Integrin signaling: roles for the cytoplasmic tails of alpha IIb beta 3 in the tyrosine phosphorylation of pp125FAK

1995 ◽  
Vol 108 (12) ◽  
pp. 3817-3825 ◽  
Author(s):  
L. Leong ◽  
P.E. Hughes ◽  
M.A. Schwartz ◽  
M.H. Ginsberg ◽  
S.J. Shattil
Keyword(s):  
Protein Tyrosine Kinase ◽  
Cytoplasmic Tail ◽  
Cell Spreading ◽  
Cellular Responses ◽  
Distal Portion ◽  
Integrin Signaling ◽  
Adherent Cells ◽  
Wild Type ◽  
Truncation Mutant ◽  
Cytoplasmic Tails

pp125FAK (focal adhesion kinase) a protein tyrosine kinase that may mediate cellular responses to adhesion, is activated and tyrosine-phosphorylated when platelets adhere to fibrinogen via the integrin, alpha IIb beta 3. To determine whether either of the cytoplasmic tails of alpha IIb beta 3 regulates FAK phosphorylation, CHO cells were stably transfected with alpha IIb beta 3 or various cytoplasmic tail truncation mutants. Cells expressing wild-type alpha IIb beta 3 or alpha IIb beta 3 that lacked the COOH-terminal 13 or 18 residues of the 20 residue alpha IIb tail adhered to and spread on fibrinogen or on an anti-alpha IIb antibody, and FAK became tyrosine-phosphorylated. FAK also became phosphorylated in adherent cells lacking the COOH-terminal 35 or 39 residues of the 47 residue beta 3 tail, although the extent of phosphorylation was reduced by about 50% in the latter mutant. Little or no FAK phosphorylation was observed if 46 residues were deleted from the beta 3 tail. None of these beta 3 truncation mutants spread on the anti-alpha IIb antibody. When cells with wild-type alpha IIb beta 3 or truncated beta 3 were detached from a surface, FAK became rapidly dephosphorylated. In contrast, FAK remained phosphorylated in the two alpha IIb truncation mutants for up to 90 minutes in suspension. This persistent phosphorylation was not due to occupancy of alpha IIb beta 3 by adhesive ligands because it was also observed with an alpha IIb tail truncation mutant that contained an additional mutation in the extracellular portion of the receptor that prevents ligand binding. These studies demonstrate that: (1) the beta 3 cytoplasmic tail, including the membrane-proximal portion, is involved in initiation of FAK phosphorylation; (2) FAK phosphorylation can be initiated by cell adhesion in the absence of cell spreading; and (3) the membrane-distal portion of the alpha IIb cytoplasmic tail may normally function to dampen FAK phosphorylation in non-anchored cells.

scholarly journals Structure and Sequence Diversity of the Membrane Distal Region of α-Cytoplasmic Tail in Integrin Activation

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3453-3453
Author(s):  
Aye Myat Myat Thinn ◽  
Jieqing Zhu
Keyword(s):  
Cytoplasmic Tail ◽  
Proximal Region ◽  
Sequence Diversity ◽  
Wild Type ◽  
Integrin Activation ◽  
Cytoplasmic Tails ◽  
Membrane Proximal Region ◽  
Inside Out ◽  
Α5β1 Integrin

