The cytoskeletal/non-muscle isoform of alpha-actinin is phosphorylated on its actin-binding domain by the focal adhesion kinase.

alpha-Actinin is tyrosine-phosphorylated in activated human platelets (Izaguirre, G., Aguirre, L., Ji, P., Aneskievich, B., and Haimovich, B. (1999) J. Biol. Chem. 274, 37012--37020). Analysis of platelet RNA by reverse transcription-polymerase chain reaction revealed that alpha-actinin expressed in platelets is identical to the cytoskeletal/non-muscle isoform. A construct of this isoform containing a His(6) tag at the amino terminus was generated. Robust tyrosine phosphorylation of the recombinant protein was detected in cells treated with the tyrosine phosphatase inhibitor vanadate. The tyrosine phosphorylation site was localized to the amino-terminal domain by proteolytic digestion. A recombinant alpha-actinin protein containing a Tyr --> Phe mutation at position 12 (Y12F) was no longer phosphorylated when expressed in vanadate-treated cells, indicating that tyrosine 12 is the site of phosphorylation. The wild type recombinant protein was not phosphorylated in cells lacking the focal adhesion kinase (FAK). Re-expression of FAK in these cells restored alpha-actinin phosphorylation. Purified wild type alpha-actinin, but not the Y12F mutant, was phosphorylated in vitro by wild type as well as a Phe-397 mutant of FAK. In contrast, no phosphorylation was detected in the presence of a kinase-dead FAK. Tyrosine phosphorylation reduced the amount of alpha-actinin that cosedimented with actin filaments. These results establish that alpha-actinin is a direct substrate for FAK and suggest that alpha-actinin mediates FAK-dependent signals that could impact the physical properties of the cytoskeleton.

Neutrophil adhesion to vascular prosthetic surfaces triggers nonapoptotic cell death.

OBJECTIVE: To test the hypothesis that neutrophil adhesion to expanded polytetrafluoroethylene (ePTFE) and Dacron triggers cell death. SUMMARY BACKGROUND DATA: Vascular prosthetic infections are intransigent clinical dilemmas associated with excessive rates of death and complications. Impaired neutrophil function has been implicated in the infection of implanted cardiovascular devices. ePTFE and Dacron are potent neutrophil stimuli able to elicit activation responses such as reactive oxygen species production independent of exogenous/soluble agonists. Reactive oxygen species that are released into the medium when neutrophils are challenged by soluble agonists are known to cause self-destruction. The authors therefore sought to examine whether neutrophil adhesion to prosthetic graft materials decreases neutrophil viability by means of reactive oxygen species production. METHODS: Neutrophils were adhered to surfaces for up to 6 hours. Cell viability was monitored with propidium iodide staining and lactate dehydrogenase release. RESULTS: Within 6 hours of adhesion to ePTFE and Dacron, respectively, 59% +/- 11% and 44% +/- 5% (n = 7) of the neutrophils were stained by propidium iodide. Indistinguishable results were obtained with plasma-coated ePTFE and Dacron. In contrast, less than 2% of the neutrophils adherent to fibrinogen-, immunoglobin-, or fetal bovine serum-coated polystyrene surfaces for 6 hours were positive for propidium iodide. The increase in membrane permeability to propidium iodide was accompanied by a two- to threefold increase in lactate dehydrogenase release. Pretreatment of neutrophils with N-acetyl-L-cysteine, cytochalasin D, or cyclosporin A significantly reduced the number of propidium iodide-positive ePTFE and Dacron adherent neutrophils. CONCLUSIONS: Neutrophil adhesion to ePTFE and Dacron triggers a rapid nonapoptotic cell death. The effect of ePTFE and Dacron on neutrophil viability appears to be caused by reactive oxygen species production. The premature death of graft-adherent neutrophils provides a novel explanation of the defect in neutrophil bacterial killing associated with vascular prosthetic grafts.

Tyrosine phosphorylation of alpha-actinin in activated platelets.

