A Glycan Wedge Between βTD and βI Domains Activates Integrin αIIbβ3,

Abstract Abstract 3259 Integrin αIIbβ3 undergoes allosteric conformational changes in its extracellular domains, resulting in integrin activation that allows high affinity binding with soluble ligands. The crystal structure of the integrin β subunit revealed an interaction of the β-tail domain (βTD) with the βI domain containing ligand-binding sites, suggesting that βTD may be involved in allosteric mechanism for integrin activation. However, previous studies have shown conflicting results on the functional role of βTD in integrin activation. In this study, we conducted site-directed mutagenesis in the βTD domain and tested ligand binding to αIIbβ3 mutants. We produced αIIbβ3 mutants in which the β3TD loop residues (DSSG) were substituted with the corresponding β1 (NGNN) or β2TD residues (DGMD). The αIIbβ3 mutants were expressed on the surface of CHO cells by cotransfection of mutant β3 and wild-type αIIb cDNAs, and were tested for binding of PAC1, a ligand-mimetic anti-αIIbβ3 antibody. The NGNN, but not DGMD mutant bound significant PAC1 binding without any stimulation, indicating a constitutively active state. To identify the residue(s) responsible for αIIbβ3 activation in the βTD, we produced αIIbβ3 mutants in which the individual residues in the β3TD loop were substituted with the corresponding β1TD residues. Among them, only G675N bound significant PAC1 binding without any stimulation. Since G675N mutation creates a sequence known to be a consensus sequence for glycosylation (Asn-X-Ser/Thr), it is possible that the insertion of glycans into the βTD loop induces conformational changes in αIIbβ3 which allow ligand binding. To test this hypothesis, we added substitution of S677 with Thr, Ala or Asp to the G675N mutation. The resultant G675N/S677T double mutant, in which the N-glycosylation site was preserved, was constitutively active. In contrast, G675N/S677A and G675N/S677D, in which the N-glycosylation site was disrupted, were in an inactive state. These results suggest that an artificial glycan wedge between βTD and βI domains activates αIIbβ3. However, our study does not provide evidence that the βTD domain constrains wild type αIIbβ3 inactive although the separation of βTD and βI domains may be able to activate integrins. Disclosures: No relevant conflicts of interest to declare.

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Integrin β3 Membrane-Proximal and -Distal Residues Cooperatively Regulate Talin-Mediated αIIbβ3 Activation.

Blood ◽

10.1182/blood.v112.11.1848.1848 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1848-1848

Author(s):

Jun Yamanouchi ◽

Takaaki Hato ◽

Hiroshi Fujiwara ◽

Yoshihiro Yakushijin ◽

Masaki Yasukawa

Keyword(s):

