Antitumor effect of E1A in ovarian cancer by cytoplasmic sequestration of activated ERK by PEA15.

The adenovirus type 5 gene E1A is known to suppress tumorigenicity by transcriptionally downregulating HER-2/neu (HER2) or by inducing apoptosis. We show here that E1A also suppressed the tumorigenicity of the low-HER2-expressing ovarian cancer cell line OVCAR-3 by decreasing cell proliferation. We further found that the mechanism responsible for this reduced proliferation is the presence of PEA15 (phosphoprotein enriched in astrocytes), which is upregulated by E1A in ovarian cancer; PEA15 promotes translocation of ERK from the nucleus to the cytoplasm, leading to inhibition of ERK-dependent transcription and proliferation. Indeed, siRNA-mediated knockdown of PEA15 expression in OVCAR-3 stable E1A transfectants resulted in a nuclear accumulation of the active form of ERK, followed by an increase in Elk-1 activity, DNA synthesis, and anchorage-independent growth. Finally, PEA15 by itself suppressed colony formation in breast and ovarian cancer cell lines, in which E1A is known to have antitumor activity. We conclude that part of the antitumor effect of E1A in ovarian cancer results from cytoplasmic sequestration of the activated form of ERK by PEA15.

Integrin activation by talin.

The development and integrity of the cardiovascular system depends on integrins, a family of adhesion receptors, vitally important for homeostasis of animal species from fruit fly to man. Integrins are critical players in cell migration, cell adhesion, cell cycle progression, differentiation, and apoptosis. Consequently, integrins have a major impact on the patterning and functions of the blood and cardiovascular system. Integrins undergo conformational changes, which alter their affinity for ligands through a process operationally defined as integrin activation. Integrin activation is important for platelet aggregation, leukocyte extravasation, and cell adhesion and migration, thus influencing such processes as hemostasis, inflammation and angiogenesis. Recently, a series of studies have begun to define the mechanism of integrin activation by demonstrating that binding of a cytoskeletal protein, talin, to integrin beta subunit cytoplasmic tail is a last common step in integrin activation. These findings indicate that talin is likely to be at the center of converging signaling pathways regulating integrin activation.

Integrin activation takes shape.

Integrins are cell surface adhesion receptors that are essential for the development and function of multicellular animals. Here we summarize recent findings on the regulation of integrin affinity for ligand (activation), one mechanism by which cells modulate integrin function. The focus is on the structural basis of integrin activation, the role of the cytoplasmic domain in integrin affinity regulation, and potential mechanisms by which activation signals are propagated from integrin cytoplasmic domains to the extracellular ligand-binding domain.

PEA-15 mediates cytoplasmic sequestration of ERK MAP kinase.

The ERK 1/2 MAP kinase pathway controls cell growth and survival and modulates integrin function. Here, we report that PEA-15, a protein variably expressed in multiple cell types, blocks ERK-dependent transcription and proliferation by binding ERKs and preventing their localization in the nucleus. PEA-15 contains a nuclear export sequence required for its capacity to anchor ERK in the cytoplasm. Genetic deletion of PEA-15 results in increased ERK nuclear localization with consequent increased cFos transcription and cell proliferation. Thus, PEA-15 can redirect the biological outcome of MAP kinase signaling by regulating the subcellular localization of ERK MAP kinase.

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.

Identification of cell signaling molecules by expression cloning.

Using expression cloning one can isolate proteins with specific biological functions. This methodology can be adapted for the identification of novel players in the regulation of cell signaling. Here, we describe an expression cloning strategy to identify suppressors of Ras signaling. This screen is based on the observation that the activation of the small guanosine triphosphate (GTP)-binding protein H-Ras initiates a mitogen-activated protein kinase (MAPK)-dependent signaling pathway that inactivates integrin ligand binding. Our strategy depends on flow cytometry and a monoclonal antibody that recognizes integrin activation states. Flow cytometry enhances the screen's sensitivity thereby allowing us to examine function quantitatively at the level of a single cell millions of times in one screen. The following protocol provides a detailed method for the isolation of proteins that regulate cell signaling.

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.

The affinity of integrin alpha(4)beta(1) governs lymphocyte migration.

