Activation of Syk protein tyrosine kinase through interaction with integrin beta cytoplasmic domains.

Syk protein tyrosine kinase is essential for immune system development and function [1]and for the maintenance of vascular integrity [2,3]. In leukocytes, Syk is activated by binding to diphosphorylated immune receptor tyrosine-based activation motifs (pITAMs)[1]. Syk can also be activated by integrin adhesion receptors [4,5], but the mechanism of its activation is unknown. Here we report a novel mechanism for Syk's recruitment and activation, which requires that Syk bind to the integrin beta3 cytoplasmic tail. We found that both Syk and the related kinase ZAP-70 bound the beta3 cytoplasmic tail through their tandem SH2 domains. However, unlike Syk binding to pITAMs, this interaction was independent of tyrosine phosphorylation and of the phosphotyrosine binding function of Syk's tandem SH2 domains. Deletion of the four C-terminal residues of the beta3 cytoplasmic tail [beta3(759X)] decreased Syk binding and disrupted its physical association with integrin alphaIIbbeta3. Furthermore, cells expressing alphaIIbbeta3(759X) failed to exhibit Syk activation or lamellipodia formation upon cell adhesion to the alphaIIbbeta3 ligand, fibrinogen. In contrast, FAK phosphorylation and focal adhesion formation were unimpaired by this mutation. Thus, the direct binding of Syk kinase to the integrin beta3 cytoplasmic tail is a novel and functionally significant mechanism for the regulation of this important non-receptor tyrosine kinase.     

The cytoplasmic tyrosines of integrin subunit beta1 are involved in focal adhesion kinase activation

We have previously shown that mutation of the two tyrosines in the cytoplasmic domain of integrin subunit beta1 (Y783 and Y795) to phenylalanines markedly reduces the capability of beta1A integrins to mediate directed cell migration. In this study, beta1-dependent cell spreading was found to be delayed in GD25 cells expressing beta1A(Y783/795F) compared to that in wild-type GD25-beta1A. Focal adhesion kinase (FAK) tyrosine phosphorylation and activation were severely impaired in response to beta1-dependent adhesion in GD25-beta1A(Y783/795F) cells compared to that in wild-type GD25-beta1A or mutants in which only a single tyrosine was altered (beta1A(Y783F) or beta1A(Y795F)). Phosphorylation site-specific antibodies selective for FAK phosphotyrosine 397 indicated that the defect in FAK phosphorylation via beta1A(Y783/795F) lies at the level of the initial autophosphorylation step. Indeed, beta1A-dependent tyrosine phosphorylation of tensin and paxillin was lost in the beta1A(Y783/795F) cells, consistent with the impairment in FAK activation. In contrast, p130(CAS) overall tyrosine phosphorylation was unaffected by the beta1 mutations. Despite the defect in beta1-mediated FAK activation, FAK was still localized to focal adhesions. Taken together, the phenotype of the GD25-beta1A(Y783/795F) cells resembles, but is distinct from, the phenotype observed in FAK-null cells. These observations argue that tyrosines 783 and 795 within the cytoplasmic tail of integrin subunit beta1A are critical mediators of FAK activation and cell spreading in GD25 cells.     

The inner world of cell adhesion: integrin cytoplasmic domains

Many of the interactions between cells and their environment are mediated by the integrin family of heterodimeric transmembrane receptors. The past decade has been a broad-based effort to decipher the rules by which integrins function. Integrins bind both intracellular and extracellular ligands and thus transfer signals across the membrane in both directions. The cytoplasmic domains of these receptors play a key role in this bidirectional flow of information and in the formation of direct physical linkages between protein structures on the inside and outside of the cell.     

