An integrin-α4–14-3-3ζ–paxillin ternary complex mediates localised Cdc42 activity and accelerates cell migration

Abstract

α4 integrins are used by leukocytes and neural crest derivatives for adhesion and migration during embryogenesis, immune responses and tumour invasion. The pro-migratory activity of α4 integrin is mediated in part through the direct binding of the cytoplasmic domain to paxillin. Here, using intermolecular FRET and biochemical analyses, we report a novel interaction of the α4 integrin cytoplasmic domain with 14-3-3ζ. This interaction depends on serine phosphorylation of α4 integrin at a site (S978) distinct from that which regulates paxillin binding (S988). Using a combination of metabolic labelling and targeted mass spectrometry by multiple reaction monitoring we demonstrate the low stoichiometry phosphorylation of S978. The interaction between α4 integrin and 14-3-3ζ is enhanced by the direct association between 14-3-3ζ and paxillin, resulting in the formation of a ternary complex that stabilises the recruitment of each component. Although pair-wise interaction between α4 integrin and paxillin is sufficient for normal Rac1 regulation, the integrity of the ternary complex is essential for focused Cdc42 activity at the lamellipodial leading edge and directed cell movement. Taken together, these data identify a key signalling nexus mediating α4 integrin-dependent migration.

14-3-3θ Facilitates plasma membrane delivery and function of mechanosensitive connexin 43 hemichannels

“…Although, 14-3-3 has been shown to interact with several integrins including b1, aLb2 and a4 (Deakin et al, 2009;Han et al, 2001;Nurmi et al, 2006), our study is the first to show that 14-3-3h interacts with integrin a5. More importantly, we show that 14-3-3h is necessary for the interaction between Cx43 and integrin a5 because reduction of 14-3-3h by siRNA impairs the association between these two proteins.…”

Ovarian cancer molecular pathology

“…14-3-3 zeta was found to have 90 %, 82.5 %, 72 %, and 94.3 % sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV), respectively, for stage I and stage II patients. 14-3-3 zeta is an adaptor protein that binds to other upstream or downstream signaling molecules containing tandem repeats of phosphoserine motifs [169,170] and has been found to form a ternary complex with integrin alpha-4 to accelerate cell migration [171]. This study highlights that metastasis of ovarian cancer is an early event since a protein involved in this behavior can be detected as a biomarker for early ovarian cancer.…”

SLK-mediated phosphorylation of paxillin is required for focal adhesion turnover and cell migration

“…This sequence conservation is also reflected in the complement of proteins that each motif is capable of interacting with and is believed to provide the specificity required for the interaction of the LD motifs with various paxillin-interacting proteins [66,86,102]. In particular, LD1 has been shown to mediate interactions with actopaxin, the integrin-linked kinase (ILK), vinculin and the papillomavirus protein E6 [66,86,102,104]. LD2 has also been shown to bind to vinculin, but like LD4, also binds FAK and the protein-tyrosine kinase 2-beta (PYK2).…”