The interaction of integrin alpha(4)beta(1) with endothelial VCAM-1 controls the trafficking of lymphocytes from blood into peripheral tissues. Cells actively regulate the affinity of alpha(4)beta(1) for VCAM-1 (activation). To investigate the biological function of alpha(4)beta(1) activation, we isolated Jurkat T cell lines with defective alpha(4)beta(1) activation. Using these cells, we found that alpha(4)beta(1)-stimulated alpha(L)beta(2)-dependent cell migration was dramatically reduced in cells with defects in alpha(4)beta(1) activation. These cells required 20 times more VCAM-1 to promote alpha(L)beta(2)-dependent cell migration. This defect was at the level of alpha(4)beta(1) affinity as an activating alpha(4)beta(1) Ab rescued alpha(4)beta(1)-stimulated alpha(L)beta(2)-dependent migration. In contrast, migration of alpha(4)beta(1) activation-defective cells on VCAM-1 alone was enhanced at higher VCAM-1 densities. Thus, alpha(4)beta(1) activation determines a set point or threshold at which VCAM-1 can regulate alpha(L)beta(2)-dependent as well as alpha(4)beta(1)-dependent cell migration. Changes in this set point may specify preferred anatomical sites of integrin-dependent leukocyte emigration from the bloodstream.

Integrin activation.

Integrins are cell surface adhesion receptors that participate in a variety of important processes throughout the vasculature. Here we summarize some recent findings on the regulation of integrin mediated cellular adhesion. Particular emphasis is placed on the regulation of integrin affinity for ligand (activation), although this is just one mechanism by which regulation of integrin-dependent cell adhesion can occur. Also discussed are recent observations on the structural basis of integrin activation, the role of the cytoplasmic domain in integrin affinity regulation, and potential mechanisms by which activation signals are propagated from integrin cytoplasmic domains to the extracellular ligand binding domain.

Binding of Paxillin to the alpha 9 Integrin Cytoplasmic Domain Inhibits Cell Spreading.

alpha(9)beta(1) integrin is a member of the beta(1) integrin family, plays an important role in extravasation of neutrophils at sites of acute inflammation, and is required for the normal development of the lymphatic system. The alpha(9) and alpha(4) integrin subunits are most closely related and form a subfamily of integrin alpha subunits. Previously, we have reported that the alpha(4) cytoplasmic domain directly and tightly binds paxillin, an intracellular signaling adaptor molecule. This interaction accounts for some of the unusual functional responses to alpha(4) integrin-mediated cell adhesion, including stimulation of cell migration and inhibition of cell spreading and focal adhesion formation. In the current studies, we have examined the interaction between the alpha(9) cytoplasmic domain and paxillin. Here we report that the alpha(9) cytoplasmic domain binds paxillin directly and tightly and that the alpha(9)-paxillin association inhibits cell spreading. We have identified amino acid residues in the alpha(9) cytoplasmic domain, Trp(999) and Trp(1001), that are critical for paxillin binding, and alanine substitution of either Trp(999) or Trp(1001) blocks paxillin binding. Furthermore, these mutations also reverse the effect of the alpha(9) cytoplasmic domain on cell spreading. Thus, the alpha(9) and alpha(4) integrin subunits form a paxillin-binding subfamily of integrin alpha subunits, and direct binding of paxillin to the alpha(9) cytoplasmic domain mediates some of the biological activities of the alpha(9)beta(1) integrin.

NMR analysis of structure and dynamics of the cytosolic tails of integrin alpha IIb beta 3 in aqueous solution.

The structural and dynamic properties of the cytosolic tails of the adhesion receptor integrin alphaIIbbeta3, fused to a coiled-coil construct via (Gly)(3) linkers, were studied in aqueous solution by nuclear magnetic resonance (NMR) spectroscopy. Both tails were largely flexible and unstructured, although, in the beta3 tail, residues Arg(724)-Ala(735) have a propensity to form a helical structure and residues Asn(744)-Tyr(747) (NPLY) have a propensity to adopt reverse-turn conformations. The mutation beta3(Y747A) disrupted this reverse-turn tendency and markedly reduced the affinity of the head domain of the cytoskeletal protein, talin for the beta3 tail. Omission of the (Gly)(3) linker connecting the coiled-coiled helices and the integrin tails lead to helix propagation into the beta3 tail extending up to eight residues. A variety of different tail constructs were made and studied to reveal tail-tail interactions, but surprisingly no significant interactions between both tails could be detected within the context of our constructs. These results provide structural insight into a highly conserved beta tail motif (NPXY/F) required for integrin signaling and highlight a second transiently structured region (residues Arg(724)-Ala(735)), which might also be of functional significance.