The integrin alpha(IIb)beta(3) mediates tyrosine phosphorylation of a 105-kDa protein (pp105) in activated platelets. We have partially purified a 105-kDa tyrosine-phosphorylated protein from platelets stimulated with phorbol 12-myristate 13-acetate and obtained the sequence of an internal 12-mer peptide derived from this protein. The sequence was identical to human alpha-actinin sequences deposited in the Swiss Protein Database. alpha-Actinin, a 105-kDa protein in platelets, was subsequently purified from activated platelets by four sequential chromatographic steps. Fractions were analyzed by Western blotting and probed with alpha-actinin and anti-phosphotyrosine antibodies. The distribution of alpha-actinin and pp105 overlapped throughout the purification. Furthermore, in the course of this purification, a 105-kDa tyrosine-phosphorylated protein was only detected in fractions that contained alpha-actinin. The purified alpha-actinin protein was immunoprecipitated with antibodies to phosphotyrosine in the absence but not in the presence of phenyl phosphate. alpha-Actinin resolved by two-dimensional gel electrophoresis of activated platelet lysates was recognized by the antibodies to phosphotyrosine, whereas pretreatment of the platelets with bisindolylmaleimide, a protein kinase C inhibitor that prevents tyrosine phosphorylation of pp105, inhibited the reactivity of the antibodies to phosphotyrosine with alpha-actinin. Taken together, these data demonstrate that a fraction of alpha-actinin is tyrosine-phosphorylated in activated platelets.

Phospholipase A2 enzymes regulate alpha IIb beta3-mediated, but not Fc gammaRII receptor-mediated, pp125FAK phosphorylation in platelets.

The alphaII(b)beta3 integrin and FcgammaRII receptors mediate, respectively, platelet adhesion and spreading on fibrinogen and immunoglobulin (IgG) coated surfaces. Platelet adhesion to fibrinogen resulted in a partial conversion of the faster to the slower migrating (phosphorylated) form of Ca(+2)-sensitive cytosolic phospholipase A2(cPLA2) but failed to trigger arachidonic acid (AA) release. Full mobility shift of cPLA2 and a massive release of AA release were stimulated by platelet adhesion to IgG or addition of thrombin to the fibrinogen adherent platelets. IgG and thrombin induced AA production were blocked by methyl arachidonyl fluorophosphonate (MAFP), an irreversible inhibitor of cPLA2 and the Ca(+2)-independent phospholipase A2 (iPLA2). In contrast, bromoenol lactone (BEL), a specific inhibitor of iPLA2 had no effect on the release of AA. MAFP and BEL prevented pp125FAK phosphorylation and platelet spreading on fibrinogen having no effect on pp125FAK phosphorylation or platelet spreading on immobilized IgG. We conclude that alpha(IIb)beta3-mediated pp125FAK phosphorylation and platelet spreading on fibrinogen are regulated by PLA2 enzymes.

Integrin alpha IIb beta 3-mediated pp125FAK phosphorylation and platelet spreading on fibrinogen are regulated by PI 3-kinase.

Activation of the focal adhesion kinase pp125FAK correlates with its phosphorylation on tyrosine residues and is mediated by multiple receptor-ligand pairs. In platelets, pp125FAK phosphorylation is triggered by alpha IIb beta 3 integrin or Fc gamma RII receptor interaction with immobilized fibrinogen and IgG, respectively. In this study we used platelets as a model system to explore the role of PI 3-kinase relative to pp125FAK phosphorylation. Treatment of the platelets with two PI 3-kinase inhibitors, wortmannin and LY294002, inhibited in a dose-dependent manner alpha IIb beta 3-mediated platelet spreading on fibrinogen having no effect on platelet spreading on IgG. Both inhibitors also completely abolished alpha IIb beta 3-mediated pp125FAK phosphorylation but not pp72syk phosphorylation. Furthermore, Fc gamma RII- and thrombin-induced pp125FAK phosphorylation were not affected by wortmannin and LY294002. Finally, the PI 3-kinase inhibitors' effect on alpha IIb beta 3-mediated spreading and pp125FAK phosphorylation was reversed by phorbol ester treatment. These results establish that the role of PI 3-kinase relative to pp125FAK phosphorylation in platelets is receptor type-specific yet essential for alpha IIb beta 3-mediated cell spreading and pp125FAK phosphorylation.

