[Show abstract][Hide abstract]ABSTRACT: Activation of Rap1 small GTPases stabilizes cell--cell junctions, and this activity requires Krev Interaction Trapped gene 1 (KRIT1). Loss of KRIT1 disrupts cardiovascular development and causes autosomal dominant familial cerebral cavernous malformations. Here we report that native KRIT1 protein binds the effector loop of Rap1A but not H-Ras in a GTP-dependent manner, establishing that it is an authentic Rap1-specific effector. By modeling the KRIT1-Rap1 interface we designed a well-folded KRIT1 mutant that exhibited a ~40-fold-reduced affinity for Rap1A and maintained other KRIT1-binding functions. Direct binding of KRIT1 to Rap1 stabilized endothelial cell-cell junctions in vitro and was required for cardiovascular development in vivo. Mechanistically, Rap1 binding released KRIT1 from microtubules, enabling it to locate to cell--cell junctions, where it suppressed Rho kinase signaling and stabilized the junctions. These studies establish that the direct physical interaction of Rap1 with KRIT1 enables the translocation of microtubule-sequestered KRIT1 to junctions, thereby supporting junctional integrity and cardiovascular development.
Full-text · Article · Jun 2011 · Molecular biology of the cell
[Show abstract][Hide abstract]ABSTRACT: Cerebral cavernous malformation (CCM), a disease associated with defective endothelial junctions, result from autosomal dominant CCM1 mutations that cause loss of KRIT-1 protein function, though how the loss of KRIT-1 leads to CCM is obscure. KRIT-1 binds to Rap1, a guanosine triphosphatase that maintains the integrity of endothelial junctions. Here, we report that KRIT-1 protein is expressed in cultured arterial and venous endothelial cells and is present in cell-cell junctions. KRIT-1 colocalized and was physically associated with junctional proteins via its band 4.1/ezrin/radixin/moesin (FERM) domain. Rap1 activity regulated the junctional localization of KRIT-1 and its physical association with junction proteins. However, the association of the isolated KRIT-1 FERM domain was independent of Rap1. Small interfering RNA-mediated depletion of KRIT-1 blocked the ability of Rap1 to stabilize endothelial junctions associated with increased actin stress fibers. Thus, Rap1 increases KRIT-1 targeting to endothelial cell-cell junctions where it suppresses stress fibers and stabilizes junctional integrity.
Full-text · Article · Nov 2007 · The Journal of Cell Biology
[Show abstract][Hide abstract]ABSTRACT: In mammals, β1 integrin adhesion receptors generate signals that mediate cell spreading, migration, proliferation, and survival.
CD98, a heterodimeric transmembrane protein, physically associates with certain integrin β subunit cytoplasmic domains (tails)
via its heavy chain, CD98hc (SLC3A2), and loss of CD98hc impairs integrin signaling. Here we have used the lack of CD98hc
interaction with the Drosophila integrin βPS tail for a homology scanning analysis that implicated the C-terminal 8 residues of β3 (Thr755-Thr802) in CD98hc binding. We then identified point mutations in the β3 C terminus (T755K and T758M) that abolish CD98hc association
and a double mutation in the corresponding residues in the βPS tail (K839T,M842T), which resulted in gain of CD98hc interaction.
Furthermore, the loss of function β3(T755K) mutation or the gain of function β3/βPS(K839T,M842T) led to a loss or gain of
integrin-mediated cell spreading, respectively. Thus, we have identified critical integrin residues required for CD98hc interaction
and in doing so have shown that CD98c interaction with the integrin β tail is required for its ability to mediate integrin
signaling. These studies also provide new insights into how CD98hc may cooperate with other cytoplasmic domain binding proteins
to modulate integrin functions and into the evolution of integrin signaling.
[Show abstract][Hide abstract]ABSTRACT: We used a TAP-tag approach to identify candidate binding proteins for the related Ras family GTPases: H-Ras, R-Ras, and Rap1A. Protein complexes were isolated from mouse fibroblasts, and component proteins were identified by a combination of nanoflow HPLC and tandem mass spectrometry. H-Ras was found to associate with numerous cytoskeletal proteins including talin-1. R-Ras and Rap1A each associated with various signaling molecules, many of which are membrane-associated. Thus, we have established the first database of potential Ras interactors in mammalian cells.