Calpain cleavage promotes talin binding to the beta 3 integrin cytoplasmic domain.

Talin links integrin beta cytoplasmic domains to the actin cytoskeleton and is involved in the clustering and activation of these receptors. To understand how talin recognizes integrin beta cytoplasmic domains, we configured surface plasmon resonance methodology to measure the interaction of talin with the beta3 integrin cytoplasmic domain. Here we report that the N-terminal approximately 47-kDa talin head domain (talin-H) has a 6-fold higher binding affinity than intact talin for the beta3 tail. The affinity difference is mainly due to a difference in k(on). Calpain cleavage of intact talin released talin-H and resulted in a 16-fold increase in apparent K(a) and a 100-fold increase in apparent k(on). The increase in talin binding after cleavage was greater than predicted for stoichiometric liberation of free talin-H. This additional increase in binding was due to cooperative binding of talin-H and talin rod domain to the beta3 tail. Talin resembles ERM (ezrin, radixin, moesin) proteins in possessing an N-terminal FERM (band four-point-one, ezrin, radixin, moesin) domain. These data show that the talin FERM domain, like that in the ERM proteins, is masked in the intact molecule. Furthermore, they suggest that talin cleavage by calpain may contribute to the effects of the protease on the clustering and activation of integrins.

A membrane-distal segment of the integrin alpha IIb cytoplasmic domain regulates integrin activation.

Previous evidence suggests that interactions between integrin cytoplasmic domains regulate integrin activation. We have constructed and validated recombinant structural mimics of the heterodimeric alpha(IIb)beta(3) cytoplasmic domain. The mimics elicited polyclonal antibodies that recognize a combinatorial epitope(s) formed in mixtures of the alpha(IIb) and beta(3) cytoplasmic domains but not present in either isolated tail. This epitope(s) is present within intact alpha(IIb)beta(3), indicating that interaction between the tails can occur in the native integrin. Furthermore, the combinatorial epitope(s) is also formed by introducing the activation-blocking beta(3)(Y747A) mutation into the beta(3) tail. A membrane-distal heptapeptide sequence in the alpha(IIb) tail ((997)RPPLEED) is responsible for this effect on beta(3). Membrane-permeant palmitoylated peptides, containing this alpha(IIb) sequence, specifically blocked alpha(IIb)beta(3) activation in platelets. Thus, this region of the alpha(IIb) tail causes the beta(3) tail to resemble that of beta(3)(Y747A) and suppresses activation of the integrin.

Integrin activation controls metastasis in human breast cancer.

Metastasis is the primary cause of death in human breast cancer. Metastasis to bone, lungs, liver, and brain involves dissemination of breast cancer cells via the bloodstream and requires adhesion within the vasculature. Blood cell adhesion within the vasculature depends on integrins, a family of transmembrane adhesion receptors, and is regulated by integrin activation. Here we show that integrin alpha v beta 3 supports breast cancer cell attachment under blood flow conditions in an activation-dependent manner. Integrin alpha v beta 3 was found in two distinct functional states in human breast cancer cells. The activated, but not the nonactivated, state supported tumor cell arrest during blood flow through interaction with platelets. Importantly, activated alpha v beta 3 was expressed by freshly isolated metastatic human breast cancer cells and variants of the MDA-MB 435 human breast cancer cell line, derived from mammary fat pad tumors or distant metastases in severe combined immunodeficient mice. Expression of constitutively activated mutant alpha v beta 3(D723R), but not alpha v beta 3(WT), in MDA-MB 435 cells strongly promoted metastasis in the mouse model. Thus breast cancer cells can exhibit a platelet-interactive and metastatic phenotype that is controlled by the activation of integrin alpha v beta 3. Consequently, alterations within tumors that lead to the aberrant control of integrin activation are expected to adversely affect the course of human breast cancer.

Platelet activation by a relapsing fever spirochaete results in enhanced bacterium-platelet interaction via integrin alphaIIbbeta3 activation.