Focal adhesion kinase activated by beta(4) integrin ligation to mCLCA1 mediates early metastatic growth

Early metastatic growth occurs at sites of vascular arrest of blood-borne cancer cells and is entirely intravascular. Here we show that lung colonization by B16-F10 cells is licensed by beta(4) integrin adhesion to the mouse lung endothelial Ca(2+)-activated chloride channel protein mCLCA1. In a manner independent of Met, beta(4) integrin-mCLCA1-ligation leads to complexing with and activation of focal adhesion kinase (FAK) and downstream signaling to extracellular signal-regulated kinase (ERK). FAK/ERK signaling is Src-dependent and is interrupted by adhesion blocking antibodies and by dominant-negative (dn)-FAK mutants. Levels of ERK activation in B16-F10 cells transfected with wild-type or mutant FAK are closely associated with rates of proliferation and bromodeoxyuridine (BrdUrd) incorporation of tumor cells grown in mCLCA1-coated dishes, the ability to form tumor cell colonies on CLCA-expressing endothelial cell monolayers, and the extent of pulmonary metastatic growth. Parallel with the transfection rates, B16-F10 cells transfected with dn-FAK mutants and injected intravenously into syngeneic mice generate approximately half the number and size of lung colonies that vector-transfected B16-F10 cells produce. For the first time, beta(4) integrin ligation to its novel CLCA-adhesion partner is shown to be associated with FAK complexing, activation, and signaling to promote early, intravascular, metastatic growth.     

The N-terminal SH2 domains of Syk and ZAP-70 mediate phosphotyrosine-independent binding to integrin beta cytoplasmic domains

Syk and ZAP-70 form a subfamily of nonreceptor tyrosine kinases that contain tandem SH2 domains at their N termini. Engagement of these SH2 domains by tyrosine-phosphorylated immunoreceptor tyrosine-based activation motifs leads to kinase activation and downstream signaling. These kinases are also regulated by beta3 integrin-dependent cell adhesion via a phosphorylation-independent interaction with the beta3 integrin cytoplasmic domain. Here, we report that the interaction of integrins with Syk and ZAP-70 depends on the N-terminal SH2 domain and the interdomain A region of the kinase. The N-terminal SH2 domain alone is sufficient for weak binding, and this interaction is independent of tyrosine phosphorylation of the integrin tail. Indeed, phosphorylation of tyrosines within the two conserved NXXY motifs in the integrin beta3 cytoplasmic domain blocks Syk binding. The tandem SH2 domains of these kinases bind to multiple integrin beta cytoplasmic domains with varying affinities (beta3 (Kd = 24 nm) > beta2 (Kd = 38 nm) > beta1 (Kd = 71 nm)) as judged by both affinity chromatography and surface plasmon resonance. Thus, the binding of Syk and ZAP-70 to integrin beta cytoplasmic domains represents a novel phosphotyrosine-independent interaction mediated by their N-terminal SH2 domains.     

GIT1 utilizes a focal adhesion targeting-homology domain to bind paxillin

The GIT proteins, GIT1 and GIT2, are GTPase-activating proteins for the ADP-ribosylation factor family of small GTP-binding proteins, but also serve as adaptors to link signaling proteins to distinct cellular locations. One role for GIT proteins is to link the PIX family of Rho guanine nucleotide exchange factors and their binding partners, the p21-activated protein kinases, to remodeling focal adhesions by interacting with the focal adhesion adaptor protein paxillin. We here identified the C-terminal domain of GIT1 responsible for paxillin binding. Combining structural and mutational analyses, we show that this region folds into an anti-parallel four-helix domain highly reminiscent to the focal adhesion targeting (FAT) domain of focal adhesion kinase (FAK). Our results suggest that the GIT1 FAT-homology (FAH) domain and FAT bind the paxillin LD4 motif quite similarly. Since only a small fraction of GIT1 is bound to paxillin under normal conditions, regulation of paxillin binding was explored. Although paxillin binding to the FAT domain of FAK is regulated by tyrosine phosphorylation within this domain, we find that tyrosine phosphorylation of the FAH domain GIT1 is not involved in regulating binding to paxillin. Instead, we find that mutations within the FAH domain may alter binding to paxillin that has been phosphorylated within the LD4 motif. Thus, despite apparent structural similarity in their FAT domains, GIT1 and FAK binding to paxillin is differentially regulated.     