Abstract Integrins are α/β heterodimeric cell adhesion receptors with each subunit comprising of a large extracellular domain, a single-spanning transmembrane domain, and usually a short cytoplasmic tail. Different combinations of 18 α and 8 β subunits make up 24 integrin members that recognize diverse extracellular ligands important in numerous biological functions such as immune responses, maintenance of hemostasis, and development. Abnormal activation of integrin is associated with many pathological conditions including thrombosis, inflammatory diseases, as well as tumor-driven cell growth, metastasis, and angiogenesis. Therefore, tight regulation is crucial in integrin activation. Recent structural and functional studies have shown that integrin activation is regulated by the cytoplasmic tails. Studies on the mechanism of integrin activation from inside the cell (namely inside-out activation) have been focused on the β cytoplasmic tail that is relatively conserved and bears binding sites for the common intracellular activators such as talin and kindlin. However, the role of α cytoplasmic tail in integrin activation remains elusive. The integrin α cytoplasmic tails share a conserved GFFKR motif at the membrane-proximal region that forms a specific interface with the membrane-proximal region of the β cytoplasmic tail. In contrast, the membrane-distal (MD) regions following the GFFKR motif are diverse significantly both in length, sequence and structure when reported, and their roles in integrin activation have not been well characterized. Our recent studies demonstrated that the α-MD region is required for talin and kindlin-induced activation of αIIb, αV, and αL integrins and suggest that the sequence diversity of the α-MD region might play a role in the regulation of integrin activation. In this study, we further examined the role of α-MD regions in integrin inside-out activation using αIIb, αL, and α5 integrins as platforms. Each MD region of αIIb, αL, and α5 was replaced with those of other α subunits that heterodimerize with β3, β2, and β1 integrins, respectively. β3 subunit forms heterodimers with αIIb and αV integrins. β2 subunit forms heterodimers with αL, αM, αD, and αX integrins. β1 subunit forms heterodimers with α1, α2, α3, α4, α5, α6, α7, α8, α9, α10, α11, and αV integrins. Thus, using these integrin α-chimeras, we were able to systemically study the role of 17 α-MD regions in integrin inside-out activation while retaining the native association of α and β subunits at the cytoplasmic domains. Ligand-mimetic mAb PAC-1, intercellular adhesion molecule-1 (ICAM-1), and human fibronectin were used to measure the talin-head-induced activation of αIIb, αL, and α5 chimeras co-expressed in HEK293FT cells with β3, β2, and β1 integrins, respectively. Conformation-specific monoclonal antibodies were used to report integrin conformational activation. The endogenous α5β1 integrin of HEK293FT cells were knocked out by the CRISPR/Cas9 technology. Our data showed that the chimeric α integrins had different levels of inside-out activation when compared with their corresponding wild-type integrins. Some chimeras such as αIIb-αV, αL-αX, αL-αD, αL-αM, α5-α2, α5-α4, and α5-α9 showed lower integrin activation than the wild types, while other chimeras such as α5-α7 and α5-α10 rendered α5β1 integrin more active than wild type. As a control, the αIIb-α1 and αIIb-αL chimeras all showed higher inside-out activation than wild-type αIIb. Our results suggest that specific amino acids of the α-MD region that immediately follow the GFFKR motif might contribute to integrin inside-out activation, probably through regulating the conformational change of the integrin α transmembrane and cytoplasmic domains. Our study demonstrates an important role of the α-MD region in integrin activation and indicates that structure and sequence diversity of the α-MD region might contribute to the diverse functions of integrins, which are determined by different integrin α subunits. Disclosures No relevant conflicts of interest to declare.

Tyrosine Phosphorylation of the Integrin β3 Subunit Regulates β3 Cleavage by Calpain.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1524-1524
Author(s):  
Aleksandra Stojanovic ◽  
Panagiotis Flevaris ◽  
Xiaodong Xi ◽  
Athar Chisti ◽  
David Phillips ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Leading Edge ◽  
Cell Spreading ◽  
Cytoplasmic Domain ◽  
Integrin Signaling ◽  
Wild Type ◽  
Sodium Vanadate ◽  
Calpain Activity ◽  
Adhesion Sites ◽  
Domain Peptide

Abstract Outside-in signaling of β3 integrins induces and requires phosphorylation at tyrosine-747 (Y747) and tyrosine-759 (Y759) of the β3 subunit, but the mechanism for this requirement is unclear. On the other hand, a key consequence of integrin signaling, cell spreading, is inhibited by calpain cleavage of β3 cytoplasmic domain. Here we show that tyrosine phosphorylation in the synthetic β3 cytoplasmic domain peptide inhibits calpain cleavage. In platelets, tyrosine phosphates inhibitor, sodium vanadate, enhances thrombin-induced phosphorylation at Y747 and Y759, which is associated with the reduced integrin cleavage by calpain. The effects of sodium vanadate is unlikely to be caused by its effects on calpain activity but is likely to be caused by the susceptibility of integrin cytoplasmic domain, because sodium vanadate did not affect the calpain cleavage of another substrate, fodrin, in platelets. To further support the protective effect of tyrosine phosphorylation against calpain cleavage, we show that mouse β3 (DiYF) with both Y759 and Y747 mutated to phenylalanine is more susceptible to calpain cleavage than wild type during thrombin-induced platelet aggregation. Furthermore, phosphorylation at Y747 and Y759 of β3 in the focal adhesion sites and the leading edge of spreading platelets was differentially regulated. Selective dephosphorylation of Y759 is associated with calpain cleavage at Y759. Thus, one mechanism by which tyrosine phosphorylation promotes integrin signaling and cell spreading is its inhibition of calpain cleavage of the β3 cytoplasmic domain.

