Tyrosine phosphorylation as a conformational switch: a case study of integrin β3 cytoplasmic tail

Reversible protein phosphorylation is vital for many fundamental cellular processes. The actual impact of adding and removing phosphate group(s) is 3-fold: changes in the local/global geometry, alterations in the electrostatic potential and, as the result of both, modified protein-target interactions. Here we present a comprehensive structural investigation of the effects of phosphorylation on the conformational as well as functional states of a crucial cell surface receptor, α(IIb)β(3) integrin. We have analyzed phosphorylated (Tyr(747) and Tyr(759)) β(3) integrin cytoplasmic tail (CT) primarily by NMR, and our data demonstrate that under both aqueous and membrane-mimetic conditions, phosphorylation causes substantial conformational rearrangements. These changes originate from novel ionic interactions and revised phospholipid binding. Under aqueous conditions, the critical Tyr(747) phosphorylation prevents β(3)CT from binding to its heterodimer partner α(IIb)CT, thus likely maintaining an activated state of the receptor. This conclusion was tested in vivo and confirmed by integrin-dependent endothelial cells adhesion assay. Under membrane-mimetic conditions, phosphorylation results in a modified membrane embedding characterized by significant changes in the secondary structure pattern and the overall fold of β(3)CT. Collectively these data provide unique molecular insights into multiple regulatory roles of phosphorylation.     

Abl regulates planar polarized junctional dynamics through β-catenin tyrosine phosphorylation

Interactions between epithelial cells are mediated by adherens junctions that are dynamically regulated during development. Here we show that the turnover of β-catenin is increased at cell interfaces that are targeted for disassembly during Drosophila axis elongation. The Abl tyrosine kinase is concentrated at specific planar junctions and is necessary for polarized β-catenin localization and dynamics. abl mutant embryos have decreased β-catenin turnover at shrinking edges, and these defects are accompanied by a reduction in multicellular rosette formation and axis elongation. Abl promotes β-catenin phosphorylation on the conserved tyrosine 667 and expression of an unphosphorylatable β-catenin mutant recapitulates the defects of abl mutants. Notably, a phosphomimetic β-catenin(Y667E) mutation is sufficient to increase β-catenin turnover and rescue axis elongation in abl deficient embryos. These results demonstrate that the asymmetrically localized Abl tyrosine kinase directs planar polarized junctional remodeling during Drosophila axis elongation through the tyrosine phosphorylation of β-catenin.     

The regulatory factor SipA is a highly stable β-II class protein with a SH3 fold

The small regulator SipA, interacts with the ATP-binding domain of non-bleaching sensor histidine kinase (NblS), the most conserved histidine kinase in cyanobacteria. NblS regulates photosynthesis and acclimation to a variety of environmental conditions. We show here that SipA is a highly stable protein in a wide pH range, with a thermal denaturation midpoint of 345 K. Circular dichroism and 1D ¹H NMR spectroscopies, as well as modelling, suggest that SipA is a β-II class protein, with short strands followed by turns and long random-coil polypeptide patches, matching the SH3 fold. The experimentally determined m-value and the heat capacity change upon thermal unfolding (ΔC_p) closely agreed with the corresponding theoretical values predicted from the structural model, further supporting its accuracy.     

As easy as flipping a switch?

Proteins that behave as switches help to establish the complex molecular logic that is central to biological systems. Aspiring to be nature's equal, researchers have successfully created protein switches of their own design; in particular, numerous and varied zinc-triggered switches have been made. Recent studies in which such switches have been readily identified from combinatorial protein libraries support the notion that proteins are primed to show allosteric behavior and that newly created ligand-binding sites will often be functionally coupled to the original activity of the protein. If true, this notion suggests that switch engineering might be more tractable than previously thought, boding well for the basic science, sensing and biomedical applications for which protein switches hold much promise.     

Tyrosine Phosphorylation of Integrin β3 Regulates Kindlin-2 Binding and Integrin Activation

Kindlins are essential for integrin activation in cell systems and do so by working in a cooperative fashion with talin via their direct interaction with integrin β cytoplasmic tails (CTs). Kindlins interact with the membrane-distal NxxY motif, which is distinct from the talin-binding site within the membrane-proximal NxxY motif. The Tyr residues in both motifs can be phosphorylated, and it has been suggested that this modification of the membrane-proximal NxxY motif negatively regulates interaction with the talin head domain. However, the influence of Tyr phosphorylation of the membrane-distal NxxY motif on kindlin binding is unknown. Using mutational analyses and phosphorylated peptides, we show that phosphorylation of the membrane-distal NITY⁷⁵⁹ motif in the β₃ CT disrupts kindlin-2 recognition. Phosphorylation of this membrane-distal Tyr also disables the ability of kindlin-2 to coactivate the integrin. In direct binding studies, peptides corresponding to the non-phosphorylated β₃ CT interacted well with kindlin-2, whereas the Tyr⁷⁵⁹-phosphorylated peptide failed to bind kindlin-2 with measurable affinity. These observations indicate that transitions between the phosphorylated and non-phosphorylated states of the integrin β₃ CT determine reactivity with kindlin-2 and govern the role of kindlin-2 in regulating integrin activation.     