Ligand Binding ◽

Double Mutant ◽

Distal Region ◽

Proximal Region ◽

Structural Rearrangements ◽

Wild Type ◽

Integrin Activation ◽

Integrin Αiibβ3 ◽

Membrane Proximal Region ◽

Constitutively Active

Abstract Integrin αIIbβ3 exists in a low affinity state in resting platelets and requires activation for high affinity binding with soluble ligands. Activation of αIIbβ3 is tightly linked to structural rearrangements of the αIIbβ3 molecule that is initiated from the cytoplasmic tails of the αIIb and β3 subunits. The β3 membrane-distal region has been shown to interact with many signaling and cytoskeletal molecules, and considered as a trigger point of integrin activation. The interaction of the β3 tail with a cytoplasmic protein, talin, largely contributes to integrin activation. In view of the link between integrin activation and allosteric structural rearrangements of integrins, one would expect that structural changes in the β3 membrane-distal region containing binding sites for intracellular proteins would be relayed to the membrane-proximal region, leading to αIIbβ3 activation. However, there has been no evidence that structural rearrangement of the β3 membrane-distal region is directly linked to integrin activation. No activating mutation has so far been reported in the β3 membrane-distal region despite numerous reports of loss-of-function mutants in this region. In this context, a previously reported αIIbβ3 mutant in which the β3 tail was replaced by the β1 tail was noteworthy. This chimeric integrin, αIIbβ3/β1, was constitutively active. Because the β1 and β3 subunits have relatively high sequence homology in their membrane-proximal regions, we reasoned that the residues differing between the β1 and β3 membrane-distal regions may be responsible for αIIbβ3 activation. To identify such residues, we produced 13 αIIbβ3 mutants in which the individual or group residues in the β3 tail were substituted with the corresponding β1 tail residues. The αIIbβ3 mutants were expressed on the surface of CHO cells by cotransfection of mutant β3 and wild-type αIIb cDNAs, and were tested for binding of fibrinogen and PAC1, a ligand-mimetic anti-αIIbβ3 antibody. Among them, only β3I719M and E749S mutants bound significant PAC1 and fibrinogen binding without any stimulation and the RGDS peptide abolished binding of these ligands, indicating a constitutively active state. The similar effect was observed with I719A and E749A mutants. Moreover, the I719M/E749S double mutant showed more PAC1 binding than the single mutants, reaching the same ligand binding activity as αIIbβ3/β1. These β3 mutations also induced αVβ3 activation. Conversely, substitution of M719 or S749 in the β1 tail with the corresponding β3 tail residue (M719I or S749E) inhibited αIIbβ3/β1 activation, and the M719I/S749E double mutant inhibited ligand binding to a level comparable with that of the wild-type αIIbβ3. Knock down of talin by short hairpin RNA inhibited the I719M- and E749S-induced αIIbβ3 activation, indicating talin-mediated activation of mutant integrins. Since I719 is located at the β3 membrane-proximal region, it is likely that the I719 mutation disrupts the well-known membrane-proximal clasp to maintain integrins at a low affinity state. On the other hand, E749 is located at the β3 membrane-distal region. This result provides experimental evidence that structural perturbation of the β3 membrane-distal region is linked to integrin activation. Moreover, our result showed that the mutational effects of the membrane-proximal I719 and the membrane-distal E749 residues were additive and talin-dependent, suggesting that the β3 membrane-proximal and –distal regions cooperatively regulate talin-mediated αIIbβ3 activation. This finding is consistent with a recent model of talin-induced αIIbβ3 activation in which talin cooperatively interacts with the β3 membrane-proximal and distal regions.

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The β-tail domain (βTD) regulates physiologic ligand binding to integrin CD11b/CD18

Blood ◽

10.1182/blood-2005-11-056689 ◽

2006 ◽

Vol 109 (8) ◽

pp. 3513-3520 ◽

Cited By ~ 29

Author(s):

Vineet Gupta ◽

Annette Gylling ◽

José Luis Alonso ◽

Takashi Sugimori ◽

Petre Ianakiev ◽

...

Keyword(s):

Electron Microscopy ◽

Ligand Binding ◽

Conformational Changes ◽

Divalent Cations ◽

Contact Region ◽

Allosteric Modulator ◽

Tail Domain ◽

Wild Type ◽

High Affinity

Abstract Crystallographic and electron microscopy studies revealed genuflexed (bent) integrins in both unliganded (inactive) and physiologic ligandbound (active) states, suggesting that local conformational changes are sufficient for activation. Herein we have explored the role of local changes in the contact region between the membrane-proximal β-tail domain (βTD) and the ligand-binding βA domain of the bent conformation in regulating interaction of integrin CD11b/CD18 (αMβ2) with its physiologic ligand iC3b. We replaced the βTD CD loop residues D658GMD of the CD18 (β2) subunit with the equivalent D672SSG of the β3 subunit, with AGAA or with NGTD, expressed the respective heterodimeric receptors either transiently in epithelial HEK293T cells or stably in leukocytes (K562), and measured their ability to bind iC3b and to conformation-sensitive mAbs. In the presence of the physiologic divalent cations Ca2+ plus Mg2+ (at 1 mM each), the modified integrins showed increased (in HEK293) or constitutive (in K562) binding to iC3b compared with wild-type receptors. K562 expressing the βTD-modified integrins bound in Ca2+Mg2+ to the βA-directed high-affinity reporter mAb 24 but not to mAb KIM127, a reporter of the genu-straightened state. These data identify a role for the membrane proximal βTD as an allosteric modulator of integrin activation.

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Identification of Critical Residues for Affinity Regulation of β3 Integrins in the β6-α7 Loop of the I-Like Domain.

