We show that SV down-regulates FA structure and function, and that the mechanism involves interactions with TRIP6. Decreased levels of either protein increase cell adhesion to fibronectin. Increased SV levels decrease cell adhesion, as well as the number of stress fibers and large, mature FAs. Although more than one region of SV deleteriously affects FAs, the SV sequence with the largest effect on FA structure and function is SV342-571, which binds directly to Tctex-1 and the C-terminal LIM domains of TRIP6. SV and TRIP6 colocalize at mature FAs, and optimal SV recruitment to FAs requires binding to TRIP6. TRIP6 and the TRIP6 LIM domains partially rescue disruptive effects of SV sequences on FAs and stress fibers. Specificity is indicated by the lack of effect of Tctex-1 or the zyxin LIM domains on SV phenotypes. Thus, binding to SV342-571 is necessary, but not sufficient, to reverse SV effects on FAs.
The TRIP6 N terminus may shield the C-terminal LIM domains from SV342-571 in the absence of a regulatory signal, as has been proposed for other LIM domain proteins (Kadrmas and Beckerle, 2004
; Lai et al., 2005
). No direct interaction between SV sequences and the TRIP6 N terminus was detected in either yeast two-hybrid or pull-down assays. Nevertheless, full-length TRIP6 rescues the disruptive effects of longer SV proteins, implying the possibility of regulatory cross-talk between the TRIP6 N terminus and SV sequences other than SV342-571.
Observations that zyxin influences motility, adhesion, and stress fiber formation (Golsteyn et al., 1997
; Hoffman et al., 2006
) are reminiscent of those observed upon overexpression of SV sequences. However, SV342-571 does not bind zyxin. In conjunction with other recent observations (Petit et al., 2005
), these results suggest that the members of the zyxin protein family have overlapping, but distinguishable, functions.
The loss of adhesion induced by SV overexpression apparently represents a gain of function because this phenotype is opposite that observed after SV knockdown. An SV-induced negative effect on large FAs is supported by the localization of SV with large FAs, which are structures that undergo dynamic remodeling (Ballestrem et al., 2001
; Carragher and Frame, 2004
). SV-mediated loss of large FAs also fits with the absence or reduced prevalence of large FAs and stress fibers in cells that contain relatively high amounts of endogenous SV, e.g., carcinomas and neutrophils (Pestonjamasp et al., 1997
; Pope et al., 1998
). Carcinomas and hematopoietic cells also express TRIP6 and/or LPP (Daheron et al., 2001
; Xu et al., 2004
), which is consistent with a physiological role for interactions with SV at dynamic FAs. Neutrophil FAs must be highly dynamic because they turn over rapidly during immune responses when little or no integrin is synthesized (Zhang et al., 2004
Although the rescue of the SV phenotype by the TRIP6 LIM domains may be attributable to a simple dominant–negative effect on TRIP6 function (Kassel et al., 2004
), the rescue by full-length TRIP6 is more interesting. In cells that overexpress both TRIP6 and wild-type SV, both proteins mislocalize into cell protrusions, sequestering SV away from FAs. Full-length SV proteins deficient in binding to TRIP6 still reduce the number of mature FAs, although these SV mutants are largely absent from FAs; TRIP6 localization at residual (or new) FAs is essentially unaffected. Thus, the FA equilibrium is disturbed by SV mutants that either contain the TRIP6-binding site out of context or contain the other SV FA-targeting sequences in the absence of high-affinity binding to TRIP6.