Novel Roles for α3β1 Integrin as a Regulator of Cytoskeletal Assembly and as a Trans-dominant Inhibitor of Integrin Receptor Function in Mouse Keratinocytes

Previously we found that α3β1 integrin–deficient neonatal mice develop micro-blisters at the epidermal–dermal junction. These micro-blisters were associated with poor basement membrane organization. In the present study we have investigated the effect of α3β1-deficiency on other keratinocyte integrins, actin-associated proteins and F-actin organization. We show that the absence of α3β1 results in an increase in stress fiber formation in keratinocytes grown in culture and at the basal face of the basal keratinocytes of α3-null epidermis. Moreover, we see a higher concentration of actin-associated proteins such as vinculin, talin, and α-actinin at focal contact sites in the α3-deficient keratinocytes. These changes in focal contact composition were not due to a change in steady-state levels of these proteins, but rather to reorganization due to α3β1 deficiency. Apart from the loss of α3β1 there is no change in expression of the other integrins expressed by the α3-null keratinocytes. However, in functional assays, α3β1 deficiency allows an increase in fibronectin and collagen type IV receptor activities. Thus, our findings provide evidence for a role of α3β1 in regulating stress fiber formation and as a trans-dominant inhibitor of the functions of the other integrins in mouse keratinocytes. These results have potential implications for the regulation of keratinocyte adhesion and migration during wound healing.     

Integrin β3 Phosphorylation Dictates Its Complex with the Shc Phosphotyrosine-binding (PTB) Domain

Adaptor protein Shc plays a key role in mitogen-activated protein kinase (MAPK) signaling pathway, which can be mediated through a number of different receptors including integrins. By specifically recognizing the tyrosine-phosphorylated integrin β₃, Shc has been shown to trigger integrin outside-in signaling, although the structural basis of this interaction remains nebulous. Here we present the detailed structural analysis of Shc phosphotyrosine-binding (PTB) domain in complex with the bi-phosphorylated β₃integrin cytoplasmic tail (CT). We show that this complex is primarily defined by the phosphorylation state of the integrin C-terminal Tyr⁷⁵⁹, which fits neatly into the classical PTB pocket of Shc. In addition, we have identified a novel binding interface which concurrently accommodates phosphorylated Tyr⁷⁴⁷ of the highly conserved NPXY motif of β₃. The structure represents the first snapshot of an integrin cytoplasmic tail bound to a target for mediating the outside-in signaling. Detailed comparison with the known Shc PTB structure bound to a target TrkA peptide revealed some significant differences, which shed new light upon the PTB domain specificity.     

Factors Associated with Nitric Oxide-mediated β2 Integrin Inhibition of Neutrophils

This investigation explored the mechanism for inhibition of β₂ integrin adhesion molecules when neutrophils are exposed to nitric oxide (^•NO). Roles for specific proteins were elucidated using chemical inhibitors, depletion with small inhibitory RNA, and cells from knock-out mice. Optimal inhibition occurs with exposures to a ^•NO flux of ∼28 nmol/min for 2 min or more, which sets up an autocatalytic cascade triggered by activating type 2 nitric-oxide synthase (NOS-2) and NADPH oxidase (NOX). Integrin inhibition does not occur with neutrophils exposed to a NOX inhibitor (Nox2ds), a NOS-2 inhibitor (1400W), or with cells from mice lacking NOS-2 or the gp91^phox component of NOX. Reactive species cause S-nitrosylation of cytosolic actin that enhances actin polymerization. Protein cross-linking and actin filament formation assays indicate that increased polymerization occurs because of associations involving vasodilator-stimulated phosphoprotein, focal adhesion kinase, and protein-disulfide isomerase in proximity to actin filaments. These effects were inhibited in cells exposed to ultraviolet light which photo-reverses S-nitrosylated cysteine residues and by co-incubations with cytochalasin D. The autocatalytic cycle can be arrested by protein kinase G activated with 8-bromo-cyclic GMP and by a high ^•NO flux (∼112 nmol/min) that inactivates NOX.     