Blood ◽

10.1182/blood.v108.11.1526.1526 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1526-1526

Author(s):

Jun Yamanouchi ◽

Takaaki Hato ◽

Yoshihiro Yakushijin ◽

Ikuya Sakai ◽

Masaki Yasukawa

Keyword(s):

Ligand Binding ◽

Cho Cells ◽

Disulfide Bonds ◽

Integrin Αvβ3 ◽

Structural Rearrangement ◽

Surface Expression ◽

Wild Type ◽

Integrin Activation ◽

Alanine Scanning Mutagenesis ◽

Constitutively Active

Abstract Integrin αIIbβ3 exists in a low-affinity state and requires activation for high-affinity binding with soluble ligands. αIIbβ3 activation is linked to rearrangements of the β3 I-like domain structure and the largest movement in the β3 I-like domain following ligand binding occurs in the β6-α7 loop. Although this loop does not comprise a ligand binding site, a recent mutational study has shown that introduction of disulfide bonds into the β6/α7 region to lock the β3 I-like domain in the open or closed conformation renders αIIbβ3 constitutively active or inactive, suggesting the regulatory role of this region in integrin activation. However, it remains to be determined which residues of the β6-α7 loop in the β I-like domain are critical for integrin activation. We therefore conducted alanine-scanning mutagenesis of the β6-α7 loop residues in the β3 I-like domain and tested for ligand binding to mutant β3 integrins. The β6-α7 loop is composed of residues from S334 to N339, and the region between L333 and V340 was targeted for mutagenesis. The mutant β3 cDNA was transfected into CHO cells together with wild-type αIIb cDNA. The expression of αIIbβ3 mutants on the cell surface was 56–116% that of the wild-type αIIbβ3. Then binding of an activation-dependent antibody. PAC1, to the αIIbβ3 mutants was examined in the presence or absence of the αIIbβ3-activating antibody PT25-2. As expected, wild-type αIIbβ3 showed PAC1 binding only after activation of αIIbβ3 by PT25-2. The L333A, S334A, M335A and V340A mutations had no effect on PAC1 binding. In contrast, the S337A and N339A mutations induced significant PAC1 binding in the absence of PT25-2, indicating a constitutively active state. Although the D336A and S338A mutations retained αIIbβ3 in an inactive state in the absence of PT25-2, they induced 3- and 4.5-fold as much PAC1 binding as wild-type αIIbβ3 in response to PT25-2, respectively. The S337A and N339A mutations, which rendered αIIbβ3 constitutively active, induced further PAC1 binding in the presence of PT25-2 (2.7~5.1-fold as much PAC1 binding as wild-type αIIbβ3). When soluble fibrinogen was used instead of PAC1, similar results were obtained. None of the mutations tested had any effect on PT25-2 binding. We next quantified adhesion of CHO cells stably expressing mutant or wild-type αIIbβ3 to immobilized fibrinogen. The S337A and N339A mutations enhanced cell adhesion to fibrinogen and the extent of adhesion of the mutants was comparable to that of wild-type αIIbβ3 activated by Mn2+. To determine whether the mutations of the β3 subunit responsible for αIIbβ3 activation also induce constitutive activation of αVβ3, CHO cells expressing αVβ3 mutants were tested for soluble fibrinogen binding. The surface expression of mutant αVβ3 was comparable to that of wild-type αVβ3. The αVβ3S337A and N339A mutants bound significantly more fibrinogen than wild-type αVβ3 without any stimulation, indicating that the S337A and N339A mutations also rendered another β3 integrin, αVβ3, constitutively active. These results suggest that the S337 and N339 residues in the β3 I-like domain are essential for constraining β3 integrins in a default low-affinity state and that structural rearrangement in the 336DSSN sequence of the β6-α7 loop alters the affinity state of αIIbβ3. Since the DSSN sequence is highly conserved among integrin β subunits, this motif may be a common regulatory component of integrin activation.

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H-Ras Is Involved in the Inside-out Signaling Pathway of Interleukin-3–Induced Integrin Activation

Blood ◽

10.1182/blood.v93.5.1540 ◽

1999 ◽

Vol 93 (5) ◽

pp. 1540-1548 ◽

Cited By ~ 29

Author(s):

Hirohiko Shibayama ◽

Naoyuki Anzai ◽

Stephen E. Braun ◽

Seiji Fukuda ◽

Charlie Mantel ◽

...

Keyword(s):