Full-text · Article · Jun 2007 · Journal of Proteome Research
[Show abstract][Hide abstract]ABSTRACT: A-kinase anchoring proteins (AKAPs) control the localization and substrate specificity of cAMP-dependent protein kinase (PKA), tetramers of regulatory (PKA-R) and catalytic (PKA-C) subunits, by binding to PKA-R subunits. Most mammalian AKAPs bind Type II PKA through PKA-RII (ref. 2), whereas dual specificity AKAPs bind both PKA-RI and PKA-RII (ref. 3). Inhibition of PKA-AKAP interactions modulates PKA signalling. Localized PKA activation in pseudopodia of migrating cells phosphorylates alpha4 integrins to provide spatial cues governing cell motility. Here, we report that the alpha4 cytoplasmic domain is a Type I PKA-specific AKAP that is distinct from canonical AKAPs in two ways: the alpha4 interaction requires the PKA holoenzyme, and is insensitive to amphipathic peptides that disrupt most PKA-AKAP interactions. We exploited type-specific PKA anchoring peptides to create genetically encoded baits that sequester specific PKA isoforms to the mitochondria and found that mislocalization of Type I, but not Type II, PKA disrupts alpha4 phosphorylation and markedly inhibits the velocity and directional persistence of cell migration.
Full-text · Article · May 2007 · Nature Cell Biology
[Show abstract][Hide abstract]ABSTRACT: Regulation of integrin affinity (activation) is essential for metazoan development and for many pathological processes. Binding of the talin phosphotyrosine-binding (PTB) domain to integrin beta subunit cytoplasmic domains (tails) causes activation, whereas numerous other PTB-domain-containing proteins bind integrins without activating them. Here we define the structure of a complex between talin and the membrane-proximal integrin beta3 cytoplasmic domain and identify specific contacts between talin and the integrin tail required for activation. We used structure-based mutagenesis to engineer talin and beta3 variants that interact with comparable affinity to the wild-type proteins but inhibit integrin activation by competing with endogenous talin. These results reveal the structural basis of talin's unique ability to activate integrins, identify an interaction that could aid in the design of therapeutics to block integrin activation, and enable engineering of cells with defects in the activation of multiple classes of integrins.
[Show abstract][Hide abstract]ABSTRACT: The regeneration of peripheral nerve is associated with a change in the alternative splicing of the fibronectin primary gene transcript to re-express embryonic isoforms containing a binding site for alpha4beta1 integrins that promote neurite outgrowth. Here we use PC12 cells to examine the role of the interaction between paxillin and the alpha4 integrin cytoplasmic domain in neurite outgrowth.
Expression of alpha4 with mutations in the paxillin-binding domain reduced neurite outgrowth on recombinant embryonic fibronectin fragments relative to wild type alpha4. Over-expression of paxillin promoted neurite outgrowth while a mutant isoform lacking the LD4 domain implicated in the regulation of ARF and Rac GTPases was less effective. Optimal alpha4-mediated migration in leucocytes requires spatial regulation of alpha4 phosphorylation at Ser988, a post-translational modification that blocks paxillin binding to the integrin cytoplasmic domain. In keeping with this alpha4(S988D), which mimics phosphorylated alpha4, did not promote neurite outgrowth. However, alpha4 was not phosphorylated in the PC12 cells, and a non-phosphorylatable alpha4(S988A) mutant promoted neurite outgrowth indistinguishably from the wild type integrin.
We establish the importance of the alpha4 integrin-paxillin interaction in a model of axonal regeneration and highlight differing dependence on phosphorylation of alpha4 for extension of neuronal growth cones and migration of non-neural cells.
[Show abstract][Hide abstract]ABSTRACT: Integrin receptors, composed of transmembrane alpha and beta subunits, are essential for the development and functioning of multicellular animals. Agonist stimulation leads cells to regulate integrin affinity ("activation"), thus controlling cell adhesion and migration, controlling extracellular-matrix assembly, and contributing to angiogenesis, tumor cell metastasis, inflammation, the immune response, and hemostasis. A final step in integrin activation is the binding of talin, a cytoskeletal protein, to integrin beta cytoplasmic domains. Many different signaling molecules that regulate integrin affinity have been described, but a pathway that connects agonist stimulation to talin binding and activation has not been mapped.