Class- and splice variant-specific association of CD98 with integrin beta cytoplasmic domains

CD98 is a type II transmembrane protein involved in neutral and basic amino acid transport and in cell fusion events. CD98 was implicated in the function of integrin adhesion receptors by its capacity to reverse suppression of integrin activation by isolated integrin beta(1A) domains. Here we report that CD98 associates with integrin beta cytoplasmic domains with a unique integrin class and splice variant specificity. In particular, CD98 interacted with the ubiquitous beta(1A) but not the muscle-specific splice variant, beta(1D), or leukocyte-specific beta(7) cytoplasmic domains. The ability of CD98 to associate with integrin cytoplasmic domains correlated with its capacity to reverse suppression of integrin activation. The association of CD98 with integrin beta(1A) cytoplasmic domains may regulate the function and localization of these membrane proteins.     

Focal adhesion kinase and Src mediate integrin regulation of insulin receptor phosphorylation.

We show here that phosphorylation of the insulin receptor and insulin receptor substrate-1 is increased when suspended cells are replated on fibronectin. This is not due to decreased numbers of cell surface receptors, alteration of insulin binding, or stimulation of a phosphatase activity in non-adherent cells. Expression of Src together with focal adhesion kinase (FAK) in suspended cells restores insulin-induced receptor autophosphorylation to levels observed in fibronectin-attached cells. Conversely, expression of dominant-negative mutants of either Src or FAK abolishes potentiation of insulin receptor phosphorylation by cell adhesion. The results suggest that both Src and FAK participate in integrin-mediated regulation of insulin receptor signal.     

Phosphorylation of the integrin alpha 4 cytoplasmic domain regulates paxillin binding

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.     

Focal adhesion kinase is not required for integrin function or viability in Drosophila

The mammalian focal adhesion kinase (FAK) family of non-receptor protein-tyrosine kinases has been implicated in controlling a multitude of cellular responses to the engagement of cell-surface integrins and G-protein-coupled receptors. The high level of sequence conservation between the mammalian proteins and the Drosophila homologue of FAK, Fak56, suggested that it would have similar functions. However, we show here that Drosophila Fak56 is not essential for integrin functions in adhesion, migration or signaling in vivo. Furthermore, animals lacking Fak56 are viable and fertile, demonstrating that Fak56 is not essential for other developmental or physiological functions. Despite this, overexpressed Fak56 is a potent inhibitor of integrins binding to the extracellular matrix, suggesting that Fak56 may play a subtle role in the negative regulation of integrin adhesion.     

Focal adhesion kinase mediates the integrin signaling requirement for growth factor activation of MAP kinase.

The mitogen-activated protein (MAP) kinase pathway is a critical regulator of cell growth, migration, and differentiation. Growth factor activation of MAP kinase in NIH 3T3 cells is strongly dependent upon integrin-mediated adhesion, an effect that contributes to the anchorage dependence of normal cell growth. We now show that expression of constructs that constitutively activate focal adhesion kinase (FAK) rescued the defect in serum activation of MAP kinase in suspended cells without directly activating MAP kinase. Dominant negative FAK blocked both the rescue of suspended cells by the activated construct and the serum activation of MAP kinase in adherent cells. MAP kinase in FAK(-/)- mouse embryo fibroblasts was adhesion-insensitive, and reexpression of FAK restored its adhesion dependence. MAP kinase activity in ras-transformed cells is still decreased in suspension, but expression of constructs that constitutively activate FAK enhanced their anchorage-independent growth without increasing adherent growth. V-src, which activates both Ras and FAK, induced MAP kinase activation that was insensitive to loss of adhesion, and that was blocked by a dominant negative FAK. These results demonstrate that FAK mediates the integrin requirement for serum activation of MAP kinase in normal cells, and that bypassing this mechanism contributes to anchorage-independent growth in transformed cells.     