Protein kinase C-delta activation and tyrosine phosphorylation in platelets.

Several protein kinase C (PKC) isoforms are expressed in human platelets. We report that PKC-delta is tyrosine phosphorylated within 30 s of platelet activation by thrombin. This correlated with a 2-3-fold increase in the kinase activity of PKC-delta relative to unstimulated platelets. The tyrosine phosphorylated PKC-delta isoform was associated with the platelet particulate (100,000 x g insoluble) fraction. Alpha(IIb)beta3 integrin mediated platelet adhesion to fibrinogen did not significantly affect PKC-delta activity. Tyrosine phosphorylation of PKC-delta was similarly not detected in fibrinogen adherent platelet lysates. Treatment of the platelets with mAb 7E3 prior to the addition of thrombin blocked aggregation having no effect on the thrombin induced PKC-delta activation. We conclude that PKC-delta is activated in platelets by an alpha(IIb)beta3 independent pathway.

Immobilized IgG and fibrinogen differentially affect the cytoskeletal organization and bactericidal function of adherent neutrophils.

The infection of a vascular prosthesis is a dreaded clinical outcome. Since fibrinogen (FBGN) and immunoglobulin (IgG) coat the implanted biomaterial surface, it is with these immobilized proteins that the neutrophil (PMN) interacts. This study tests the hypothesis that PMN are impaired in their ability to kill bacteria when bound to immobilized IgG or FBGN. Isolated human PMN were bound to FBGN or IgG and then left untreated or exposed to phorbol myristate acetate (PMA; 10(-7) M). PMN adhered loosely, but did not spread, onto FBGN. In contrast, PMN spread fully onto IgG, exhibiting polarized pseudopodia. PMA treatment induced spreading of the FBGN bound cells. Suspended and adherent PMN were incubated 1 h with 14C-labeled Staphylococcus aureus; then, phagocytosis was assessed by radioactive uptake or bacterial kill was determined by plating recovered bacteria and colony counting. Data were analyzed by unpaired t test. We observed that both the phagocytic and the killing ability of FBGN-bound PMN were similar to that of suspended PMN. Conversely, IgG-bound PMN displayed a 62 +/- 6% (P < 0.01) decrease in phagocytosis and 33 +/- 7% (P < 0.05) reduction in kill vs suspended cells. PMA induced a 74 +/- 6% (P < 0.01) reduction in phagocytosis and 68 +/- 5% (P < 0.01) reduction in kill of bacteria for PMN bound to FBGN with no further effect on IgG-bound PMN. Using fluorescent vital dyes and confocal microscopy we determined that 33% fewer PMN were engaged in phagocytosis when bound to IgG vs FBGN. We conclude that Fc receptor ligation by immobilized IgG or PMA treatment of the FBGN-adherent PMN triggers cell spreading and reduced bactericidal activity. These results indicate that excessive cytoskeletal organization may impair the ability of PMN to kill bacteria and result in vascular graft infections.

The FcgammaRII receptor triggers pp125FAK phosphorylation in platelets.

Platelets express a single low affinity receptor for immunoglobulin, FcgammaRII, that triggers multiple cellular responses upon interaction with multivalent immune complexes. In this study we show that immobilized IgG is also a potent stimulant of platelet activation triggering adhesion, aggregation, massive dense granule secretion, and thromboxane production. Platelet adhesion to IgG was blocked by the FcgammaRII receptor-specific monoclonal antibody, IV. 3. Pretreatment of the platelets with cytochalasin D to inhibit actin polymerization similarly prevented cell binding to IgG having no effect on platelet binding to fibrinogen. Platelet adhesion to IgG also led to the induction of tyrosine phosphorylation of multiple proteins including pp125(FAK) and p72(SYK). These proteins were also tyrosine-phosphorylated in alphaIIbbeta3-deficient IgG-adherent platelets from patients with Glanzmann's thrombasthenia. These data demonstrate that FcgammaRII mediates pp125(FAK) phosphorylation and platelet adhesion to IgG independent of the integrin alphaIIbbeta3. Treatment of the platelets with bisindolylmaleimide to inhibit protein kinase C prevented phosphorylation of pp125(FAK) as well as several other proteins, but not p72(SYK) phosphorylation. This study establishes that the FcgammaRII receptor mediates pp125(FAK) phosphorylation via protein kinase C.

