CDC14 is an essential dual-specificity phosphatase that counteracts CDK1 activity during anaphase to promote mitotic exit in Saccharomyces cerevisiae. Surprisingly, human CDC14A is not essential for cell cycle progression. Instead, it regulates cell migration and cell adhesion. Little is known about the substrates of hCDC14A and the counteracting kinases. Here, we combine phospho-proteome profiling and proximity-dependent biotin identification to identify hCDC14A substrates. Among these targets were actin regulators, including the tumor suppressor eplin. hCDC14A counteracts EGF-induced rearrangements of actin cytoskeleton by dephosphorylating eplin at two known extracellular signal-regulated kinase sites, serine 362 and 604. hCDC14A(PD) and eplin knockout cell lines exhibited down-regulation of E-cadherin and a reduction in alpha/beta-catenin at cell-cell adhesions. Reduction in the levels of hCDC14A and eplin mRNA is frequently associated with colorectal carcinoma and is correlated with poor prognosis. We therefore propose that eplin dephosphorylation by hCDC14A reduces actin dynamics to restrict tumor malignancy
Integrins are α/β heterodimers that interconvert between inactive and active states. In the active state the α/β cytoplasmic domains recruit integrin-activating proteins and separate the transmembrane and cytoplasmic (TMcyto) domains (unclasped TMcyto), while in the inactive state the α/β TMcyto domains bind integrin-inactivating proteins resulting in the association of the TMcyto domains (clasped TMcyto). Here, we report the isolation of integrin cytoplasmic tail interactors using either lipid bicelle-incorporated integrin TMcyto domains (α5, αM, αIIb, β1, β2 and β3 integrin TMcyto) or a clasped, lipid bicelle-incorporated αMβ2 TMcyto. Among the proteins found to preferentially bind clasped rather than the isolated αM and β2 subunits was L-plastin (LCP1), which binds to and maintains the inactive state of αMβ2 integrin in vivo and thereby regulates leukocyte adhesion to integrin ligands under flow. Our findings offer a global view on cytoplasmic proteins interacting with different integrins and provide evidence for the existence of conformation-specific integrin interactors.
In a paper published in PNAS, Li et al. (1) solve the long-awaited crystal structure of kindlin-2, which plays a central role in integrin activation, clustering, and signaling (2, 3). Integrins are a large family of heterodimeric adhesion molecules composed of αand β-subunits and expressed on almost all cells. They mediate cell matrix adhesion by binding extracellular matrix proteins, such as collagens, fibronectin, and laminins, and cell-cell adhesion by interacting with counterreceptors such as VCAM, ICAM, and cadherins (Fig. 1A). Upon ligand binding, integrins cluster and assemble an enormous number of proteins at their short cytoplasmic domains, which are collectively called the adhesome (4). The adhesome transmits biochemical signals into the cell and connects integrins to the actin cytoskeleton, which, in turn, enables the transduction of myosin-II-generated traction forces to ligated integrins. The integrin α/β-subunits have large ectodomains that associate at their head domains to form the ligand-binding site, single-span transmembrane domains, and short cytoplasmic tails. A hallmark of integrins is that the affinity of their ligand-binding sites is tightly regulated by complex conformational changes affecting the entire integrin molecule: They reversibly shift from a low-affinity (or inactive) state, in which they adopt a bent-closed (BC) or extended-closed (EC) conformation, to the high-affinity (or active) state characterized by an extended-open (EO) conformation (2, 5) (Fig. 1A). The BC and EC conformations are characterized by a "closed" ligand-binding site, a loose connection between the transmembrane helices and cytoplasmic tails of αand β-subunits, and either an association of the ectodomain headpiece with the legs (BC) or an extension of the α/β-subunits (EC). The conversion of integrins into the EO conformation is characterized by the separation of the legs, transmembrane, and tail domains; the swing-away of the hybrid domain from the α-subunit; and the opening of the ligandbinding site, which results in a massive increase in affinity for ligand. A recent study measured the intrinsic affinity of α5β1 integrin on K562 cells for fibronectin and the free energy for each activation state of α5β1 (6). The
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