Focal adhesion proteins FAK and paxillin increase in hypertrophied skeletal muscle

Components of signaling pathways for mechanotransduction duringload-induced enlargement of skeletal muscle have not been completelydefined. We hypothesized that loading of skeletal muscle would resultin an adaptive increase in the expression of two focal adhesion complex(FAC)-related proteins, focal adhesion kinase (FAK) and paxillin, aswell as increased FAK activity. FAK protein was immunolocalized to thesarcolemmal region of rooster anterior latissimus dorsi (ALD) myofibersin the middle of the ALD muscle. FAK (77 and 81%) and paxillin (206 and 202%) protein concentrations per unit of total protein in Westernblots increased significantly after 1.5 and 7 days, but not after 13 days, of stretch-induced hypertrophy-hyperplasia of the ALD muscle. FAK autokinase activity in immunoprecipitates was increased after 1.5, 7, and 13 days in stretched ALD muscles. To determine whether increasedFAK and paxillin protein concentrations are associated with hypertrophyand/or new fiber formation, two additional experiments were performed.First, during formation of primary chicken myotubes (a model of newfiber formation), FAK protein concentration (63%), FAK activity(157%), and paxillin protein concentration (97%) increased comparedwith myoblasts. Second, FAK (112% and 611%) and paxillin (87% and431%) protein concentrations per unit of total protein in the soleusmuscle increased at 1 and 8 days after surgical ablation of thesynergistic gastrocnemius muscle (a model of hypertrophy withouthyperplasia). Thus increases in components of the FAC occur inhypertrophying muscle of animals and in newly formed muscle fibers inculture. Furthermore, increased FAK activity suggests a possibleconvergence of signaling at the FAC in load-induced growth of skeletal muscle.

     

Assembly and function of integrin receptors is dependent on opposing alpha and beta cytoplasmic domains.

The membrane proximal regions of integrin alpha and beta subunits are highly conserved in evolution. In particular, all integrin alpha subunits share the KXGFFKR sequence at the beginning of their cytoplasmic domains. Previous work has shown that this domain is important in integrin receptor assembly. Using chimeric integrin alpha and beta subunits, we show that the native cytoplasmic domains of both subunits must be present for efficient assembly. Most strikingly, chimeric alpha 1 and beta 1 subunits with reciprocally swapped intracellular domains dimerize selectively into collagen IV receptors expressed at high levels on the surface. However, these receptors, which bind ligand efficiently, are deficient in a variety of post-ligand binding events, including cytoskeletal association and induction of tyrosine phosphorylation. Furthermore, deletion of the distal alpha cytoplasmic domain in the swapped heterodimers leads to ligand-independent focal contact localization, which also occurs in wild-type subunits when the distal cytoplasmic domain is deleted. These results show that proper integrin assembly requires opposed alpha and beta cytoplasmic domains, and this opposition prevents ligand-independent focal contact localization. Our working hypothesis is that these two domains may associate during receptor assembly and provide the mechanism for integrin receptor latency.     

Contrasting roles for integrin beta 1 and beta 5 cytoplasmic domains in subcellular localization, cell proliferation, and cell migration

To carry out a detailed comparison of the roles of integrin beta 1 and beta 5 cytoplasmic domains, we expressed both wild type beta 1 and chimeric beta 1/5 constructs in CHO cells. In the latter, the cytoplasmic domain of beta 1 was replaced with that of beta 5. The human beta 1 and beta 1/5 constructs appeared at similar levels at the cell surface (mostly as alpha 5 beta 1 heterodimers) and contributed equally to CHO cell adhesion to fibronectin. However, beta 1 but not beta 1/5 localized to focal adhesion-like structures when CHO cells were spread on fibronectin. Furthermore, only the beta 1-CHO cells showed increased proliferation in response to fibronectin plus an integrin-activating anti-beta 1 antibody, and showed increased appearance of 32P-labeled protein (p90) that correlated with proliferation. In sharp contrast, the beta 1/5-CHO cells were notably more migratory than beta 1-CHO cells in a transwell haptotactic migration assay. These results indicate that the beta 1 and beta 5 integrin subunit cytoplasmic domains can translate similar adhesive information into highly contrasting subsequent events. Thus, we have established that "inside-out" and "outside-in" integrin signaling pathways are regulated by fundamentally distinct mechanisms. In addition, we suggest that the same properties of the beta 1 cytoplasmic domain that promote recruitment to visible focal adhesion-like structures may also be conductive to cell proliferation. Conversely, the properties of the beta 5 tail that make it less likely to localize into focal adhesion-like structures may contribute to enhanced cell migration.     