The simplest interpretation of these results is that TRIP6 and SV, together with other associated proteins, act during a FA assembly/disassembly cycle to control the rate of FA turnover. In this working model, TRIP6 helps recruit SV to FAs; SV then, directly or indirectly, either blocks later stages in FA maturation and/or increases the rate of FA turnover. When SV or TRIP6 levels are limiting, cell–substrate adhesion increases because FAs are locked “on.” Increasing amounts of the limiting protein decrease cell adhesion by increasing the rate of maturation of adhesive nascent FAs into less adhesive mature FAs and/or by increasing the rate of FA disassembly. In this model, TRIP6 overexpression helps restore the balance between FA assembly and disassembly in SV-overexpressing cells by (a) accelerating the formation of new FAs, (b) sequestering SV and proteins required for FA disassembly away from FAs, and/or (c) promoting the recycling of SV-associated proteins that are required for FA assembly. Overexpressed full-length SV that is deficient for binding to TRIP6 may disrupt FAs by interacting with other proteins involved in FA turnover in such a way that they become rate limiting for FA reassembly.
This model is consistent with the conflicting observations about the role of TRIP6 during cell migration. The prediction is that cellular responses to exogenous changes in TRIP6 are dependent on endogenous TRIP6 levels, relative to the levels of SV and other interactors in the proposed FA dis/assembly pathway, and on cell type–specific regulation.
We suggest that the SV–TRIP6 interaction demarcates a FA subdomain, perhaps a signaling scaffold, which controls FA integrity and/or turnover. One possibility is that the SV–TRIP6 interaction brings other SV-binding proteins into proximity with other TRIP6 partners. In addition to TRIP6, the SV N terminus (SV1-830) binds to the S2 subdomain of nonmuscle myosin II, to F-actin, and to the plasma membrane (Wulfkuhle et al., 1999
; Chen et al., 2003
). SV binding to TRIP6 may recruit myosin II in stress fibers into the vicinity of TRIP6-binding proteins that destabilize FAs. Candidate TRIP6 interactors include CAS/CasL (Yi et al., 2002
), c-Src (Xu et al., 2004
), Crk (Lai et al., 2005
), the LPA2
receptor (Xu et al., 2004
), the tyrosine phosphatase PTPN13/PTP-BL/FAP-1, and the adaptor protein RIL/PDLIM4 (Cuppen et al., 2000
), which is known to increase stress fiber dynamics (Vallenius et al., 2004
). In agreement with this hypothesis, both SV and the LPA–TRIP6–CAS pathway positively regulate ERK signaling (Gangopadhyay et al., 2004
; Lai et al., 2005
), a process implicated in FA turnover (Webb et al., 2004
Alternatively, SV binding to TRIP6 may displace a positive regulator of FA stability through steric hindrance or binding to a shared site on the TRIP6 C terminus. For instance, SV may disrupt the interaction of TRIP6 with endoglin/CD105, which is a transmembrane component of the TGF-β complex that promotes stress fiber formation and the localization of TRIP6 and zyxin to FAs (Conley et al., 2004
; Sanz-Rodriguez et al., 2004
Finally, we cannot exclude the possibility that SV indirectly influences FA structure through potentiation of TRIP6 effects in the nucleus. Despite its lack of a canonical NLS, TRIP6 can accumulate in the nucleus and modulate transcription (Kassel et al., 2004
; Li et al., 2005
). The TRIP6 LIM domains are sufficient for both SV binding () and nuclear transport (Wang and Gilmore, 2001
). SV contains functional NLS (Wulfkuhle et al., 1999
) and, like TRIP6, is implicated in steroid hormone signaling (Ting et al., 2002
). Thus, some of the effects reported here might be caused by changes in transcriptional activity induced by an SV–TRIP6 complex.
In summary, we show that the myosin II– and actin-binding protein SV regulates FA function through binding to TRIP6, a LIM domain–containing protein associated with signaling scaffolds that control cell motility. This is the first evidence for a myosin II–binding protein at FAs and for an explicit role for TRIP6 in adhesion. The direct binding of SV and TRIP6 suggest several specific, testable hypotheses by which TRIP6-associated scaffolds may control FA function. The SV–TRIP6 interaction may provide a “missing link” for actin-independent attachment of myosin II to the membrane at FAs and insight into molecular mechanisms for FA disassembly and/or recycling.