We used forward, reverse, and synthetic genetics to engineer and order an integrin activation pathway in cells expressing a prototype activatable integrin, platelet alphaIIbbeta3. Phorbol myristate acetate (PMA) activated alphaIIbbeta3 only after the increased expression of both recombinant protein kinase Calpha (PKCalpha) and talin to levels approximating those in platelets. Inhibition of Rap1 GTPase reduced alphaIIbbeta3 activation, whereas activated Rap1A(G12V) bypassed the requirement for PKC, establishing that Rap1 is downstream of PKC. Talin binding to integrins mediates Rap1-induced activation because Rap1A(G12V) failed to activate alphaIIbbeta3 in cells expressing integrin binding-defective talin (W359A). Rap1 activated integrins by forming an integrin-associated complex containing talin in combination with the Rap effector, RIAM. Furthermore, siRNA-mediated knockdown of RIAM blocked integrin activation.
We have, for the first time, ordered a pathway from agonist stimulation to integrin activation and established the Rap1-induced formation of an "integrin activation complex," containing RIAM and talin, that binds to and activates the integrin.
[Show abstract][Hide abstract]ABSTRACT: Antagonists to alpha4 integrin show promise for several autoimmune and inflammatory diseases but may exhibit mechanism-based toxicities. We tested the capacity of blockade of alpha4 integrin signaling to perturb functions involved in inflammation, while limiting potential adverse effects. We generated and characterized mice bearing a Y991A mutation in alpha4 integrin [alpha4(Y991A) mice], which blocks paxillin binding and inhibits alpha4 integrin signals that support leukocyte migration. In contrast to the embryonic-lethal phenotype of alpha4 integrin-null mice, mice bearing the alpha4(Y991A) mutation were viable and fertile; however, they exhibited defective recruitment of mononuclear leukocytes into thioglycollate-induced peritonitis. Alpha4 integrins are essential for definitive hematopoiesis; however, the alpha4(Y991A) mice had intact lymphohematopoiesis and, with the exception of reduced Peyer's patches, normal architecture and cellularity of secondary lymphoid tissues. We conclude that interference with alpha4 integrin signaling can selectively impair mononuclear leukocyte recruitment to sites of inflammation while sparing vital functions of alpha4 integrins in development and hematopoiesis.
Full-text · Article · Mar 2006 · Journal of Clinical Investigation
[Show abstract][Hide abstract]ABSTRACT: Formation of a stable lamellipodium at the front of migrating cells requires localization of Rac activation to the leading edge. Restriction of alpha4 integrin phosphorylation to the leading edge limits the interaction of alpha4 with paxillin to the sides and rear of a migrating cell. The alpha4-paxillin complex inhibits stable lamellipodia, thus confining lamellipod formation to the cell anterior. Here we report that binding of paxillin to the alpha4 integrin subunit inhibits adhesion-dependent lamellipodium formation by blocking Rac activation. The paxillin LD4 domain mediates this reduction in Rac activity by recruiting an ADP-ribosylation factor GTPase-activating protein (Arf-GAP) that decreases Arf activity, thereby inhibiting Rac. Finally, the localized formation of the alpha4-paxillin-Arf-GAP complex mediates the polarization of Rac activity and promotes directional cell migration. These findings establish a mechanism for the spatial localization of Rac activity to enhance cell migration.
Preview · Article · May 2005 · Nature Cell Biology
[Show abstract][Hide abstract]ABSTRACT: alpha 4 integrins mediate increased cell migration and decreased cell spreading because the alpha 4 cytoplasmic domain (tail) binds tightly to paxillin, a signaling adaptor protein. Paxillin binding to the alpha 4 tail is blocked by alpha 4 phosphorylation at Ser988. To establish the biological role of alpha 4 phosphorylation, we reconstituted alpha 4-deficient Jurkat T cells with phosphorylation-mimicking (alpha 4(S988D)) or non-phosphorylatable (alpha 4(S988A)) mutants. alpha 4(S988D) disrupted paxillin binding and also inhibited cell migration and promoted cell spreading. In contrast, the non-phosphorylatable alpha 4(S988A) resulted in a further reduction in cell spreading; however, this mutation led to an unexpected suppression of cell migration. The suppression of cell migration by alpha 4(S988A) was ascribable to enhanced alpha 4-paxillin association, because enforced association by an alpha 4-paxillin fusion led to a phenotype similar to that of the non-phosphorylatable alpha 4(S988A) mutant. These data establish that optimal alpha 4-mediated cell migration requires both phosphorylation and dephosphorylation of the alpha 4 cytoplasmic domain to regulate the reversible binding of paxillin.