Induction of regulatory T cells by a murine β-defensin

β-Defensins are antimicrobial peptides of the innate immune system produced in the skin by various stimuli, including proinflammatory cytokines, bacterial infection, and exposure to UV radiation (UVR). In this study we demonstrate that the UVR-inducible antimicrobial peptide murine β-defensin-14 (mBD-14) switches CD4(+)CD25(-) T cells into a regulatory phenotype by inducing the expression of specific markers like Foxp3 and CTLA-4. This is functionally relevant because mBD-14-treated T cells inhibit sensitization upon adoptive transfer into naive C57BL/6 mice. Accordingly, injection of mBD-14, comparable to UVR, suppresses the induction of contact hypersensitivity and induces Ag-specific regulatory T cells (Tregs). Further evidence for the ability of mBD-14 to induce Foxp3(+) T cells is provided using DEREG (depletion of Tregs) mice in which Foxp3-expressing cells can be depleted by injecting diphtheria toxin. mBD-14 does not suppress sensitization in IL-10 knockout mice, suggesting involvement of IL-10 in mBD-14-mediated immunosuppression. However, unlike UVR, mBD-14 does not appear to mediate its immunosuppressive effects by affecting dendritic cells. Accordingly, UVR-induced immunosuppression is not abrogated in mBD-14 knockout mice. Together, these data suggest that mBD-14, like UVR, has the capacity to induce Tregs but does not appear to play a major role in UVR-induced immunosuppression. Through this capacity, mBD-14 may protect the host from microbial attacks on the one hand, but tame T cell-driven reactions on the other hand, thereby enabling an antimicrobial defense without collateral damage by the adaptive immune system.     

Apparent Involvement of a β1 Type Integrin in Coral Fertilization

Integrins are involved in a wide variety of cell adhesion processes, and have roles in gamete binding and fusion in mammals. Integrins have been also discovered in the scleractinian coral Acropora millepora (Cnidaria: Anthozoa). As a first step toward understanding the molecular basis of fertilization in corals, we examined the effect of polyclonal antisera raised against recombinant coral integrins on gamete interactions in A. millepora. Antiserum raised against integrin βcn1 dramatically decreased the binding of Acropora sperm to eggs and significantly decreased fertilization rates relative to preimmune serum and seawater controls. However, the antiserum against AmIntegrin α1 did not affect significantly either sperm–egg binding or fertilization. One possible explanation for this is that AmIntegrin α1 may preferentially mediate interactions with RGD-containing ligands, whereas mammalian α6 integrin (which is most directly implicated in gamete interactions) preferentially interacts with laminin-related ligands. Our results suggest that β1 type integrins are involved in the fertilization process in Acropora and that some functions of these molecules may have been conserved between corals and mammals. A. Iguchi and L. M. Márquez contributed equally to this work.     

The tyrosine phosphatase SHP2 regulates recovery of endothelial adherens junctions through control of β-catenin phosphorylation

Impaired endothelial barrier function results in a persistent increase in endothelial permeability and vascular leakage. Repair of a dysfunctional endothelial barrier requires controlled restoration of adherens junctions, comprising vascular endothelial (VE)-cadherin and associated β-, γ-, α-, and p120-catenins. Little is known about the mechanisms by which recovery of VE-cadherin–mediated cell–cell junctions is regulated. Using the inflammatory mediator thrombin, we demonstrate an important role for the Src homology 2-domain containing tyrosine phosphatase (SHP2) in mediating recovery of the VE-cadherin–controlled endothelial barrier. Using SHP2 substrate-trapping mutants and an in vitro phosphatase activity assay, we validate β-catenin as a bona fide SHP2 substrate. SHP2 silencing and SHP2 inhibition both result in delayed recovery of endothelial barrier function after thrombin stimulation. Moreover, on thrombin challenge, we find prolonged elevation in tyrosine phosphorylation levels of VE-cadherin–associated β-catenin in SHP2-depleted cells. No disassembly of the VE-cadherin complex is observed throughout the thrombin response. Using fluorescence recovery after photobleaching, we show that loss of SHP2 reduces the mobility of VE-cadherin at recovered cell–cell junctions. In conclusion, our data show that the SHP2 phosphatase plays an important role in the recovery of disrupted endothelial cell–cell junctions by dephosphorylating VE-cadherin–associated β-catenin and promoting the mobility of VE-cadherin at the plasma membrane.     

Identification of an 80kD Protein Associated with the α3β1 Integrin as a Proteolytic Fragment of the α3 Subunit: Studies with Human Keratinocytes

We have characterised a protein of approximately 80kD previously observed to co-immunoprecipitate with the α₃β₁ integrin in lysates of surface labelled human epiderrnalkerati-nocytes. The 80kD protein only appeared when keratinocytes were harvested with trypsin/EDTA prior to lysis and a protein of similar molecular mass could be immunoprecipitated from human dermal fibroblasts following treatment of the cells with trypsin/EDTA. N terminal sequencing established that the 80kD protein had homology with the as integrin subunit. Peptide-mass fingerprinting was used to confirm that the protein comprised the amino terminus of α₃ and established that the site of cleavage was after amino acid 629. The 80kD fragment could be coimmunoprecipitated with α₃β₁ using an antibody to the cytoplasmic domain of the α₃ subunit, showing that the fragment remained complexed with intact α₃β₁. When antibodies to the cytoplasmic and extracellular domains of α₃ were used to label human epidermis by immunofluorescence, the staining patterns were indistinguishable and there is therefore no evidence that proteolysis of α₃ plays a role in keratinocyte detachment from the basement membrane during terminal differentiation. Whether the 80kD fragment has any effects, positive or negative, on α₃β₁-mediated adhesion remains to be determined.     

