contain sufficient information to trigger FAK phosphorylation when expressed in single-subunit chimeric receptors connected to an extracellular reporter. In the present study, ␤ 3 cytoplasmic domain deletion and substitution mutants were constructed to identify amino acids within the integrin ␤ 3 cytoplasmic domain that regulate its ability to trigger FAK phosphorylation. Cells transiently expressing chimeric receptors containing these mutant cytoplasmic domains were magnetically sorted and assayed for the tyrosine phosphorylation of FAK. Analysis of these mutants indicated that structural information in both the membrane-proximal and C-terminal segments of the ␤ 3 cytoplasmic domain is important for triggering FAK phosphorylation. In the C-terminal segment of the ␤ 3 cytoplasmic domain, the highly conserved NPXY motif was found to be required for the ␤ 3 cytoplasmic domain to trigger FAK phosphorylation. However, the putative FAK binding domain within the N-terminal segment of the ␤ 3 cytoplasmic domain was found to be neither required nor sufficient for this signaling event. We also demonstrate that the serine 752 to proline mutation, known to cause a variant of Glanzmann's thrombasthenia, inhibits the ability of the ␤ 3 cytoplasmic domain to signal FAK phosphorylation, suggesting that a single mutation in the ␤ 3 cytoplasmic domain can inhibit both "inside-out" and "outside-in" integrin signaling.

Ancient origin of the integrin-mediated adhesion and signaling machinery

“…S3), which accounts for their longer size relative to metazoan integrin β proteins. Other key motifs in metazoan integrin β proteins are the cytoplasmic integrin α-interacting motif and the NPXY motif, which plays a key role in protein interactions (17,20,37,38). Both motifs are well conserved in C. owczarzaki integrin β1-β3 and Amastigomonas sp.…”

Restoration of positioning control following Disabled-2 expression in ovarian and breast tumor cells

“…Alternatively, all of the integrin beta subunits also contain NPXY motif in their intracellular domain. The NPXY motif in beta-integrin subunit is important in intracellular signaling (Law et al, 1999;Vignoud et al, 1997;Tahiliani et al, 1997;O'Toole et al, 1995), and Dab2 may mediate contact signaling of integrin. Whether Dab2 bind to these NPXY motifs and is involved in LDL-family receptors or integrin signaling is to be determined.…”

Renal Ischemia/Reperfusion Injury

“…One important component of this adhesion complex is the p125 FAK that can be activated by autophosphorylation, and this activation is required for the FAC formation and function (revised in 32). Phosphorylated FAK can bind the cytoplasmic domain of ␤1 integrins and activate actin cytoskeleton assembly (33)(34)(35). Activation/deactivation of ␤1 integrins can be estimated indirectly by FAK phosphorylation, as assessed in this study.…”