scholarly journals Kindlin-2 directly binds actin and regulates integrin outside-in signaling

2016 ◽  
Vol 213 (1) ◽  
pp. 97-108 ◽  
Author(s):  
Kamila Bledzka ◽  
Katarzyna Bialkowska ◽  
Khalid Sossey-Alaoui ◽  
Julia Vaynberg ◽  
Elzbieta Pluskota ◽  
...  
Keyword(s):  
Cell Spreading ◽  
Cellular Responses ◽  
Cell System ◽  
Actin Binding ◽  
Wild Type ◽  
Cho Cell ◽  
Actin Organization ◽  
Support Cell ◽  
Direct Binding ◽  
Actin Binding Site

Reduced levels of kindlin-2 (K2) in endothelial cells derived from K2+/− mice or C2C12 myoblastoid cells treated with K2 siRNA showed disorganization of their actin cytoskeleton and decreased spreading. These marked changes led us to examine direct binding between K2 and actin. Purified K2 interacts with F-actin in cosedimentation and surface plasmon resonance analyses and induces actin aggregation. We further find that the F0 domain of K2 binds actin. A mutation, LK47/AA, within a predicted actin binding site (ABS) of F0 diminishes its interaction with actin by approximately fivefold. Wild-type K2 and K2 bearing the LK47/AA mutation were equivalent in their ability to coactivate integrin αIIbβ3 in a CHO cell system when coexpressed with talin. However, K2-LK47/AA exhibited a diminished ability to support cell spreading and actin organization compared with wild-type K2. The presence of an ABS in F0 of K2 that influences outside-in signaling across integrins establishes a new foundation for considering how kindlins might regulate cellular responses.

scholarly journals The Extracellular Domain of the β2Integrin β Subunit (CD18) Is Sufficient forEscherichia coliHemolysin andAggregatibacter actinomycetemcomitansLeukotoxin Cytotoxic Activity

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Laura C. Ristow ◽  
Vy Tran ◽  
Kevin J. Schwartz ◽  
Lillie Pankratz ◽  
Andrew Mehle ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Urinary Tract Infections ◽  
Integrin Signaling ◽  
Β Subunit ◽  
Wild Type ◽  
Α Subunit ◽  
Content Type ◽  
Animal Pathogens ◽  
Tract Infections