Integrin β3 is not critical for neutrophil recruitment in a mouse model of pneumococcal pneumonia

Streptococcus pneumoniae is one of the most common causes of bacterial pneumonias in humans. Neutrophil migration into lungs infected with S. pneumoniae is central to the host defense but the mechanisms of neutrophil recruitment, as mediated by S. pneumoniae, into lungs are incompletely understood. Therefore, we have assessed the role of integrin αvβ3 by evaluating its subunit β3 in a mouse model of lung inflammation induced by S. pneumonia. Integrin subunit β3 knockout (β3^-/-) and wild-type (WT) mice were intratracheally instilled with either S. pneumoniae or saline. Other groups of WT mice were treated intraperitoneally with 25 μg or 50 μg of antibody against integrin β3 or with isotype-matched antibody at 1 h before instillation of S. pneumoniae. Mice were killed 24 h after infection. Flow cytometry confirmed the absence or presence of integrin subunit β3 on peripheral blood neutrophils in β3^-/- or WT mice, respectively. Neutrophil numbers in bronchoalveolar lavage (BAL) from infected β3^-/- and WT mice showed no differences. Neutrophil numbers in BAL of infected WT mice treated with β3 antibody were lower compared with those without antibody but similar to those of mice administered isotype-matched antibody. Many neutrophils were present in the perivascular spaces of the lungs in β3^-/- mice. Lungs from infected β3^-/- mice had negligible mitogen-activated protein kinase expression compared with those of infected WT mice. Thus, integrin β3 or its heterodimer αvβ3 is not critical for neutrophil migration into lungs infected with S. pneumoniae.     

Alzheimer's β-secretase cleaves a glycosyltransferase as a physiological substrate

Alzheimer's beta-secretase (BACE1) is a membrane-bound protease that cleaves the amyloid precursor protein (APP) in the trans-Golgi network, an initial step in the pathogenesis of Alzheimer's disease. Although BACE1 is distributed among various tissues including brain, its physiological substrate other than APP have not been identified. We have recently found that when BACE1 was overexpressed in COS cells together with α2,6-sialyltransferase (ST6Gal I), the secretion of ST6Gal I markedly increased, suggesting that BACE1 cleaves ST6Gal I as a physiological substrate. Thus BACE1 is the first identified protease that is responsible for the cleavage and secretion of glycosyltransferases. Published in 2004. This revised version was published online in August 2006 with corrections to the Cover Date.     

Spermine, a molecular switch regulating EGFR, integrin β3, Src, and FAK scaffolding

Intracellular polyamine levels are highly regulated by the activity of ornithine decarboxylase (ODC), which catalyzes the first rate-limiting reaction in polyamine biosynthesis, producing putrescine, which is subsequently converted to spermidine and spermine. We have shown that polyamines regulate proliferation, migration, and apoptosis in intestinal epithelial cells. Polyamines regulate key signaling events at the level of the EGFR and Src. However, the precise mechanism of action of polyamines is unknown. In the present study, we demonstrate that ODC localizes in lamellipodia and in adhesion plaques during cell spreading. Spermine regulates EGF-induced migration by modulating the interaction of the EGFR with Src. The EGFR interacted with integrin β3, Src, and focal adhesion kinase (FAK). Active Src (pY418-Src) localized with FAK during spreading and migration. Spermine prevented EGF-induced binding of the EGFR with integrin β3, Src, and FAK. Activation of Src and FAK was necessary for EGF-induced migration in HEK293 cells. EGFR-mediated Src activation in live HEK293 cells using a FRET based Src reporter showed that polyamine depletion significantly increased Src kinase activity. In vitro binding studies showed that spermine directly binds Src, and preferentially interacts with the SH2 domain of Src. The physical interaction between Src and the EGFR was severely attenuated by spermine. Therefore, spermine acts as a molecular switch in regulating EGFR-Src coupling both physically and functionally. Upon activation of the EGFR, integrin β3, FAK and Src are recruited to EGFR leading to the trans-activation of both the EGFR and Src and to the Src-mediated phosphorylation of FAK. The activation of FAK induced Rho-GTPases and subsequently migration. This is the first study to define mechanistically how polyamines modulate Src function at the molecular level.