The NPIY motif in the integrin β1 tail dictates the requirement for talin-1 in outside-in signaling

Abstract

SummaryProtein interactions with the integrin b-subunit cytoplasmic domain (b-tail) are essential for adhesion-dependent processes, including cell spreading and the connection of integrins with actin filaments at adhesion sites. Talin-1 binds to the conserved membrane-proximal NPxY motif of b-tails (NPIY in b1 integrin) promoting the inside-out activation of integrins and providing a linkage between integrins and the actin cytoskeleton. Here, we characterize the role of interactions between talin-1 and b-tail downstream of integrin activation, in the context of recombinant integrins containing either the wild type (WT) or the (YA) mutant b1A tail, with a tyrosine to alanine substitution in the NPIY motif. In addition to inhibiting integrin activation, the YA mutation suppresses cell spreading, integrin signaling, focal adhesion and stress-fiber formation, as well as microtubule assembly. Constitutive activation of the mutant integrin restores these integrin-dependent processes, bringing into question the importance of the NPIY motif downstream of integrin activation. Depletion of talin-1 using TLN1 siRNA demonstrated that talin-1 is required for cell spreading, focal adhesion and stress-fiber formation, as well as microtubule assembly, even when cells are adhered by constitutively activated WT integrins. Depletion of talin-1 does not inhibit these processes when cells are adhered by constitutively activated mutant integrins, suggesting that the binding of an inhibitory protein to the NPIY motif negatively regulates integrin function when talin-1 is depleted. We identified filamin A (FLNa) as this inhibitory protein; it binds to the b1A tail in an NPIY-dependent manner and inhibition of FLNa expression in talin-1-depleted cells restores integrin function when cells are adhered by constitutively activated WT integrins. FLNa binds FilGAP, which is a negative regulator of Rac activation. Expression of the dominant inhibitory mutant, FilGAP DGAP , which lacks GAP activity restores spreading in cells adhered by constitutively activated integrins containing the b1A tail, but not by integrins containing the b1D tail, which is known to bind poorly to FLNa. Together, these results suggest that the binding of talin-1 to the NPIY motif is required downstream of integrin activation to promote cell spreading by preventing the inappropriate recruitment of FLNa and FilGAP to the b1A tail. Our studies emphasize the importance of understanding the mechanisms that regulate the differential binding FLNa and talin-1 to the b1 tail downstream of integrin activation in promoting integrin function.

Rac1 is deactivated at integrin activation sites via an IQGAP1/filamin-A/RacGAP1 pathway

“…While we discovered that RacGAP1 was recruited to a complex containing FLNa and IQGAP1 upon integrin activation, it has been reported previously that FLNa can recruit an alternative suppressor of Rac1 activity, FilGAP, and this association is modulated by mechanical strain (Ehrlicher et al, 2011;Nieves et al, 2010;Ohta et al, 2006). Although we were unable to detect the association of FilGAP with GFP-FLNa in our mass spectrometric analyses, the fact that multiple mechanisms exist to limit Rac1 activity upon integrin activation, or during integrin-mediated mechanosensation, highlights the importance of constraining Rac1 activity during these processes.…”

Quantitative Proteomics Profiling of Murine Mammary Gland Cells Unravels Impact of Annexin-1 on DNA Damage Response, Cell Adhesion, and Migration

“…In the absence of ANXA1, a number of proteins involved in cellular adhesion are highly up-regulated, making the "on" movement of a cell more difficult. For example, talin, one of the up-regulated proteins identified in our ANXA1 Ϫ/Ϫ mammary gland cells, is especially important in sustaining cell spreading and adhesion (50,51). ␤-Parvin, a protein that directly interacts with integrin-linked kinase and is important for stabilizing focal adhesions (52)(53)(54), has been shown to be expressed in normal mammary gland but down-regulated in advanced breast cancer and breast cancer cell lines (55).…”

Integrins Promote Cytokinesis through the RSK Signaling Axis

“…We isolated the function of recombinant integrins by adhering cells to fibrinogen in CCM1, a serum-free growth-promoting medium, as previously described (Colello et al, 2012;Nieves et al, 2010;Reverte et al, 2006). To assay cytokinesis, mitotic cells, isolated by mitotic shake off, were plated on fibrinogen-coated coverslips and their progression through cytokinesis was assessed at 30 minute intervals by immunofluorescence microscopy (Fig.…”

“…Previous studies from our laboratory demonstrate that a tyrosine-to-alanine substitution in the membrane-proximal NPIY motif (Y783A) in the integrin b1 cytoplasmic domain (b1 tail) that suppresses integrin activity also inhibits cytokinesis (Reverte et al, 2006). Constitutive activation of the YA (Y783A) mutant rescued cytokinesis (Reverte et al, 2006), but did not restore stress fibers and focal adhesions (Nieves et al, 2010). Thus, the ability to form focal adhesions and stress fibers is not required for successful cytokinesis, at least not in CHO cells.…”