Borrelia hermsii, a spirochaete responsible for relapsing fever in humans, grows to high density in the bloodstream and causes thrombocytopenia. We show here that B. hermsii binds to human platelets. Extended culture in bacteriological medium resulted in both diminished infectivity in vivo and diminished platelet binding in vitro. Platelet binding was promoted by the platelet integrin alphaIIbbeta3: the bacterium bound to purified integrin alphaIIbbeta3, and bacterial binding to platelets was diminished by alphaIIbbeta3 antagonists or by a genetic defect in this integrin. Integrin alphaIIbbeta3 undergoes a conformational change upon platelet activation, and bacteria bound more efficiently to activated rather than resting platelets. Nevertheless, B. hermsii bound at detectable levels to preparations of resting platelets. The bacterium did not recognize a point mutant of alphaIIbbeta3 that cannot acquire an active conformation. Rather, B. hermsii was capable of triggering platelet and integrin alphaIIbbeta3 activation, as indicated by the expression of the platelet activation marker P-selectin and integrin alphaIIbbeta3 in its active conformation. The degree of platelet activation varied depending upon bacterial strain and growth conditions. Prostacyclin I2, an inhibitor of platelet activation, diminished bacterial attachment, indicating that activation enhanced bacterial binding. Thus, B. hermsii signals the host cell to activate a critical receptor for the bacterium, thereby promoting high-level bacterial attachment.

Distinct domains of CD98hc regulate integrins and amino acid transport.

CD98 is a cell surface heterodimer formed by the covalent linkage of CD98 heavy chain (CD98hc) with several different light chains to form amino acid transporters. CD98hc also binds specifically to the integrin beta(1A) cytoplasmic domain and regulates integrin function. In this study, we examined the relationship between the ability of CD98hc to stimulate amino acid transport and to affect integrin function. By constructing chimeras with CD98hc and a type II transmembrane protein (CD69), we found that the cytoplasmic and transmembrane domains of CD98hc are required for its effects on integrin function, while the extracellular domain is required for stimulation of isoleucine transport. Consequently, the capacity to promote amino acid transport is not required for CD98hc's effect on integrin function. Furthermore, a mutant of CD98hc that lacks its integrin binding site can still promote increased isoleucine transport. Thus, these two functions of CD98hc are separable and require distinct domains of the protein.

Increased filamin binding to beta-integrin cytoplasmic domains inhibits cell migration.

Multicellular animal development depends on integrins. These adhesion receptors link to the actin cytoskeleton, transmitting biochemical signals and force during cell migration and interactions with the extracellular matrix. Many integrin-cytoskeleton connections are formed by filamins and talin. The beta7 integrin tail binds strongly to filamin and supports less migration, fibronectin matrix assembly and focal adhesion formation than either the beta1D tail, which binds strongly to talin, or the beta1A tail, which binds modestly to both filamin and talin. To probe the role of filamin binding, we mapped the filamin-binding site of integrin tails and identified amino acid substitutions that led to selective loss of filamin binding to the beta7 tail and gain of filamin binding to the beta1A tail. These changes affected cell migration and membrane protrusions but not fibronectin matrix assembly or focal adhesion formation. Thus, tight filamin binding restricts integrin-dependent cell migration by inhibiting transient membrane protrusion and cell polarization.

Integrin cytoplasmic domain-binding proteins.

Integrins are a large family of cell surface receptors that mediate cell adhesion and influence migration, signal transduction, and gene expression. The cytoplasmic domains of integrins play a pivotal role in these integrin-mediated cellular functions. Through interaction with the cytoskeleton, signaling molecules, and other cellular proteins, integrin cytoplasmic domains transduce signals from both the outside and inside of the cell and regulate integrin-mediated biological functions. Identification and functional analyses of integrin cytoplasmic domain-binding proteins have been pursued intensively. In recent years, more cellular proteins have been reported to directly interact with integrin cytoplasmic domains and some of these interactions may play important roles in integrin-mediated biological responses. Integrin (&bgr;) chains, for example, interact with actin-binding proteins (e.g. talin and filamin), which form mechanical links to the cytoskeleton. These and other proteins (e.g. FAK, ILK and novel proteins such as TAP20) might also link integrins to signaling mechanisms and, in some cases (e.g. JAB1) mediate integrin-dependent gene regulation.