Substrate-Dependent Activity of ERK and MEK Proteins in Breast Cancer (MCF7), and Kidney Embryonic (Hek-293) Cell Lines, Cultured on Different Substrates.

BACKGROUND: Breast cancer has been one of the most common types of cancer, as the leading cause of women death in world. Breast cancer has known as a heterogenic disease that the clinical path in different patients would be very different. Since the current classification has not covered the diverse clinical course of breast cancer, lots of efforts has done to find new biological markers. Integrins are hetero dimmer proteins of α and ß subunits on cell membrane. After binding to extra cellular matrix (ECM), integrins activate MAPK pathway that regulated different activities like survival, differentiation, migration, immunologic response. The interaction of integrins and ECM have a key role in cancer cell activities like survival and metastasis. OBJECTIVES: In this study the expression of αvß3 integrin, substrate -dependent morphology and ERK and p-ERK activation was compared in MCF7 and Hek-293 cells lines. MATERIALS AND METHODS: The expression of αvß3 integrin was assayed by flow cytometry. These cell lines were cultured on pre-covered plates with fibronectin (FN), fibrinogen (Fg) or collagen (Col) and the expression of ERK and p-ERK proteins was assessed in attached and free cells for each substrate after 1 hour incubation. The morphology of the cells have examined under an inverted phase contrast microscope at 15 min, 1 hour, 3 hours, 5 hours and 1 day of incubatioon. RESULTS: Different substrate induced the expression ERK or p-ERK differently in the two cell lines. In MCF7 cells, substrates induced the expression of ERK in all the attached cells but free cells in BSA, collagen and Fg showed a lower expression of ERK. In comparison with Hek-293 cells althought all the attached cells have expressed ERK peotein but only free cells in collagen plates showed the expression of ERK. None of the cell lines has shown any expression of ERK and p-ERK in attached or free cells except for the Hek-293 free cells in collagen platees that have shown a weak signal for p-ERK. CONCLUSIONS: Overall the breast cancer cell lines MCF7 and Hek-293 cells have differently responded on similar substrates regarding morpology or ERK and MEK expressions.

Ligand recognition specificity of leukocyte integrin αMß2 (Mac-1, CD11b/CD18) and its functional consequences.

The broad recognition specificity exhibited by integrin α(M)ß2 (Mac-1, CD11b/CD18) has allowed this adhesion receptor to play innumerable roles in leukocyte biology, yet we know little about how and why α(M)ß2 binds its multiple ligands. Within α(M)ß2, the α(M)I-domain is responsible for integrin's multiligand binding properties. To identify its recognition motif, we screened peptide libraries spanning sequences of many known protein ligands for α(M)I-domain binding and also selected the α(M)I-domain recognition sequences by phage display. Analyses of >1400 binding and nonbinding peptides derived from peptide libraries showed that a key feature of the α(M)I-domain recognition motif is a small core consisting of basic amino acids flanked by hydrophobic residues. Furthermore, the peptides selected by phage display conformed to a similar pattern. Identification of the recognition motif allowed the construction of an algorithm that reliably predicts the α(M)I-domain binding sites in the α(M)ß2 ligands. The recognition specificity of the α(M)I-domain resembles that of some chaperones, which allows it to bind segments exposed in unfolded proteins. The disclosure of the α(M)ß2 binding preferences allowed the prediction that cationic host defense peptides, which are strikingly enriched in the α(M)I-domain recognition motifs, represent a new class of α(M)ß2 ligands. This prediction has been tested by examining the interaction of α(M)ß2 with the human cathelicidin peptide LL-37. LL-37 induced a potent α(M)ß2-dependent cell migratory response and caused activation of α(M)ß2 on neutrophils. The newly revealed recognition specificity of α(M)ß2 toward unfolded protein segments and cationic proteins and peptides suggests that α(M)ß2 may serve as a previously proposed "alarmin" receptor with important roles in innate host defense.

Peptides derived from central turn motifs within integrin αIIb and αV cytoplasmic tails inhibit integrin activation.