Protein kinase C regulates tyrosine phosphorylation of pp125FAK in platelets adherent to fibrinogen.

Platelet adhesion to immobilized fibrinogen stimulates the induction of tyrosine phosphorylation of multiple proteins. However, platelet spreading and tyrosine phosphorylation of three proteins, the focal adhesion kinase pp125FAK and proteins of 101 and 105 kD (pp101 and pp105), require a second adenosine diphosphate (ADP)-dependent costimulatory event. In this study we show that protein kinase C (PKC) inhibitors prevented the induction of tyrosine phosphorylation of pp125FAK, pp101 and pp105, and abolished spreading. These inhibitory effects were not observed after treatment of the platelets with the intracellular Ca2+ chelator BAPTA-AM. This suggested that in platelets, PKC regulates spreading and related protein tyrosine phosphorylation. In addition, the inhibitory effects of apyrase, an ADP scavenger, on spreading and tyrosine phosphorylation of pp125FAK, pp101, and pp105, were not observed in the presence of phorbol 12-myristate 13-acetate (PMA). These data implied that in fibrinogen-adherent platelets integrin ligation and an agonist receptor occupancy are required for the functional association of PKC and the alpha IIb beta 3-mediated signaling pathways. Taken together these results show that PKC plays a central role in the transduction of intracellular signals downstream from alpha IIb beta 3 that regulate spreading and pp125FAK phosphorylation.

Magainin-induced cytotoxicity in eukaryotic cells: kinetics, dose-response and channel characteristics.

Magainin 1 and magainin 2 are broad-spectrum antimicrobial and antifungal peptides initially purified from Xenopus laevis skin glands. The mechanism of cytotoxicity of the naturally occurring magainin 2 and a potent all-D amino acid analogue, MSI-238, was examined for eukaryotic cells using flow cytometric analysis with propidium iodide (PI). Exposure to MSI-238 resulted in cell death within seconds to minutes, depending on the concentration of the peptide. Several cell types were examined including a mouse fibroblast cell line Balb/3T3 and a Rous sarcoma virus Balb/3T3-transformed cell line, SRD/3T3, primary chick embryo fibroblasts and cells derived from a human ovarian carcinoma, OVCA-3. The K0.5 values determined from 5 min exposures ranged from 24 to 80 micrograms/ml for MSI-238 and approximately 600 micrograms/ml for magainin 2. Molecular properties of MSI-238 induced channels were studied in excised membrane patch recordings from Balb/3T3 and SRD/3T3 cells. At low concentrations of 0.1 micrograms/ml, occasional, brief, multiple-level current fluctuations were seen suggesting channels with multiple, rapidly changing conductance levels. At 5 or 10 micrograms/ml of MSI-238, the current fluctuations were larger in magnitude and occurred more frequently producing a general disruption of the membrane similar to the effects of melittin on membranes.

Fc gamma RII, Fc gamma RIII, and CD18 receptors mediate in part neutrophil activation on a plasma coated expanded polytetrafluoroethylene surface.