ABSTRACTTheEscherichia colihemolysin (HlyA) is a pore-forming exotoxin associated with severe complications of human urinary tract infections. HlyA is the prototype of the repeats-in-toxin (RTX) family, which includes LtxA fromAggregatibacter actinomycetemcomitans, a periodontal pathogen. The existence and requirement for a host cell receptor for these toxins are controversial. We performed an unbiased forward genetic selection in a mutant library of human monocytic cells, U-937, for host factors involved in HlyA cytotoxicity. The top candidate was the β2integrin β subunit. Δβ2cell lines are approximately 100-fold more resistant than wild-type U-937 cells to HlyA, but remain sensitive to HlyA at high concentrations. Similarly, Δβ2cells are more resistant than wild-type U-937 cells to LtxA, as Δβ2cells remain LtxA resistant even at >1,000-fold-higher concentrations of the toxin. Loss of any single β2integrin α subunit, or even all four α subunits together, does not confer resistance to HlyA. HlyA and LtxA bind to the β2subunit, but not to αL, αM, or αXin far-Western blots. Genetic complementation of Δβ2cells with either β2or β2with a cytoplasmic tail deletion restores HlyA and LtxA sensitivity, suggesting that β2integrin signaling is not required for cytotoxicity. Finally, β2mutations do not alter sensitivity to unrelated pore-forming toxins, as wild-type or Δβ2cells are equally sensitive toStaphylococcus aureusα-toxin andProteus mirabilisHpmA. Our studies show two RTX toxins use the β2integrin β subunit alone to facilitate cytotoxicity, but downstream integrin signaling is dispensable.IMPORTANCEUrinary tract infections are one of the most common bacterial infections worldwide. UropathogenicEscherichia colistrains are responsible for more than 80% of community-acquired urinary tract infections. Although we have known for nearly a century that severe infections stemming from urinary tract infections, including kidney or bloodstream infections are associated with expression of a toxin, hemolysin, from uropathogenicEscherichia coli, how hemolysin functions to enhance virulence is unknown. Our research defines the interaction of hemolysin with the β2integrin, a human white cell adhesion molecule, as a potential therapeutic target during urinary tract infections. TheE. colihemolysin is the prototype for a toxin family (RTX family) produced by a wide array of human and animal pathogens. Our work extends to the identification and characterization of the receptor for an additional member of the RTX family, suggesting that this interaction may be broadly conserved throughout the RTX toxin family.

scholarly journals Focal adhesions are sites of integrin extension

2010 ◽  
Vol 188 (6) ◽  
pp. 891-903 ◽  
Author(s):  
Janet A. Askari ◽  
Christopher J. Tynan ◽  
Stephen E.D. Webb ◽  
Marisa L. Martin-Fernandez ◽  
Christoph Ballestrem ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Resonance Energy Transfer ◽  
Focal Adhesions ◽  
Resonance Energy ◽  
Cell Spreading ◽  
Integrin Subunit ◽  
Adherent Cells ◽  
Extended Form ◽  
Integrin Α5β1 ◽  
Mediate Cell

Integrins undergo global conformational changes that specify their activation state. Current models portray the inactive receptor in a bent conformation that upon activation converts to a fully extended form in which the integrin subunit leg regions are separated to enable ligand binding and subsequent signaling. To test the applicability of this model in adherent cells, we used a fluorescent resonance energy transfer (FRET)–based approach, in combination with engineered integrin mutants and monoclonal antibody reporters, to image integrin α5β1 conformation. We find that restricting leg separation causes the integrin to adopt a bent conformation that is unable to respond to agonists and mediate cell spreading. By measuring FRET between labeled α5β1 and the cell membrane, we find extended receptors are enriched in focal adhesions compared with adjacent regions of the plasma membrane. These results demonstrate definitely that major quaternary rearrangements of β1-integrin subunits occur in adherent cells and that conversion from a bent to extended form takes place at focal adhesions.

Deletion of the kinase insert sequence of the platelet-derived growth factor beta-receptor affects receptor kinase activity and signal transduction

1990 ◽  
Vol 10 (2) ◽  
pp. 801-809
Author(s):  
L Severinsson ◽  
B Ek ◽  
K Mellström ◽  
L Claesson-Welsh ◽  
C H Heldin
Keyword(s):  
Growth Factor ◽  
Protein Tyrosine Kinase ◽  
Catalytic Efficiency ◽  
Kinase Domain ◽  
Platelet Derived Growth Factor ◽  
Tyrosine Kinase Domain ◽  
Wild Type ◽  
Mutant Receptor ◽  
Beta Receptor ◽  
Type Receptor

A characteristic feature of the platelet-derived growth factor (PDGF) beta-receptor is the presence of an insert sequence in the protein tyrosine kinase domain. A receptor mutant which lacks the entire insert of 98 amino acids was expressed in CHO cells, and its functional characteristics were compared with those of the wild-type receptor. The mutant receptor bound PDGF-BB with high affinity and mediated internalization and degradation of the ligand with efficiency similar to that of the wild-type receptor but did not transduce a mitogenic signal. It was found to display a decreased autophosphorylation after ligand stimulation and had a decreased ability to phosphorylate exogenous substrates; phosphofructokinase was not phosphorylated at all, whereas a peptide substrate was phosphorylated, albeit at a lower rate compared with phosphorylation by the wild-type receptor. Furthermore, the mutant receptor did not mediate actin reorganization but mediated an increase in c-fos expression. The data indicate that the insert in the kinase domain of the PDGF beta-receptor is important for the substrate specificity or catalytic efficiency of the kinase; the deletion of the insert interferes with the transduction of some, but not all, of the signals that arise after activation of the receptor.