We previously found that peptides derived from the full length of integrin αIIb and αV cytoplasmic tails inhibited their parent integrin activation, respectively. Here we showed that the cell-permeable peptides corresponding to the conserved central turn motif within αIIb and αV cytoplasmic tails, myr-KRNRPPLEED (αIIb peptide) and myr-KRVRPPQEEQ (αV peptide), similarly inhibited both αIIb and αV integrin activation. Pre-treatment with αIIb or αV peptides inhibited Mn(2+)-activated αIIbß3 binding to soluble fibrinogen as well as the binding of αIIbß3-expressing Chinese Hamster Ovary cells to immobilized fibrinogen. Our turn peptides also inhibited adhesion of two breast cancer cell lines (MDA-MB-435 and MCF7) to αV ligand vitronectin. These results suggest that αIIb and αV peptides share a same mechanism in regulating integrin function. Using αIIb peptide as a model, we found that replacement of RPP with AAA significantly attenuated the inhibitory activity of αIIb peptide. Furthermore, we found that αIIb peptide specifically bound to ß-tubulin in cells. Our work suggests that the central motif of α tails is an anchoring point for cytoskeletons during integrin activation and integrin-mediated cell adhesion, and its function depends on the turn structure at RPP. However, post-treatment of peptides derived from the full-length tail or from the turn motif did not reverse αIIb and αV integrin activation.

Skelemin in integrin α(IIb)ß(3) mediated cell spreading.

Skelemin, a myosin-associated protein in skeletal muscle, has been demonstrated to interact with integrin α(IIb)ß(3) in nonmuscle cells during initial stages of cell spreading. The significance of this interaction and the role of skelemin in integrin signaling and cytoskeletal reorganization were investigated in this study. We established a series of Chinese hamster ovary cell lines expressing wild-type or mutant α(IIb)ß(3) receptors in which skelemin binding residues at the membrane proximal region of integrin tails were mutated to alanine. Most cells displayed unimpaired adhesive capacity and spreading on immobilized fibrinogen at the early stages of cell spreading. In addition, they formed normal focal adhesions and stress fibers with no indication of impaired cell spreading. R995A/R997A/L1000A, H722A, and K716A exhibited the greatest cell spreading, which was associated with enhanced p-Src activation but was independent of FAK activation. Transfection of the cells with GFP-skelemin, containing only the C2 integrin binding domain, caused wild-type cells to round up, but had no effect on R995A/R997A/L1000A, H722A, and K716A cell spreading. Furthermore, the protrusions of the leading edge of K716A cells showed strong colocalization of talin with α(IIb)ß(3) which was associated with a loss in skelemin binding. Thus, we propose that during early stages of cell spreading, skelemin exerts contractile force on cell spreading and modulates the attachment of cytoskeletal proteins and Src to integrin clusters.

Differences in integrin expression and signaling within human breast cancer cells.

BACKGROUND: Integrins are used as prognostic indicators in breast cancer. Following engagement with extracellular matrix proteins, their signaling influences numerous cellular processes including migration, proliferation, and death. Integrin signaling varies between cell types through differential expression of integrin subunits, and changes within a given cell upon exposure to a cell agonist or through changes in its surroundings. These variations in signaling can profoundly affect the phenotypic, tumorogenecity and metastatic properties of cancer cells. In the present study, we investigated if there were differences in the expression of integrins, integrin structures, and integrin co-receptors within three breast cancer cells and if these differences effected integrin signaling. METHODS: Expression of integrins, urokinase receptor and vascular endothelial cell growth factor receptor (VEGFR) in metastatic MDA-MB-435 and MDA-MB-231, non-metastatic MCF7 and non-breast cancer Hek-293 cells was measured by flow cytometry. Cell adhesion was assessed using collagen, fibrinogen, fibronectin and vitronectin coated plates. Changes in kinase levels following PMA stimulation, and cell adhesion-induced activation of kinases were determined by western blot analysis. Distribution of actin stress fibers and focal adhesions was assessed by immunocytochemistry. RESULTS: All cells expressed αv integrins, while high ß5 and αvß5 expression was restricted to the cancer cells and high ß3 and αvß3 expression was restricted to MDA-MB-435 cells. The two metastatic cells were the least adhesive, but all cells adhered well to most proteins in the absence of PMA. All proliferating cells expressed activated pSrc, but only proliferating metastatic cells expressed high pMEK levels. PMA treatment resulted in time-dependent changes in activated kinase levels, and only MDA-MB-231 cells constitutively expressed high levels of activated pMEK. MDA-MB-435 cells formed more stress fibers and focal adhesions and only exhibited adhesion-induced activation of pMEK and pFAK. All cells expressed the urokinase receptor, but MCF7 cells had markedly higher VEGFR expression. Adhesion induced differential expression of pFAK, pMEK and pERK. CONCLUSIONS: This study demonstrates that breast cancers vary in their expression of integrins, their capacity to form focal adhesion and to signal through integrins. These differences likely contribute to phenotypic variations between cancer lines and account for some of the heterogeneity of breast cancer.