Distinct functions of integrin alpha and beta subunit cytoplasmic domains in cell spreading and formation of focal adhesions

Integrin-mediated cell adhesion often results in cell spreading and the formation of focal adhesions. We exploited the capacity of recombinant human alpha IIb beta 3 integrin to endow heterologous cells with the ability to adhere and spread on fibrinogen to study the role of integrin cytoplasmic domains in initiation of cell spreading and focal adhesions. The same constructs were also used to analyze the role of the cytoplasmic domains in maintenance of the fidelity of the integrin repertoire at focal adhesions. Truncation mutants of the cytoplasmic domain of alpha IIb did not interfere with the ability of alpha IIb beta 3 to initiate cell spreading and form focal adhesions. Nevertheless, deletion of the alpha IIb cytoplasmic domain allowed indiscriminate recruitment of alpha IIb beta 3 to focal adhesions formed by other integrins. Truncation of the beta 3 subunit cytoplasmic domain abolished cell spreading mediated by alpha IIb beta 3 and also abrogated recruitment of alpha IIb beta 3 to focal adhesions. This truncation also dramatically impaired the ability of alpha IIb beta 3 to mediate the contraction of fibrin gels. In contrast, the beta 3 subunit cytoplasmic truncation did not reduce the fibrinogen binding affinity of alpha IIb beta 3. Thus, the integrin beta 3 subunit cytoplasmic domain is necessary and sufficient for initiation of cell spreading and focal adhesion formation. Further, the beta 3 cytoplasmic domain is required for the transmission of intracellular contractile forces to fibrin gels. The alpha subunit cytoplasmic domain maintains the fidelity of recruitment of the integrins to focal adhesions and thus regulates their repertoire of integrins.     

The Talin head domain binds to integrin beta subunit cytoplasmic tails and regulates integrin activation.

The beta subunit cytoplasmic domains of integrin adhesion receptors are necessary for the connection of these receptors to the actin cytoskeleton. The cytoplasmic protein, talin, binds to beta integrin cytoplasmic tails and actin filaments, hence forming an integrin-cytoskeletal linkage. We used recombinant structural mimics of beta(1)A, beta(1)D and beta(3) integrin cytoplasmic tails to characterize integrin-binding sites within talin. Here we report that an integrin-binding site is localized within the N-terminal talin head domain. The binding of the talin head domain to integrin beta tails is specific in that it is abrogated by a single point mutation that disrupts integrin localization to talin-rich focal adhesions. Integrin-cytoskeletal interactions regulate integrin affinity for ligands (activation). Overexpression of a fragment of talin containing the head domain led to activation of integrin alpha(IIb)beta(3); activation was dependent on the presence of both the talin head domain and the integrin beta(3) cytoplasmic tail. The head domain of talin thus binds to integrins to form a link to the actin cytoskeleton and can thus regulate integrin function.     

Focal adhesion kinase stabilizes the cytoskeleton

Focal adhesion kinase (FAK) is a central focal adhesion protein that promotes focal adhesion turnover, but the role of FAK for cell mechanical stability is unknown. We measured the mechanical properties of wild-type (FAKwt), FAK-deficient (FAK−/−), FAK-silenced (siFAK), and siControl mouse embryonic fibroblasts by magnetic tweezer, atomic force microscopy, traction microscopy, and nanoscale particle tracking microrheology. FAK-deficient cells showed lower cell stiffness, reduced adhesion strength, and increased cytoskeletal dynamics compared to wild-type cells. These observations imply a reduced stability of the cytoskeleton in FAK-deficient cells. We attribute the reduced cytoskeletal stability to rho-kinase activation in FAK-deficient cells that suppresses the formation of ordered stress fiber bundles, enhances cortical actin distribution, and reduces cell spreading. In agreement with this interpretation is that cell stiffness and cytoskeletal stability in FAK−/− cells is partially restored to wild-type level after rho-kinase inhibition with Y27632.     