Signaling Pathway ◽

Dominant Negative ◽

The Other ◽

Wild Type ◽

Integrin Activation ◽

Mapk Kinase ◽

Interleukin 3 ◽

Other Hand ◽

Inside Out ◽

Constitutively Active

Abstract The proto-oncogene product, p21ras, has been implicated in the cellular mechanism of adhesion, although its precise role has been controversial. Numerous cytokines and growth-factors activate Ras, which is an important component of their growth-promoting signaling pathways. On the other hand, the role of Ras in cytokine-induced adhesion has not been elucidated. We therefore investigated the function of H-Ras in the inside-out signaling pathway of interleukin-3 (IL-3)–induced integrin activation in the murine Baf3 cell line after transfection of cells with either constitutively active, dominant-negative, or wild-type H-Ras cDNAs. Adhesion of Baf3 cells to fibronectin was induced by IL-3 in a dose-dependent manner via very late antigen-4 (VLA-4; 4β1 integrins) and VLA-5 (5β1 integrins) activation. On the other hand, IL-4 did not induce the adhesion of Baf3 cells to fibronectin, although IL-4 did stimulate the cell proliferation of Baf3 cells. Constitutively active H-Ras–transfected Baf3 cells adhered to fibronectin without IL-3 stimulation through VLA-4 and VLA-5, whereas dominant-negative H-Ras–transfected Baf3 cells showed significantly less adhesion induced by IL-3 compared with wild-type and constitutively active H-Ras–transfected Baf3 cells. Anti-β1 integrin antibody (clone; 9EG7), which is known to change integrin conformation and activate integrins, induced the adhesion of dominant-negative H-Ras–transfected Baf3 cells as much as the other types of H-Ras–transfected Baf3 cells. 8-Br-cAMP, Dibutyryl-cAMP, Ras-Raf-1 pathway inhibitors, and PD98059, a MAPK kinase inhibitor, suppressed proliferation and phosphorylation of MAPK detected by Western blotting with anti–phospho-MAPK antibody, but not adhesion of any type of H-Ras–transfected Baf3 cells, whereas U-73122, a phospholipase C (PLC) inhibitor, suppressed adhesion of these cells completely. These data indicate that H-Ras and PLC, but not Raf-1, MAPK kinase, or the MAPK pathway, are involved in the inside-out signaling pathway of IL-3–induced VLA-4 and VLA-5 activation in Baf3 cells.

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The CSF3R T618I Mutation Found In Chronic Neutrophilic Leukemia Removes An O-Linked Glycosylation Site and Increases Receptor Dimerization

Blood ◽

10.1182/blood.v122.21.270.270 ◽

2013 ◽

Vol 122 (21) ◽

pp. 270-270

Author(s):

Julia E. Maxson ◽

Jason Gotlib ◽

Daniel A. Pollyea ◽

Angela G. Fleischman ◽

Anupriya Agarwal ◽

...

Keyword(s):