The lethality of p60v-src in Saccharomyces cerevisiae and the activation of p34CDC28 kinase are dependent on the integrity of the SH2 domain

1993 ◽  
Vol 105 (2) ◽  
pp. 519-528
Author(s):  
F. Boschelli ◽  
S.M. Uptain ◽  
J.J. Lightbody
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Protein Tyrosine Kinase ◽  
Kinase Activity ◽  
Cell Cycle Control ◽  
Sh2 Domain ◽  
Wild Type ◽  
Type V ◽  
Cdc28 Kinase ◽  
In Cells

The lethal effects of the expression of the oncogenic protein tyrosine kinase p60v-src in Saccharomyces cerevisiae are associated with a loss of cell cycle control at the G1/S and G2/M checkpoints. Results described here indicate that the ability of v-Src to kill yeast is dependent on the integrity of the SH2 domain, a region of the Src protein involved in recognition of proteins phosphorylated on tyrosine. Catalytically active v-Src proteins with deletions in the SH2 domain have little effect on yeast growth, unlike wild-type v-Src protein, which causes accumulation of large-budded cells, perturbation of spindle microtubules and increased DNA content when expressed. The proteins phosphorylated on tyrosine in cells expressing v-Src differ from those in cells expressing a Src protein with a deletion in the SH2 domain. Also, unlike the wild-type v-Src protein, which drastically increases histone H1-associated Cdc28 kinase activity, c-Src and an altered v-Src protein have no effect on Cdc28 kinase activity. These results indicate that the SH2 domain is functionally important in the disruption of the yeast cell cycle by v-Src.

scholarly journals Characterization of calcium- and integrin-binding protein 1 (CIB1) knockout platelets: Potential compensation by CIB family members

2008 ◽  
Vol 100 (05) ◽  
pp. 847-856 ◽  
Author(s):  
Brenda R. Temple ◽  
Holly R. Gentry ◽  
Jan C. DeNofrio ◽  
Weiping Yuan ◽  
Leslie V. Parise
Keyword(s):  
Thrombus Formation ◽  
Mrna Level ◽  
Family Members ◽  
Cytoplasmic Tail ◽  
Binding Pocket ◽  
Vitro Binding ◽  
Cytoplasmic Tails ◽  
Hydrophobic Binding

SummaryPlatelet aggregation requires activation of the αIIbβ3 integrin,an event regulated by the integrin cytoplasmic tails. CIB1 binds to the cytoplasmic tail of the integrin αIIb subunit. Previous overexpression and knockdown studies in murine megakaryocytes demonstrated that CIB1 inhibits integrin αIIbβ3 activation.Here we analyzed Cib1-/- mice to determine the function of CIB1 in platelets in vitro and in vivo. We found that although these mice had no overt platelet phenotype, mRNA level of CIB1 homolog CIB3 was increased in Cib1-/- megakaryocytes. In vitro binding experiments showed that recombinant CIB1, -2 and -3 bound specifically to an αIIb cytoplasmic tail peptide. Subsequent protein modeling experiments indicated that CIBs 1–3 each have a highly conserved hydrophobic binding pocket. Therefore, the potential exists for compensation for the loss of CIB1 by these CIB family members, thereby preventing pathologic thrombus formation in Cib1-/- mice.