BACKGROUND: A biomaterial-induced polymorphonuclear neutrophil (PMN) defect may predispose the implanted vascular graft to infection. PMNs bind, activate, and undergo morphologic changes when exposed to uncoated or plasma coated expanded polytetrafluoroethylene (ePTFE) surfaces. The purpose of this study was to investigate whether the CD18 integrin receptor or the immunoglobulin receptors Fc gamma RII and Fc gamma RIII mediate either PMN binding or activation on ePTFE. METHODS: PMN binding and activation were determined after incubation of these cells on human immunoglobulin (IgG) or fibrinogen coated surfaces and uncoated or plasma coated ePTFE. PMN activation was measured by using the ferricytochrome reduction assay. Binding was determined with chromium 51-labeled PMNs. To block the Fc gamma RII, Fc gamma RIII, and CD18 receptors, PMNs were preincubated with the monoclonal antibodies (mAbs) IV.3, 3G8, and IB4, respectively. Irrelevant isotype matched mAbs were used as control. RESULTS: Monoclonal antibody IB4 inhibited binding of activated PMNs to fibrinogen coated surfaces. Binding to IgG was affected by either mAb IB4 or IV.3, but the greatest degree of inhibition was achieved when mAbs IB4 and IV.3 were used in combination. IgG-induced activation was partially inhibited by mAb IV.3 but was fully inhibited by a combination of mAbs IB4 and IV.3 The mAbs did not affect PMN binding to uncoated or plasma coated ePTFE, nor was PMN activation on the uncoated ePTFE surface inhibited. PMN activation on the plasma coated ePTFE surface was, however, partially inhibited by the combination of mAb IB4 with either mAb IV.3 or 3G8. CONCLUSIONS: A synergistic interaction between the PMN Fc gamma RII receptor and the CD18 integrin receptor accounts for surface bound IgG-induced cell activation. Both receptors also play a role in mediating PMN activation on the plasma-coated ePTFE surface.

Neutrophil activation by expanded polytetrafluoroethylene is dependent on the induction of protein phosphorylation.

BACKGROUND: Polymorphonuclear leukocyte (PMN) activation after interaction with implantable surfaces has been previously reported. The purpose of this study was to examine the mechanism of PMN activation in response to expanded polytetrafluoroethylene (ePTFE). METHODS: To demonstrate PMN activation, the cumulative production of superoxide was measured on uncoated, plasma coated, or albumin coated ePTFE discs. Chromium 51-labeled PMNs were used to measure binding. Cell structure was examined by scanning electron microscopy. RESULTS: By 4 hours, PMN activation on either uncoated or plasma coated ePTFE was approximately 30% of phorbol 12-myristate 13-acetate-induced activation. Albumin inhibited PMN activation by ePTFE. No apparent correlation existed between chromium 51-labeled PMN binding and cell activation on the surfaces. Pretreatment of the cells with the protein kinase inhibitors bisindolylmaleimide or genistein resulted in marked inhibition of superoxide production on the uncoated and plasma coated ePTFE surfaces, whereas binding to these surfaces was not affected. PMNs spread on the uncoated surface and transmigrated into the plasma coated ePTFE surface. These effects of ePTFE on cell structure were inhibited by bisindolylmaleimide and genistein. CONCLUSIONS: ePTFE induced PMN activation, as measured by superoxide production, and changes in cell behavior are dependent on the activation of signaling pathways that involve protein phosphorylation events.

Multiple platelet surface receptors mediate platelet adhesion to surfaces coated with plasma proteins.

The interactions between platelets and plasma proteins previously shown to adhere to biomaterials were evaluated, using monoclonal antibodies (mAbs) against specific platelet surface glycoprotein (GP) receptors. Purified 51Cr-labeled human platelets in plasma-free medium were incubated with each of the following antibodies: mAb 10E5 [anti-GP IIb/IIIa; fibrinogen, von Willebrand factor (vWF), and fibronectin receptor]; mAb 6D1 (anti-GP Ib-IX; vWF receptor); mAb IV.3 (anti-Fc gamma RII; IgG receptor); polyclonal antiserum A108 or mAb BIIG4 (anti-GP Ic-IIa; fibronectin receptor). Antibody-treated platelets were added to microtiter wells coated with fibronectin, fibrinogen, vWF, IgG, vitronectin, albumin, or platelet-poor plasma (PPP). 51Cr-labeled platelet adhesion to matrix proteins was expressed as a percentage of that measured on PPP-coated surface. Platelets adhered to fibrinogen, fibronectin, vWF, or IgG immobilized on polystyrene. Limited binding to either vitronectin or albumin was detected. Binding to fibrinogen and IgG was blocked by mAb 10E5. Binding to IgG was also blocked by mAb IV.3. Binding to fibronectin, reduced in the presence of mAb 10E5, mAb BIIG4, or the polyclonal antiserum A108 alone, was further reduced by combined 10E5 and BIIG4 or 10E5 and A108. Neither mAb 10E5 nor 6D1 alone blocked adhesion to vWF; however, the combination of 10E5 and 6D1 significantly reduced platelet adhesion to this matrix. Finally, platelet adhesion to the plasma-coated surface was reduced by mAbs 10E5 and BIIG4. These results indicate that multiple adhesion receptors can mediate platelet adhesion to matrix proteins immobilized on surfaces.