Clinical Trials ◽

Board Of Directors ◽

Research Funding ◽

Conflicts Of Interest ◽

Glycosylation Site ◽

Wild Type ◽

Advisory Committees ◽

Advisory Boards ◽

Financial Interest ◽

Truncation Mutant

Abstract Background We have recently identified mutations in Colony Stimulating Factor 3 Receptor (CSF3R, aka GCSFR) in ∼60% of chronic neutrophilic leukemia (CNL) and atypical chronic myeloid leukemia (aCML) patients (Maxson et al, NEJM 2013). These mutations fall into two categories: membrane proximal point mutations (the most common of which is T618I) and truncation mutations. Drug and siRNA screening of primary patient samples revealed that the two classes of CSF3R mutations exhibit differential sensitivity to inhibition of SRC or JAK kinases. CSF3R truncation mutations conferred sensitivity to SRC family kinase inhibition, while CSF3R membrane proximal mutations (T618I) conferred sensitivity to JAK kinase inhibition. A patient with the T618I membrane proximal mutation responded to treatment with the FDA approved JAK inhibitor, ruxolitinib. CSF3R truncation mutations have also been observed in a subset of severe congenital neutropenia patients who are at high risk for development of acute myeloid leukemia. Prior studies in this context have shown that truncation mutations result in loss of endocytic and degradation motifs, leading to increased expression of the receptor. The differences in signaling and drug sensitivity of these mutation classes suggest that membrane proximal mutations may activate CSF3R signaling through a distinct, as-yet unknown mechanism. Furthermore, a subset of CNL patients harbor both membrane proximal and truncation mutations on the same allele, though the consequences of these compound mutations are not yet known. Methods CSF3R expression level and banding pattern were assessed by immunoblot of lysates from 293T17 cells transfected with wild type, membrane proximal mutant, or truncation mutant CSF3R. O-linked glycosylation was removed from the receptor by treatment with O-glycosidase and neuraminidase. Ligand independence of the CSF3R mutants was analyzed in murine interleukin-3 (IL3)-dependent Ba/F3 cells. CSF3R dimerization was assessed by co-transfecting CSF3R-Flag and CSF3R-V5 tagged constructs and then immunoprecipitating CSF3R-Flag and detecting co-immunoprecipitation of the CSF3R-V5 by immunoblot. Transforming potential of the CSF3R compound mutations relative to the corresponding point or truncation mutations was assessed by analyzing IL3-independent growth of Ba/F3 cells or mouse bone marrow colony formation. Results To better understand the functional and biochemical differences between membrane proximal and truncation mutant CSF3R, we examined transformation potential, requirement for ligand, and expression patterns in Ba/F3 and 293T17 cells. We found membrane proximal mutations to exhibit rapid transformation potential and ligand independence, while truncation mutations exhibited delayed transformation and ligand hypersensitivity. Unlike the truncation mutations, which induce dramatic overexpression of CSF3R, the T618I mutation did not result in overexpression of the receptor but instead induced a shifted banding pattern, indicative of altered protein modification. We examined the amino acid sequence surrounding the membrane proximal mutations and found residue T618 to be part of a consensus motif for O-glycosylation, wherein wild type CSF3R is O-glycosylated and the T618I mutation abrogates this O-glycosylation event. Furthermore, the T618I mutation exhibited increased receptor dimerization compared to wild type CSF3R, which likely explains its ligand independence. Finally, we found that CSF3R compound mutations have increased transforming potential in Ba/F3 and murine bone marrow colony assays compared with either class of single mutation, further underscoring the different mechanisms of action of the membrane proximal and truncation mutations. Conclusion CSF3R represents a promising therapeutic target for patients with CNL. We show that T618I, the most common CSF3R mutation in CNL, is part of an O-linked glycosylation site. Mutation of this residue leads to loss of O-linked glycosylation and represents a novel mechanism of homodimeric cytokine receptor activation. CSF3R compound mutations are more rapidly transforming relative to the membrane proximal or truncation mutations alone, warranting their further investigation for patient prognosis and therapy. Disclosures: Off Label Use: Ruxolitinib - a JAK1/2 inhibitor that we propose can be used off-label for disease management of CSF3R-mutant neutrophilic leukemia. Gotlib:Incyte: Membership on an entity’s Board of Directors or advisory committees, Research Funding, Travel Support Other. Fleischman:Incyte: Speakers Bureau. Collins:Genoptix: Membership on an entity’s Board of Directors or advisory committees. Oh:Incyte Corporation: Membership on an entity’s Board of Directors or advisory committees, Research Funding, Speakers Bureau. Deininger:Novartis: Advisory Boards, Advisory Boards Other, Consultancy, Research Funding; Ariad Pharmaceuticals: Advisory Boards, Advisory Boards Other, Consultancy; Bristol-Myers Squibb: Advisory Boards Other, Consultancy, Research Funding; Celgene: Research Funding; Gilead Sciences: Research Funding. Druker:Bristol-Myers Squibb: PI or co-investigator on BMS clinical trials. OHSU and Dr. Druker have a financial interest in MolecularMD. OHSU has licensed technology used in some of these clinical trials to MolecularMD. Potential conflicts of interest are managed by OHSU. Other; Novartis: PI or co-investigator on Novartis clinical trials. OHSU and Dr. Druker have a financial interest in MolecularMD. OHSU has licensed technology used in some of these clinical trials to MolecularMD. Potential conflicts of interest are managed by OHSU., PI or co-investigator on Novartis clinical trials. OHSU and Dr. Druker have a financial interest in MolecularMD. OHSU has licensed technology used in some of these clinical trials to MolecularMD. Potential conflicts of interest are managed by OHSU. Other; Incyte: PI or co-investigator on clinical trials., PI or co-investigator on clinical trials. Other. Tyner:Incyte Corporation: Research Funding.

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Agonist-Induced Kindlin-3 Phsphorylation Regulates αIIbβ3 Integrin Activation In HEL Cells and Platelets

Blood ◽

10.1182/blood.v122.21.22.22 ◽

2013 ◽

Vol 122 (21) ◽

pp. 22-22

Author(s):

Katarzyna Bialkowska ◽

Eugene Podrez ◽

Tatiana V. Byzova ◽

Edward F. Plow

Keyword(s):