Focal adhesion kinase modulates tension signaling to control actin and focal adhesion dynamics

In response to alphabeta1 integrin signaling, transducers such as focal adhesion kinase (FAK) become activated, relaying to specific machineries and triggering distinct cellular responses. By conditionally ablating Fak in skin epidermis and culturing Fak-null keratinocytes, we show that FAK is dispensable for epidermal adhesion and basement membrane assembly, both of which require alphabeta1 integrins. FAK is also dispensible for proliferation/survival in enriched medium. In contrast, FAK functions downstream of alphabeta1 integrin in regulating cytoskeletal dynamics and orchestrating polarized keratinocyte migration out of epidermal explants. Fak-null keratinocytes display an aberrant actin cytoskeleton, which is tightly associated with robust, peripheral focal adhesions and microtubules. We find that without FAK, Src, p190RhoGAP, and PKL-PIX-PAK, localization and/or activation at focal adhesions are impaired, leading to elevated Rho activity, phosphorylation of myosin light chain kinase, and enhanced tensile stress fibers. We show that, together, these FAK-dependent activities are critical to control the turnover of focal adhesions, which is perturbed in the absence of FAK.     

Differential role of beta(1C) and beta(1A) integrin cytoplasmic variants in modulating focal adhesion kinase, protein kinase B/AKT, and Ras/Mitogen-activated protein kinase pathways

The integrin cytoplasmic domain modulates cell proliferation, adhesion, migration, and intracellular signaling. The beta(1) integrin subunits, beta(1C) and beta(1A), that contain variant cytoplasmic domains differentially affect cell proliferation; beta(1C) inhibits proliferation, whereas beta(1A) promotes it. We investigated the ability of beta(1C) and beta(1A) to modulate integrin-mediated signaling events that affect cell proliferation and survival in Chinese hamster ovary stable cell lines expressing either human beta(1C) or human beta(1A). The different cytodomains of either beta(1C) or beta(1A) did not affect either association with the endogenous alpha(2), alpha(V), and alpha(5) subunits or cell adhesion to fibronectin or TS2/16, a mAb to human beta(1). Upon engagement of endogenous and exogenous integrins by fibronectin, cells expressing beta(1C) showed significantly inhibited extracellular signal-regulated kinase (ERK) 2 activation compared with beta(1A) stable cell lines. In contrast, focal adhesion kinase phosphorylation and Protein Kinase B/AKT activity were not affected. Selective engagement of the exogenously expressed beta(1C) by TS2/16 led to stimulation of Protein Kinase B/AKT phosphorylation but not of ERK2 activation; in contrast, beta(1A) engagement induced activation of both proteins. We show that Ras activation was strongly reduced in beta(1C) stable cell lines in response to fibronectin adhesion and that expression of constitutively active Ras, Ras 61 (L), rescued beta(1C)-mediated down-regulation of ERK2 activation. Inhibition of cell proliferation in beta(1C) stable cell lines was attributable to an inhibitory effect of beta(1C) on the Ras/MAP kinase pathway because expression of activated MAPK kinase rescued beta(1C) antiproliferative effect. These findings show that the beta(1C) variant, by means of a unique signaling mechanism, selectively inhibits the MAP kinase pathway by preventing Ras activation without affecting either survival signals stimulated by integrins or cellular interactions with the extracellular matrix. These findings highlight a role for beta(1)-specific cytodomain sequences in maintaining an intracellular balance of proliferation and survival signals.