Tyrosine phosphorylation of pp125FAK in platelets requires coordinated signaling through integrin and agonist receptors.

FAK is a focal adhesion kinase that is phosphorylated on tyrosine in activated platelets. Induction of FAK phosphorylation requires both fibrinogen binding to integrin alpha IIb beta 3 and post-occupancy events during agonist-induced platelet aggregation or platelet spreading on a fibrinogen matrix. To identify the signaling pathways necessary for tyrosine phosphorylation of FAK, we have examined the conditions that stimulate or inhibit this phosphorylation in platelets in which fibrinogen binding to alpha IIb beta 3 and platelet aggregation were induced directly with an anti-beta 3 Fab fragment (anti-LIBS6). Apyrase was added to prevent effects of the endogenous platelet agonist, ADP. Under these conditions, neither fibrinogen binding nor primary platelet aggregation was sufficient to induce FAK phosphorylation, suggesting that a second "costimulatory" event was required. Indeed, when epinephrine was added with fibrinogen and anti-LIBS6, large platelet aggregates formed and FAK phosphorylation occurred. This response was prevented by blockade of cyclooxygenase with indomethacin or thromboxane A2 receptors with SQ 30,741. A stable thromboxane A2 analogue (U46619) could substitute for epinephrine as the costimulus. Epinephrine costimulation of FAK phosphorylation was also prevented by chelation of intracellular Ca2+ with BAPTA or selective inhibition of protein kinase C (PKC) with bisindolylmaleimide, indicating that Ca2+ and PKC are necessary for FAK phosphorylation under these conditions. Epinephrine also promoted FAK phosphorylation and adhesive spreading of apyrase-treated platelets on a fibrinogen matrix. Cytochalasin D, an inhibitor of actin polymerization, blocked FAK phosphorylation under all these conditions. Thus, tyrosine phosphorylation of FAK in platelets requires coordinated signaling through occupied integrin and agonist receptors. These separate pathways may converge to increase free Ca2+ and activate PKC and thus promote the cytoskeletal reorganization required for activation of FAK.

Tyrosine phosphorylation and cytoskeletal reorganization in platelets are triggered by interaction of integrin receptors with their immobilized ligands.

Agonist-induced platelet activation causes fibrinogen binding to integrin alpha IIb beta 3 (glycoprotein (GP) IIb-IIIa) and platelet aggregation. This is associated with the phosphorylation of specific platelet proteins on tyrosine residues. Since fibrinogen immobilized on a solid matrix can bind platelet GP IIb-IIIa without the need for exogenous agonists, we examined whether platelet adhesion to a fibrinogen matrix induces tyrosine phosphorylation. Platelets adhered to fibrinogen in a GP IIb-IIIa-dependent manner and assumed a spread morphology. This change in cell shape was associated with tyrosine phosphorylation of multiple proteins, most prominently two unidentified proteins (101 and 105 kDa) and pp125FAK, a focal adhesion protein-tyrosine kinase. Pretreatment of platelets with prostaglandin I2 to increase cAMP, with cytochalasin D to inhibit actin polymerization, or with ADP scavengers to remove ADP did not affect initial adhesion, but inhibited both platelet spreading and tyrosine phosphorylation of pp125FAK and the 101- and 105-kDa proteins. This suggested that adhesion to fibrinogen caused cytoskeletal reorganization and the local release of ADP from platelet-dense granules, which potentiated the biochemical and morphological responses of the platelets to fibrinogen. Platelet adhesion to a collagen matrix also led to the induction of tyrosine phosphorylation of the 101- and 105-kDa proteins and pp125FAK. In this case, tyrosine phosphorylation was dependent on the interaction of collagen with integrin alpha 2 beta 1 (GP Ia-IIa), and it was independent of both GP IIb-IIIa and ADP. These results indicate that platelet adhesion to fibrinogen or collagen induces signal transduction that is initiated through integrins GP IIb-IIIa and alpha 2 beta 1, respectively. In both cases, tyrosine phosphorylation is accompanied by cytoskeletal reorganization and changes in cell shape. However, different regulatory components may be interposed between each of these integrins and the enzymes that control the level of protein tyrosine phosphorylation.