Conflicts Of Interest ◽

Focal Adhesions ◽

Human Platelets ◽

Phosphorylation Sites ◽

Integrin Activation ◽

Suspension Cells ◽

Integrin Αiibβ3 ◽

Hel Cells ◽

Β3 Integrin

Abstract The contributions of integrins to platelet responses depend upon the dynamic regulation of their activation status, which in turn depends on engagement of binding partners by their cytoplasmic tails. It is well-established that not only talin but also kindlin family members are essential for integrin activation, and both must present for optimal integrin function. Recent studies in humans have specifically emphasized the vital role of kindlin-3 in integrin functions in hematopoietic cells, including platelets, where kindlin-3 deficiency can lead to episodic bleeding, frequent infections and osteopetrosis, consequences of an inability to activate β1, β2 and β3 integrins. Despite this evidence, little is known about kindlin-3 structure-function relationship. Here, we used human platelets and human erythroleukemic HEL cell line that expresses integrin αIIbβ3 to investigate whether posttranslational modification(s) of kindlin-3 occurs and can influence its integrin activity. Non-stimulated HEL cells are suspension cells, and they do not adhere to fibrinogen or bind soluble fibrinogen and PAC-1 antibody (specific for activated αIIbβ3) readily. Thrombopoietin or PMA stimulation activated αIIbβ3 such that the cells adhered and spread on fibrinogen and increased their binding of PAC-1 and soluble fibrinogen. β3 integrin and kindlin-3 colocalized in focal adhesions in the adherent cells, and there was enhanced β3 integrin-kindlin-3 association as detected by coimmunoprecipitation. Kindlin-3 knockdown impaired agonist-stimulated adhesion and spreading on fibrinogen. Since, as we have shown previously, β3 integrin phosphorylation regulates kindlin and integrin interaction, we sought to determine whether kindlin-3 is also phosphorylated. Human platelets were stimulated with thrombin and HEL cells with PMA, and kindlin-3 was immunoprecipitated from lysates of control and stimulated cells. A kindlin-3 peptide showing significant increase in phosphorylation upon agonist stimulation was identified in both platelets and HEL cells by mass spectrometry. T482 or S484 were identified as phosphorylation sites in sequence that resides in the kindlin-3 variable region, which is not present either in kindlin-1 or kindlin-2 but is conserved across all species in which kindlin-3 has been sequenced. When expressed in HEL cells, TS/AA kindlin-3 mutant displayed decreased soluble fibrinogen binding and cell spreading on immobilized fibrinogen when compared to wild-type kindlin-3. Membrane-permeable, poly-arginine tagged kindlin-3 peptide containing the candidate phosphorylation sites kindlin-3 was introduced into HEL cells and platelets. HEL cell adhesion and spreading was blunted by the kindlin-3 peptide when compared to a scramble poly-arginine control peptide. Moreover, thrombin-induced platelet aggregation was inhibited by kindlin-3 peptide but not by the scramble peptide. Thus, our data emphasizes a role of previously unknown, agonist-induced kindlin-3 phosphorylation, in integrin αIIbβ3 activation in HEL cells and platelets and provides a basis for functional differences between kindlin-3 and its other two paralogs, kindlin-1 and kindlin-2. Disclosures: No relevant conflicts of interest to declare.

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Tailless keratins assemble into regular intermediate filaments in vitro

Journal of Cell Science ◽

10.1242/jcs.97.2.317 ◽

1990 ◽

Vol 97 (2) ◽

pp. 317-324

Author(s):

M. Hatzfeld ◽

K. Weber

Keyword(s):

Intermediate Filaments ◽

Consensus Sequence ◽

In Vitro Mutagenesis ◽

Type I ◽

Type Ii ◽

Tail Domain ◽

Filament Formation ◽

Wild Type ◽

Keratin 8

To study the influence of the non alpha-helical tail domain of keratins in filament formation, we prepared a truncated keratin 8 mutant, K8/tailless. Using site-directed in vitro mutagenesis we introduced a stop codon in the position coding for amino acid number 417 of the K8/wild-type sequence, thereby deleting 86 amino acids of the non alpha-helical tail domain but leaving the consensus sequence at the end of the rod domain intact. Expression of the truncated keratin 8 in Escherichia coli allowed us to purify the protein by a two-step procedure. The filament-forming capacity of the truncated K8 with wild-type K18 and K19 was analyzed using in vitro reconstitution. The in vitro assembly studies with K8/tailless and K18 wild-type indicate that the C-terminal tail domain of a type II keratin, including the homologous subdomain H2, is not required for filament formation. Moreover, reconstitution experiments with K8/tailless and K19, a naturally occurring tailless keratin I, show that the tail domains of type I as well as type II keratins are not an essential requirement for in vitro filament formation. Our results suggest that in vitro filament elongation does not depend on interactions between head and tail domains, although the tail domain might have a role in stabilization of intermediate filaments arising from certain keratin pairs.