Preview · Article · Oct 2003 · Journal of Biological Chemistry
[Show abstract][Hide abstract]ABSTRACT: Integrins coordinate spatial signaling events essential for cell polarity and directed migration. Such signals from alpha4 integrins regulate cell migration in development and in leukocyte trafficking. Here, we report that efficient alpha4-mediated migration requires spatial control of alpha4 phosphorylation by protein kinase A, and hence localized inhibition of binding of the signaling adaptor, paxillin, to the integrin. In migrating cells, phosphorylated alpha4 accumulated along the leading edge. Blocking alpha4 phosphorylation by mutagenesis or by inhibition of protein kinase A drastically reduced alpha4-dependent migration and lamellipodial stability. alpha4 phosphorylation blocks paxillin binding in vitro; we now find that paxillin and phospho-alpha4 were in distinct clusters at the leading edge of migrating cells, whereas unphosphorylated alpha4 and paxillin colocalized along the lateral edges of those cells. Furthermore, enforced paxillin association with alpha4 inhibits migration and reduced lamellipodial stability. These results show that topographically specific integrin phosphorylation can control cell migration and polarization by spatial segregation of adaptor protein binding.
Full-text · Article · Sep 2003 · The Journal of Cell Biology
[Show abstract][Hide abstract]ABSTRACT: Engagement of very late Ag-4 (integrin alpha(4)beta(1)) by ligands such as VCAM-1 markedly stimulates leukocyte migration mediated by LFA-1 (integrin alpha(L)beta(2)). This form of integrin trans-regulation in T cells requires the binding of paxillin to the alpha(4) integrin cytoplasmic domain. This conclusion is based on the abolition of trans-regulation in Jurkat T cells by an alpha(4) mutation (alpha(4)(Y991A)) that disrupts paxillin binding. Furthermore, cellular expression of an alpha(4)-binding fragment of paxillin that blocks the alpha(4)-paxillin interaction, selectively blocked VCAM-1 stimulation of alpha(L)beta(2)-dependent cell migration. The alpha(4)-paxillin association mediates trans-regulation by enhancing the activation of tyrosine kinases, focal adhesion kinase (FAK) and/or proline-rich tyrosine kinase-2 (Pyk2), based on two lines of evidence. First, disruption of the paxillin-binding site in the alpha(4) tail resulted in much less alpha(4)beta(1)-mediated phosphorylation of Pyk2 and FAK. Second, transfection with cDNAs encoding C-terminal fragments of Pyk2 and FAK, which block the function of the intact kinases, blocked alpha(4)beta(1) stimulation of alpha(L)beta(2)-dependent migration. These results define a proximal protein-protein interaction of an integrin cytoplasmic domain required for trans-regulation between integrins, and establish that augmented activation of Pyk2 and/or FAK is an immediate signaling event required for the trans-regulation of integrin alpha(L)beta(2) by alpha(4)beta(1).
Preview · Article · Jul 2003 · The Journal of Immunology
[Show abstract][Hide abstract]ABSTRACT: The alpha4 integrins (alpha4beta1 and alpha4beta7) play multiple roles in the immune system. Alpha4 integrins impact hematopoiesis, leukocyte trafficking in immune surveillance and inflammation, and leukocyte activation and survival. To perform these functions, alpha4 integrins act as both adhesive and signaling receptors. Paxillin, a signaling adapter molecule, binds directly to the alpha4 subunit cytoplasmic domain, and its binding is regulated by serine phosphorylation of the alpha4 subunit. This regulated interaction of paxillin with the alpha4 subunit is likely to regulate the diverse functions of alpha4 integrins in the immune system. Furthermore, this protein-protein interaction may provide novel targets for the modulation of the immune response.