Integrin-dependent phosphorylation and activation of the protein tyrosine kinase pp125FAK in platelets.

We have investigated mechanisms involved in integrin-mediated signal transduction in platelets by examining integrin-dependent phosphorylation and activation of a newly identified protein tyrosine kinase, pp125FAK (FAK, focal adhesion kinase). This kinase was previously shown to be localized in focal adhesions in fibroblasts, and to be phosphorylated on tyrosine in normal and Src-transformed fibroblasts. We show that thrombin and collagen activation of platelets causes an induction of tyrosine phosphorylation of pp125FAK and that pp125FAK molecules isolated from activated platelets display enhanced levels of phosphorylation in immune-complex kinase assays. pp125FAK was not phosphorylated on tyrosine after thrombin or collagen treatment of Glanzmann's thrombasthenic platelets deficient in the fibrinogen receptor GPIIb-IIIa, or of platelets pretreated with an inhibitory monoclonal antibody to GP IIb-IIIa. Fibrinogen binding to GP IIb-IIIa was not sufficient to induce pp125FAK phosphorylation because pp125FAK was not phosphorylated on tyrosine in thrombin-treated platelets that were not allowed to aggregate. These results indicate that tyrosine phosphorylation of pp125FAK is dependent on platelet aggregation mediated by fibrinogen binding to the integrin receptor GP IIb-IIIa. The induction of tyrosine phosphorylation of pp125FAK was inhibited in thrombin- and collagen-treated platelets preincubated with cytochalasin D, which prevents actin polymerization following activation. Under all of these conditions, there was a strong correlation between the induction of tyrosine phosphorylation of pp125FAK in vivo and stimulation of the phosphorylation of pp125FAK in vitro in immune-complex kinase assays. This study provides the first genetic evidence that tyrosine phosphorylation of pp125FAK is dependent on integrin-mediated events, and demonstrates that there is a strong correlation between tyrosine phosphorylation of pp125FAK in platelets, and the activation of pp125FAK-associated phosphorylating activity in vitro.

Phosphorylation of integrin in differentiating ts-Rous sarcoma virus-infected myogenic cells.

The differentiation of primary myogenic cultures requires the attachment of the cells to an extracellular matrix substrate using an integrin family receptor. These integrin receptors can be phosphorylated on both their alpha and beta chains, and it has been postulated that phosphorylation regulates the receptor function. Quail myogenic clones transformed with ts-LA24A differentiated into mature myotubes following a temperature shift to nonpermissive temperature which inactivates the viral src kinas. Phosphorylation of integrin beta-1 chain and of at least one alpha chain was detected on both serine and tyrosine. An additional alpha chain(s) with a mobility similar to alpha 5 was not phosphorylated at either temperature. Following the induction of differentiation by a temperature shift, there was a marked decrease in integrin phosphorylation of both alpha and beta integrin chains. This decrease was more prominent for serine than for tyrosine, suggesting that src could not be the only kinase involved. The drop in integrin phosphorylation correlated with the initiation of differentiation, suggesting that integrin phosphorylation could be at least part of the mechanism by which myogenic differentiation is blocked by v-src.

Cellular partitioning of beta-1 integrins and their phosphorylated forms is altered after transformation by Rous sarcoma virus or treatment with cytochalasin D.