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The Role of Akt3 in Mediating Outside-in Signaling of the Platelet Integrin αIIbβ3

Blood ◽

10.1182/blood.v118.21.1134.1134 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1134-1134

Author(s):

Kelly A O'Brien ◽

Nissim Hay ◽

Xiaoping Du

Keyword(s):

Kinase Inhibitor ◽

Conflicts Of Interest ◽

Wild Type ◽

Akt Inhibitor ◽

Integrin Αiibβ3 ◽

Akt Activation ◽

Downstream Effector ◽

Platelet Spreading ◽

Human And Mouse

Abstract Abstract 1134 Ligand binding to integrins mediates cell adhesion and transmits “outside in” signals that lead to cell spreading, migration, and proliferation. In platelets, the prototype integrin aIIbb3-mediated outside-in signaling is required for platelet spreading and retraction, and greatly amplifies platelet activation. Previous studies suggest that phosphoinositide 3-Kinases (PI3K) are activated upon binding of integrin αIIbβ3 to its ligand fibrinogen, and is important in outside-in signaling leading to platelet spreading. However, the mechanism by which PI3K transmits outside-in signals has been unclear. A major known downstream effector of PI3K is the Akt (protein kinase B) family of serine/threonine kinases, including Akt1, Akt2, and Akt3. We have recently shown that platelets not only express Akt1 and Akt2 as previously reported, but also express a substantial amount of Akt3. To investigate whether Akt3 is a downstream effector mediating PI3K-dependent integrin outside-in signaling, platelets from Akt3 knockout mice were compared with wild type platelets for their spreading on fibrinogen. Platelets from Akt3−/− mice showed partially, but significantly reduced spreading on fibrinogen, indicating that Akt3 is important in integrin-mediated outside-in signaling leading to platelet spreading. Consistent with the results of Akt3 knockout, treatment of platelets with a pan Akt inhibitor also significantly inhibited spreading of human and mouse platelets on fibrinogen. Akt becomes phosphorylated upon platelet spreading on fibrinogen, which is significantly reduced in Akt3 knockout platelets, and is abolished by PI3 Kinase inhibitor, wortmannin, or Src Family Kinase (SFK) Inhibitor, PP2, suggesting that Akt activation is downstream from PI3K, and SFK during integrin outside-in signaling. Thus, our data reveals that Akt3 is an important downstream effector of PI3K-dependent integrin outside-in signaling promoting platelet spreading. Disclosures: No relevant conflicts of interest to declare.

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Talin activates integrins by altering the topology of the β transmembrane domain

The Journal of Cell Biology ◽

10.1083/jcb.201112141 ◽

2012 ◽

Vol 197 (5) ◽

pp. 605-611 ◽

Cited By ~ 71

Author(s):

Chungho Kim ◽

Feng Ye ◽

Xiaohui Hu ◽

Mark H. Ginsberg

Keyword(s):

Amino Acid ◽

Ligand Binding ◽

Outer Membrane ◽

Transmembrane Domain ◽

Side Chain ◽

Amino Acid Side Chain ◽

Integrin Activation ◽

Integrin Αiibβ3 ◽

Environmentally Sensitive ◽

Extracellular Side

Talin binding to integrin β tails increases ligand binding affinity (activation). Changes in β transmembrane domain (TMD) topology that disrupt α–β TMD interactions are proposed to mediate integrin activation. In this paper, we used membrane-embedded integrin β3 TMDs bearing environmentally sensitive fluorophores at inner or outer membrane water interfaces to monitor talin-induced β3 TMD motion in model membranes. Talin binding to the β3 cytoplasmic domain increased amino acid side chain embedding at the inner and outer borders of the β3 TMD, indicating altered topology of the β3 TMD. Talin’s capacity to effect this change depended on its ability to bind to both the integrin β tail and the membrane. Introduction of a flexible hinge at the midpoint of the β3 TMD decoupled the talin-induced change in intracellular TMD topology from the extracellular side and blocked talin-induced activation of integrin αIIbβ3. Thus, we show that talin binding to the integrin β TMD alters the topology of the TMD, resulting in integrin activation.

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The Interaction of Coagulation Factor XI with Polyphosphate

Blood ◽

10.1182/blood.v120.21.498.498 ◽

2012 ◽

Vol 120 (21) ◽

pp. 498-498

Author(s):

Yipeng Geng ◽

Ingrid M. Verhamme ◽

Qiufang Cheng ◽

Anton Matafonov ◽

Stephen B. Smith ◽

...