Adhesion-induced unclasping of cytoplasmic tails of integrin alpha(IIb)beta3.

Integrin alpha(IIb)beta(3) plays a pivotal role in hemostasis and thrombosis by mediating adhesive interactions of platelets. Binding of alpha(IIb)beta(3) to its physiological ligands, immobilized fibrinogen and fibrin, induces outside-in signaling in platelets, leading to their adhesion and spreading even without prior stimulation by agonists. Implicit in these phenomena is a requirement for the linkage between integrins' cytoplasmic tails and intracellular proteins. However, the nature of the initiating signal has not been established. In this study, we examined whether binding of alpha(IIb)beta(3) to immobilized fibrin(ogen), per se, triggers interaction of the integrin with cytoplasmic proteins. Using the integrin-binding skelemin fragment as a marker of exposure of residues involved in the clasp between alpha(IIb) and beta(3) cytoplasmic tails, we showed that its binding site in the membrane-proximal beta(3) 715-730 segment is cryptic and becomes exposed as a result of binding of isolated alpha(IIb)beta(3) to immobilized ligands. Furthermore, the skelemin-like protein present in platelets and CHO cells does not associate with alpha(IIb)beta(3) in resting platelets or suspended alpha(IIb)beta(3)-expressing CHO cells but is recruited to integrin during cell adhesion. In addition, not only beta(3) but also the membrane-proximal 989-1000 segment of the alpha(IIb) cytoplasmic tail binds the skelemin fragment. Finally, the same residues, alpha(IIb) Val(990), alpha(IIb) Arg(995), and beta(3) His(722), involved in the formation of the clasp between the tails are also required for skelemin binding. These studies suggest that ligation of alpha(IIb)beta(3) by immobilized ligands during platelet adhesion induces a transmembrane conformation change in the integrin, resulting in unclasping of the complex between the membrane-proximal parts of cytoplasmic tails, thereby unmasking residues involved in binding the skelemin-like protein. Thus, the junction between alpha(IIb) and beta(3) cytoplasmic tails may contain the critical structural information for the initiation of outside-in signaling.

Allosteric regulation of alpha(IIb)beta(3) by beta(3) 95-105.

The linear recognition sequences of an anti-beta(3) antibody that blocked platelet aggregation were identified using beta(3) tryptic peptides. Two of these recognition sequence-containing peptides were mapped to beta(3) 92-105, and antibodies affinity purified using these peptides blocked platelet aggregation. Examining the structure of alpha(IIb)beta(3) identified beta(3) 95-105 as the solvent accessible sequence within beta(3) 92-105. A peptide corresponding to beta(3) 95-105 was synthesized and used to affinity purify the beta(3) antibody. Anti-beta(3) 95-105 completely blocked platelet aggregation and agonist-induced fibrinogen binding to platelets, but had no effect on cyclic-RGD binding. Binding of anti-beta(3) 95-105 to alpha(IIb)beta(3) also did not alter the structure of the alpha(IIb) cap subdomain, as measured by anti-alpha(IIb) 201-217 binding. beta(3) 95-105 and peptides spanning two adjacent sequences in the structure of beta(3) did not bind fibrinogen and were ineffectual in blocking agonist-induced platelet aggregation. Structure analysis revealed that beta(3) 95-105 is adjacent to one of the two hinges in beta(3) that allows for the outward swing of the hybrid and PSI domains which is central to the conversion of alpha(IIb)beta(3) from a low into a high affinity state. Thus, the binding of an antibody to beta(3) 95-105 could serve as a fulcrum for allosteric regulation of alpha(IIb)beta(3) by regulating the movement of the hybrid-PSI domain.

Identification of residues of functional importance within the central turn motifs present in the cytoplasmic tails of integrin alphaIIb and alphaV subunits.