No preview · Article · Sep 2002 · Immunological Reviews
[Show abstract][Hide abstract]ABSTRACT: This chapter describes the protocols designed to analyze the role of both Ras and R-Ras in regulating integrin function. The chapter describes specifically, a fluorescence activated cell sorting (FACS)-based assay to determine the effect of activated H-Ras and R-Ras on integrin ligand-binding affinity. Integrins are heterodimeric cell-cell and cell-matrix adhesion receptors that play a central part in controlling cell growth, survival, migration, and tumor metastasis. A characteristic feature of certain integrins is their ability to dynamically modulate their affinity for ligand in response to extracellular cues, a process termed inside-out signaling or activation. At present, the signal transduction pathways controlling integrin activation are poorly understood. However, studies have demonstrated that the members of the Ras family of small GTP-binding proteins and their downstream effectors are key players in regulating integrin activation. In contrast to R-Ras, activated H-Ras and its downstream effector kinase Raf-1 were found to suppress the activation of integrins in Chinese hamster ovary (CHO) cells.
[Show abstract][Hide abstract]ABSTRACT: alpha4 integrins are essential for embryogenesis, hematopoiesis, inflammation, and immune response possibly because alpha4 integrins have distinct signaling properties from other integrins. Specifically, the alpha4 cytoplasmic domain binds tightly to paxillin, a signaling adaptor protein, leading to increased cell migration and an altered cytoskeletal organization that results in reduced cell spreading. The alpha4 tail contains potential phosphorylation sites clustered in its core paxillin binding region. We now report that the alpha4 tail is phosphorylated in vitro and in vivo. Furthermore, Ser(988) is a major phosphorylation site. Using antibodies specific for Ser(988)-phosphorylated alpha4, we found the stoichiometry of alpha4 phosphorylation varied in different cells. However, >60% of alpha4 was phosphorylated in Jurkat T cells. Phosphorylation at Ser(988) blocked paxillin binding to the alpha4 tail. A phosphorylation-mimicking mutant of alpha4 (alpha4S988D) blocked paxillin binding and reversed the inhibitory effect of alpha4 on cell spreading. Consequently, alpha4 phosphorylation is a biochemical mechanism to modulate paxillin binding to alpha4 integrins with consequent regulation of alpha4 integrin-dependent cellular functions.
Preview · Article · Nov 2001 · Journal of Biological Chemistry
[Show abstract][Hide abstract]ABSTRACT: The closely related small GTP-binding proteins H-Ras and R-Ras have opposing effects on the regulation of integrin cell adhesion receptors. To gain insight into the properties of R-Ras with respect to the regulation of integrin function and interactions with downstream effectors we performed an analysis of R-Ras variants containing mutations in the effector binding domain and C-terminal prenylation site. We found that the activation of the downstream effector PI 3-kinase was sensitive to mutations in the effector binding domain, as was the binding to the effectors, Ral-GDS, Raf-1 and the novel effector Nore1. Furthermore, specific mutations in the effector binding loop and C-terminal prenylation motif impaired the ability of R-Ras to regulate integrin function in CHO cells. However, the ability of the R-Ras effector loop mutants to bind, and activate known effectors did not correlate with their ability to regulate integrin function. Thus, the known R-Ras effectors are not critical for regulating integrin activation, at least in CHO cells. Consequently, these studies provide insight into the structural basis of the interactions between R-Ras and its candidate effectors and suggest the existence of novel mechanisms through which this GTPase could regulate cell adhesion.
[Show abstract][Hide abstract]ABSTRACT: Alpha4 integrins (alpha4beta1 and alpha4beta7) have a restricted distribution pattern and are critical for the development and diseases of the cardiovascular system. alpha4 integrins support unique biological properties such as promoting cell migration and inhibiting cell spreading and focal adhesion formation. We have found that the alpha4 integrin subunit directly and tightly binds to a signaling adapter molecule, paxillin, and disruption of the alpha4-paxillin interaction interferes with many of alpha4-dependent biological functions. Consequently, the interaction of alpha4 integrins with paxillin may play an important role in regulating alpha4-mediated functions. This review focuses on what we have known about the alpha4-paxillin interaction and discusses the possible mechanism of regulation for this interaction.
No preview · Article · Sep 2000 · Trends in Cardiovascular Medicine