Sorting of rat liver and ileal sodium-dependent bile acid transporters in polarized epithelial cells

1998 ◽  
Vol 275 (5) ◽  
pp. G1045-G1055 ◽  
Author(s):  
An-Qiang Sun ◽  
Meenakshisundaram Ananthanarayanan ◽  
Carol J. Soroka ◽  
Sundararajah Thevananther ◽  
Benjamin L. Shneider ◽  
...  
Keyword(s):  
Bile Acid ◽  
Amino Acid ◽  
Mdck Cells ◽  
Cytoplasmic Tail ◽  
Apical Surface ◽  
Acid Transport ◽  
Transport Activity ◽  
Wild Type ◽  
Bile Acid Transport ◽  
Apical Domain

The rat ileal apical Na+-dependent bile acid transporter (ASBT) and the liver Na+-taurocholate cotransporting polypeptide (Ntcp) are members of a new family of anion transporters. These transport proteins share limited sequence homology and almost identical predicted secondary structures but are localized to the apical surface of ileal enterocytes and the sinusoidal surface of hepatocytes, respectively. Stably transfected Madin-Darby canine kidney (MDCK) cells appropriately localized wild-type ASBT and Ntcp apically and basolaterally as assessed by functional activity and immunocytochemical localization studies. Truncated and chimeric transporters were used to determine the functional importance of the cytoplasmic tail in bile acid transport activity and membrane localization. Two cDNAs were created encoding a truncated transporter in which the 56-amino-acid COOH-terminal tail of Ntcp was removed or substituted with an eight-amino-acid epitope FLAG. For both mutants there was some loss of fidelity in basolateral sorting in that ∼75% of each protein was delivered to the basolateral surface compared with ∼90% of the wild-type Ntcp protein. In contrast, deletion of the cytoplasmic tail of ASBT led to complete loss of transport activity and sorting to the apical membrane. An Ntcp chimera in which the 56-amino-acid COOH-terminal tail of Ntcp was replaced with the 40-amino-acid cytoplasmic tail of ASBT was largely redirected (82.4 ± 3.9%) to the apical domain of stably transfected MDCK cells, based on polarity of bile acid transport activity and localization by confocal immunofluorescence microscopy. These results indicate that a predominant signal for sorting of the Ntcp protein to the basolateral domain is located in a region outside of the cytoplasmic tail. These studies have further shown that a novel apical sorting signal is localized to the cytoplasmic tail of ASBT and that it is transferable and capable of redirecting a protein normally sorted to the basolateral surface to the apical domain of MDCK cells.

scholarly journals Lamellipodium is a myosin-independent mechanosensor

2018 ◽  
Vol 115 (11) ◽  
pp. 2646-2651 ◽  
Author(s):  
Patrick W. Oakes ◽  
Tamara C. Bidone ◽  
Yvonne Beckham ◽  
Austin V. Skeeters ◽  
Guillermina R. Ramirez-San Juan ◽  
...  
Keyword(s):  
Cell Attachment ◽  
Myosin Ii ◽  
Adhesion Formation ◽  
Leading Edge ◽  
Cell Spreading ◽  
Substrate Stiffness ◽  
Adherent Cells ◽  
Matrix Stiffness ◽  
Biophysical Properties ◽  
Independent Mechanism

The ability of adherent cells to sense changes in the mechanical properties of their extracellular environments is critical to numerous aspects of their physiology. It has been well documented that cell attachment and spreading are sensitive to substrate stiffness. Here, we demonstrate that this behavior is actually biphasic, with a transition that occurs around a Young’s modulus of ∼7 kPa. Furthermore, we demonstrate that, contrary to established assumptions, this property is independent of myosin II activity. Rather, we find that cell spreading on soft substrates is inhibited due to reduced myosin-II independent nascent adhesion formation within the lamellipodium. Cells on soft substrates display normal leading-edge protrusion activity, but these protrusions are not stabilized due to impaired adhesion assembly. Enhancing integrin–ECM affinity through addition of Mn2+ recovers nascent adhesion assembly and cell spreading on soft substrates. Using a computational model to simulate nascent adhesion assembly, we find that biophysical properties of the integrin–ECM bond are optimized to stabilize interactions above a threshold matrix stiffness that is consistent with the experimental observations. Together, these results suggest that myosin II-independent forces in the lamellipodium are responsible for mechanosensation by regulating new adhesion assembly, which, in turn, directly controls cell spreading. This myosin II-independent mechanism of substrate stiffness sensing could potentially regulate a number of other stiffness-sensitive processes.

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