INTRODUCTION: Previous studies demonstrated that cell-permeable alphaIIb cytoplasmic peptides can modulate the activation of alphaIIbbeta3. An integrin activation motif was mapped to its membrane proximal region and a double proline mutant peptide and receptor indicated that its central turn motif had inhibitory capacity. However, the residues critical for inhibition of alphaIIbbeta3 activation were not identified. Using central turn peptides derived from alphaIIb and alphaV, residues critical for suppression of integrin activation were identified and the importance of these residues in protein-protein interactions was assessed. MATERIALS AND METHODS: Cell-permeable peptides were used to determine the capacity of the central turn peptides to suppress alphaIIbbeta3 and alphaVbeta3 activation. Far Western analysis was used to characterize the capacity of the peptides to interact with CIB1 and surface plasmon resonance was used to characterize the binding of an antibody to the cytoplasmic tails of alphaIIb and alphaV. RESULTS AND CONCLUSIONS: The central turn peptide from alphaV, alphaV(993-1001), has full inhibitory capacity while that derived from alphaIIb requires additional residues located adjacent to alphaIIb(995-1003). Within these two sequences there is a switch in the position of an asparaginine and leucine residue for a valine and glutamine (alphaIIb, RNRPPLEED; alphaV, RVRPPQEEQ). This switch had a dramatic effect on their inhibitory capacity and on protein-protein interactions. The two arginine and glutamic residues, juxtapositioned at identical locations in both subunits, appeared to be important in specifying the orientation by which proteins can dock to this region in alphaIIb and alphaV.

Discrete functional motifs reside within the cytoplasmic tail of alphaV integrin subunit.

Previous studies have demonstrated that cell-permeable cytoplasmic tail (CT) alpha(II)beta peptides can modulate the activation of alpha(IIb)b(3). As alpha(V) CT contains an alpha(II)beta homologous region, a series of cell-permeable alpha(V) and alpha(IIb) peptides were generated to determine if alpha(V) CT can modulate the activation of beta(3) integrins in comparison to alpha(IIb), and to identify the minimal bioactive sequences in alpha(V) CT. Using NMR structures and molecular models as guides, the initial peptides for study encompassed the alpha(II)beta homologous sequences of alpha(V) CT (alpha(V)(987-1006); V-1), its amino-terminus (alpha(V)(987-993); V-2), a turn motif (alpha(V)(993-1001); V-3), the carboxyl-terminus (alpha(V)(999-1006); V-4), and corresponding homologous alpha(IIb) peptides. Treatment of platelets and alpha(V)beta(3)-expressing cells with the peptides revealed that IIb-1 inhibited alpha(IIb)beta(3) activation and V-1 inhibited alpha(V)beta(3) activation, but not vice versa. The inhibitory capacity of these peptides was mapped to the central turn-motif region which was encompassed by V-3, but only partially by IIb-3. V-2 and IIb-2 activated both beta(3) integrins, while V-4 and IIb-4 were inactive. The use of truncation and mutant peptides confirmed the importance of the turn motif for inhibitory activity and identified the side-chain of alpha(V)(Q1001) as a critical inhibitory residue. The difference in the integrin inhibitory capacity of alpha(V) and alpha(IIb) peptides and their capacity to influence the assembly of kinases with integrin CTs, reveals a possible divergence in the regulatory control of the two beta(3) integrins.

N-acyl-3,5-bis(arylidene)-4-piperidones and related compounds which stimulate fyn kinase.

This study is part of a long term project designed to explore the hypothesis that stimulation of cancer cells followed by treatment with one or more cytotoxic agents may create greater damage to tumours than to the corresponding normal tissues. The aim of the present investigation was to discover various compounds which stimulate a protein tyrosine kinase, namely fyn kinase. The N-acyl-3,5-bis(arylidene)-4-piperidones and related analogues activated this enzyme using concentrations of 25 microM while representative molecules achieved this result at 0.1 microM. Molecular modelling suggested that the compounds interact transiently with the ATP binding site of fyn kinase thereby enhancing the catalytic phosphorylation of proteins. In the future, candidate antineoplastic agents will be designed which incorporate the structural features of these enzyme stimulators with the goal of their being formed in vitro and in vivo prior to the release of cytotoxins.

ARL1 plays a role in the binding of the GRIP domain of a peripheral matrix protein to the Golgi apparatus in plant cells.