A sequential extraction procedure of 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS) buffer followed by RIPA or Laemmli sample buffer was developed to define two distinct subpopulations of beta-1 integrins in primary chicken embryo fibroblasts. Extraction of cells in culture revealed an association of adhesion plaque-localized integrin with the CHAPS-insoluble fraction. Phosphorylated integrins were found in both fractions, but the specific phosphorylation was 12-fold higher in the CHAPS insoluble fraction. The phosphorylation was evenly distributed between phosphoserine and phosphotyrosine. Transformation by Rous sarcoma virus caused a redistribution of integrin to rosettes and an increase in total integrin phosphorylation. Treatment with cytochalasin D caused a redistribution of the adhesion plaque-associated integrin into lacelike structures and reduced the level of integrin phosphorylation. These treatments also caused an altered distribution of phosphorylated integrin between the CHAPS soluble and insoluble fractions. These results suggest a role for integrin phosphorylation in the assembly and disassembly of cellular adhesion structures.

Expression and function of chicken integrin beta 1 subunit and its cytoplasmic domain mutants in mouse NIH 3T3 cells.

Chicken integrin beta 1 cDNA and its site-directed mutants were cloned into a mammalian expression vector and introduced into mouse NIH 3T3 cells. Stable transfectants expressing the chicken beta 1 subunit or its site-directed mutants were identified by immunostaining with antibodies specific for the chicken integrin beta 1 subunit. The chicken beta 1 proteins were expressed predominately in the endoplasmic reticulum of transfectants and to a lesser degree in the plasma membrane. Immunoblots and immunoprecipitations, using anti-chicken integrin antibodies, revealed three different sizes of the chicken subunit (90, 95, and 120 kD) and a mouse 140-kD alpha subunit. Immunoprecipitations of the cell surface receptors showed only two peptides, an 120-kD beta 1 and an 140-kD alpha subunit. Antibodies perturbing mouse and chicken integrin-specific cell adhesions were used to demonstrate that the chimeric receptors functioned in adhesion to both laminin and fibronectin. Immunofluorescent staining with antibodies specific for either the chicken or mouse receptors showed that both the wild type and the chimeric receptors localized in focal contacts. Several mutations in the cytoplasmic domain were synthesized and used in the transfection experiments. In one mutant the tyrosine (Tyr 788) in the consensus sequence for phosphorylation was replaced by a phenylalanine. In another the lysine (Lys 757) at the end of the membrane spanning region was replaced by a leucine. Both of these mutants formed dimers with mouse alpha subunits, participated in adhesion, localized in focal contacts, and displayed biological properties indistinguishable from the wild-type transfection. In contrast, mutants containing deletions greater than 5-15 amino acids nearest the carboxyl end in the cytoplasmic domain neither promoted adhesion nor localized in focal contacts. They did, however, form heterodimers that were expressed on the cell surface.

Localization of sodium channel subtypes in adult rat skeletal muscle using channel-specific monoclonal antibodies.

Five monoclonal antibodies specific for the 260 kDa subunit of the rat skeletal muscle sodium channel were used to probe the distribution of this channel in adult muscle. All the antibodies reacted with the surface membrane of fast- and slow-twitch fibers in the rat anterior tibial and soleus muscles. Immunoreactivity was also present in the endplate region; this was significantly more intense than that in the surrounding extrajunctional membrane. At the electron microscopic level, this junctional immunoreactivity could be traced uniformly throughout the secondary folds of the post-synaptic membrane. Three of the monoclonal antibodies (A/B2, F/E4, and I/E3) exhibited an additional distinct immunoreactivity pattern, staining the interior of selected fibers in the anterior tibial muscle that were subsequently identified as slow-twitch fibers. An identical reactivity pattern was observed with most of the soleus muscle fibers. In longitudinal sections of slow fibers examined at the light microscopic level, transversely oriented, regularly spaced doublets of fluorescence were localized at the junction of the A and I bands in each sarcomere. In permeabilized slow fibers exposed to A/B2 and examined at the electron microscopic level, internal reactivity was associated exclusively with the membranes of the T-tubular system. A/B2 also strongly stained a transversely oriented pattern within cardiac muscle fibers exhibiting the characteristics of the T-tubular system in that tissue. We conclude that at least 3 subpopulations of sodium channels are present in adult skeletal muscle: those in the sarcolemma of fast and slow fibers, those in slow-twitch fiber T-tubular membranes, and those in the T-tubular system of fast fibers. The channels in the slow fiber T-system apparently share common epitopes with those in the T-system of cardiac fibers.