Keyword(s):

Ferric Chloride ◽

Conformational Changes ◽

Conflicts Of Interest ◽

Thrombus Formation ◽

Coagulation Factor ◽

Wild Type ◽

Zymogen Activation ◽

Factor Xi ◽

Dimeric Structure

Abstract Abstract 498 The plasma zymogen factor XI (FXI) is a homodimer of 80 kDa subunits. During blood coagulation, each subunit is activated by cleavage of the Arg369-Ile370 bond by factor XIIa (FXIIa) or thrombin. Initially, one subunit of the FXI dimer is activated to create the species 1/2FXIa, followed by activation of the second subunit, generating FXIa. Initial rates of activation of the first subunit are relatively low (∼8000 M−1.sec−1 for FXIIa and 120 M−1.sec−1 for thrombin). Rates for activation of the second subunit are even lower (1200 M−1.sec−1 for FXIIa and 35 M−1.sec−1 for thrombin), suggesting that conformational changes accompanying activation of a subunit reduce the efficiency of activation of its partner. The slow rate of FXI activation in solution strongly suggests that a cofactor is required for protease activation in vivo. FXI activation by FXIIa or thrombin is enhanced by polyanions such as dextran sulfate (DS). In addition, polyanions induce FXI activation by FXIa (autoactivation). Polymers of inorganic phosphate (polyP) released from platelet dense granules accelerate FXI activation by thrombin or FXIa (Blood 2011;118:6963), and likely represent the physiologic counterpart to DS. PolyP (4 μM) increased the initial rate of FXI activation by FXIIa ∼30-fold (300,000 M−1.sec−1), and by a-thrombin ∼3600-fold (440,000 M−1.sec−1). Furthermore, polyP induced FXI autoactivation in a manner similar to DS. Each FXI subunit contains two polyanion binding sites (residues Arg250, Lys252, Lys253, Lys255 on the A3 domain, and Lys529, Arg530, Arg532 on the catalytic domain). Both sites bind heparin, and are required for normal heparin-mediated enhancement of FXIa inhibition by antithrombin. FXI lacking the A3 domain site (FXIΔA3), but not FXI lacking the protease domain site (FXIΔCD), is activated slowly in the presence of DS compared to wild type FXI (FXIWT). Interestingly, both FXIΔA3 and FXIΔCD are activated slowly compared to FXIWT in the presence of polyP, and a species lacking both sites (FXIΔA3/CD) has an even greater defect, indicating FXI's interaction with polyP is different from its interaction with DS. The FXI gene arose from a duplication of the gene for the monomeric protein prekallikrein (PK). The observations that the dimeric structure of FXI is highly conserved across species, and that the ancestral molecule is a monomer, strongly indicate that the dimer is important for a specific aspect of FXI function. It was recently reported that monomeric forms of FXI are activated slowly compared to dimeric FXI in solution or in the presence of DS (J Biol Chem 2008;283:18655). FXI dimer formation is mediated through a hydrophobic interface involving Leu284, Ile290, and Tyr329, and an interchain disulfide bond involving Cys321. A Ser substitution for Cys321 in combination with an Ala substitution for Leu284 or Ile290 results in the monomeric species FXIC321S, L284A and FXIC321S, I290A. Rates of FXIC321S, L284A or FXIC321S, I290A activation by FXIIa were significantly lower than for FXIWT in the presence of polyP. However, this defect was not observed during activation by thrombin or FXIa, demonstrating that the dimeric structure is not a prerequisite for zymogen activation on polyP. FXI-deficient (FXI−/−) mice are more resistant to arterial thrombus formation induced by vessel injury with ferric chloride than are wild type mice. FXIWT, FXIC321S, L284A and FXIC321S, I290A were transiently expressed in FXI−/− mice by hydrodynamic tail vein injection. While the three proteins were expressed at comparable levels, only FXIWT completely reconstituted the wild type phenotype in the ferric chloride thrombosis model. In summary, polyP is a strong candidate for a cofactor to support FXI activation in vivo. The interaction of FXI with this polyanion differs from its interaction with DS. The dimeric structure of FXI appears to be required for normal protease function in vivo, and for FXIIa-mediated FXI activation, but not for thrombin- or FXIa-mediated activation in the presence of polyP. Considering that the FXI homolog PK is a monomer that is activated efficiently by FXIIa, and that FXII deficiency is not associated with a significant phenotype, our results suggest that the FXI dimeric structure is required for a function distinct from zymogen activation. Disclosures: No relevant conflicts of interest to declare.

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