ARF GTPases play a central role in regulating membrane dynamics and protein transport in eukaryotic cells. ARF-like (ARL) proteins are close relatives of the ARF regulators of vesicular transport, but their function in plant cells is poorly characterized. Here, by means of live cell imaging and site-directed mutagenesis, we have investigated the cellular function of the plant GTPase ARL1. We provide direct evidence for a role of this ARL family member in the association of a plant golgin with the plant Golgi apparatus. Our data reveal the existence of key residues within the conserved GRIP-domain of the golgin and within the GTPase ARL1 that are central to ARL1-GRIP interaction. Mutations of these residues abolish the interaction of GRIP with the GTP-bound ARL1 and induce a redistribution of GRIP into the cytosol. This indicates that the localization of GRIP to the Golgi apparatus is strongly influenced by the interaction of GRIP with Golgi-localized ARL1. Our results assign a cellular role to a member of the Arabidopsis ARL family in the plant secretory pathway and propose mechanisms for localization of peripheral golgins to the plant Golgi apparatus.

Membrane-mediated structural transitions at the cytoplasmic face during integrin activation.

Cytoplasmic face-mediated integrin inside-out activation remains a paradigm in transmembrane signal transduction. Emerging evidence suggests that this process involves dissociation of the complex between the integrin cytoplasmic tails; however, a dynamic image of how it occurs on the membrane surface remains elusive. We show here that, whereas membrane-proximal helices of integrin alpha/beta cytoplasmic tails associate in cytoplasm-like aqueous medium, they become partially embedded into membrane-mimetic micelles when unclasped. Membrane embedding induces substantial structural changes of the cytoplasmic tails as compared to their aqueous conformations and suggests there may be an upward movement of the membrane-proximal helices into the membrane during their separation. We further demonstrate that the beta3 tail exhibits additional membrane binding site at its C terminus containing the NPLY motif. Talin, a key intracellular integrin activator, recognizes this site as well as the membrane-proximal helix, thereby promoting cytoplasmic tail separation along the membrane surface. These data provide a structural basis of membrane-mediated changes at the cytoplasmic face in regulating integrin activation and signaling.

Sequence gamma 377-395(P2), but not gamma 190-202(P1), is the binding site for the alpha MI-domain of integrin alpha M beta 2 in the gamma C-domain of fibrinogen.

The interaction between the leukocyte integrin alpha(M)beta(2) (CD11b/CD18, Mac-1, CR3) and fibrinogen mediates the recruitment of phagocytes during the inflammatory response. Previous studies demonstrated that peptides P2 and P1, duplicating gamma 377-395 and gamma 190-202 sequences in the gamma C domain of fibrinogen, respectively, blocked the fibrinogen-binding function of alpha(M)beta(2), implicating these sequences as possible binding sites for alpha(M)beta(2). To determine the role of these sequences in integrin binding, recombinant wild-type and mutant gamma C domains were prepared, and their interactions with the alpha(M)I-domain, a ligand recognition domain within alpha(M)beta(2), were tested. Deletion of gamma 383-411 (P2-C) and gamma 377-411 produced gamma C mutants which were defective in binding to the alpha(M)I-domain. In contrast, alanine mutations of several residues in P1 did not affect alpha(M)I-domain binding, and simultaneous mutations in P1 and deletion of P2 did not decrease the binding function of gamma C further. Verifying the significance of P2, inserting P2-C and the entire P2 into the homologous position of the beta C-domain of fibrinogen imparted the higher alpha(M)I-domain binding ability to the chimeric proteins. To further define the molecular requirements for the P2-C activity, synthetic peptides derived from P2-C and a peptide array covering P2-C have been analyzed, and a minimal recognition motif was localized to gamma(390)NRLTIG(395). Confirming a critical role of this sequence, the cyclic peptide NRLTIG retained full activity inherent to P2-C, with Arg and Leu being important residues. Thus, these data demonstrate the essential role of the P2, but not P1, sequence for binding of gamma C by the alpha(M)I-domain and suggest that the adhesive function of P2 depends on the minimal recognition motif NRLTIG.

Identification of functional segments within the beta2I-domain of integrin alphaMbeta2.

The alpha(M)beta(2) integrin plays an important role in leukocyte biology through its interactions with a diverse set of ligands. Efficient ligand binding requires the involvement of both the alpha(M) and beta(2) subunits. Past ligand binding studies have focused mainly on the alpha(M) subunit, with the beta(2) subunit being largely unexplored. Therefore, in this study we conducted homolog-scanning mutagenesis on the I-domain (residues 125-385) within the beta(2) subunit. We identified four noncontiguous sequences (Arg(144)-Lys(148), Gln(199)-Ala(203), Leu(225)-Leu(230), and Gly(305)-His(309)) that are critical for fibrinogen and C3bi binding to alpha(M)beta(2). Molecular modeling revealed that these four sequences reside within a narrow region on the surface of the beta(2)I-domain, in close proximity to three potential cation-binding sites. Among these sequences, Gln(199)-Ala(203), Leu(225)-Leu(230), and Gly(305)-His(309) are important for the binding of both ligands, whereas Arg(144)-Lys(148) is more critical for fibrinogen than for C3bi binding. These sequences within the beta(2)I-domain are directly involved in ligand binding, since 1) switching these segments to their corresponding beta(1) sequences destroyed ligand binding; 2) loss of function was not due to a nonspecific gross conformational change, since the defective alpha(M)beta(2) mutants reacted well with a panel of conformation-dependent mAbs; 3) mutation of these functional sequences did not effect Ca(2+) binding; and 4) synthetic peptides corresponding to sequences Gln(199)-Ala(203) and Gly(305)-His(309) blocked ligand binding to alpha(M)beta(2), and the peptides interacted directly with fibrinogen and C3bi. Given the similarity among all integrin beta subunits, our results may help us to understand the underlying mechanism of integrin-ligand interactions in general.

Myelin proteolipid protein forms a complex with integrins and may participate in integrin receptor signaling in oligodendrocytes.

Myelination of axons in the CNS by oligodendrocytes is a process critical to rapid and efficient impulse conduction. A new role for the myelin proteolipid protein (PLP), the most abundant protein of CNS myelin, has been identified, in studies showing PLP interaction with signaling proteins in oligodendrocytes. In particular, these studies suggest that the PLP protein may be involved in signaling through integrins in oligodendrocytes. Stimulation of muscarinic acetylcholine receptors on oligodendrocytes induced formation of a tripartite complex containing PLP, calreticulin, and alpha(v)-integrin. PLP interacted directly with the cytoplasmic domain of the alpha(v)-integrin. Complex formation was mediated by phospholipase C and Ca2+ binding to the high affinity binding site on calreticulin. This complex appears important for binding of fibronectin to oligodendrocytes. These data establish a novel function for PLP as a part of the integrin signaling complex in oligodendrocytes and suggest that neurotransmitter-mediated integrin receptor signaling may be involved in myelinogenesis.

Identification and characterization of two cation binding sites in the integrin beta 3 subunit.

The midsegment of the beta(3) subunit has been implicated in the ligand and cation binding functions of the beta(3) integrins. This region may contain a metal ion-dependent adhesion site (MIDAS) and fold into an I domain-like structure. Two recombinant fragments, beta(3)-(95-373) and beta(3)-(95-301), were expressed and found to bind fibrinogen. Whereas 0.1 mm Ca(2+) supported ligand binding to both recombinant fragments, 1.0 mm Ca(2+) suppressed binding to the longer but not the shorter fragment. These properties suggest that beta(3)-(95-373) contains both the ligand-competent (LC) and inhibitory (I) cation binding sites, and beta(3)-(95-301) lacks the I site. In equilibrium dialysis experiments, beta(3)-(95-373) contained two divalent cation binding sites, one reactive with either Mg(2+) or Ca(2+) and one Ca(2+)-specific, whereas beta(3)-(95-301) lacked the Ca(2+)-specific site. Mutant forms of beta(3)-(95-373) suggested that the LC site is a MIDAS motif involving Asp(119), Ser(121), Ser(123), Asp(217), and/or Glu(220) as coordination sites, and the I site was dependent upon residues within beta(3)-(301-323). In a molecular model of beta(3)-(95-373), a second Ca(2+) could be docked onto a flexible loop in close proximity to the MIDAS. These results indicate that the ligand competent and Ca(2+)-specific inhibitory cation binding sites are distinct and reside in beta(3)-(95-373).