scholarly journals SRC-dependent outside-in signalling is a key step in the process of autoregulation of beta2 integrins in polymorphonuclear cells

2004 ◽  
Vol 380 (1) ◽  
pp. 57-65 ◽  
Author(s):  
Paola PICCARDONI ◽  
Stefano MANARINI ◽  
Lorenzo FEDERICO ◽  
Zsuzsa BAGOLY ◽  
Romina PECCE ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Actin Polymerization ◽  
Protein Tyrosine Kinase ◽  
Polymorphonuclear Cells ◽  
High Avidity ◽  
Β2 Integrin ◽  
High Affinity ◽  
Tyrosine Kinase 2 ◽  
Oncogenic Protein ◽  
Β2 Integrins

In human PMN (polymorphonuclear cells), challenged by P-selectin, the β2-integrin Mac-1 (macrophage antigen-1) promoted the activation of the SRC (cellular homologue of Rous sarcoma virus oncogenic protein) family members HCK (haematopoietic cell kinase) and LYN (an SRC family protein tyrosine kinase) and phosphorylation of a P-110 (110 kDa protein). SRC kinase activity in turn was necessary for macrophage antigen-1-mediated adhesion [Piccardoni, Sideri, Manarini, Piccoli, Martelli, de Gaetano, Cerletti and Evangelista (2001) Blood 98, 108–116]. This suggested that an SRC-dependent outside-in signalling strengthens the β2-integrin interaction with the ligand. To support this hypothesis further, in the present study, we used the monoclonal antibody KIM127 or manganese to lock β2 integrins in a high-affinity state, and homotypic PMN adhesion was analysed to monitor β2-integrin adhesive function. KIM127 or manganese induced PMN homotypic adhesion and P-110 phosphorylation. Both these processes were abolished by blocking antibodies against the common β2 chain, by a combination of antibodies against αL and αM or by inhibitors of SRC activity. Confocal microscopy showed that activation epitopes were expressed by β2 integrins co-localized with patches of F-actin at the adhesion sites. Blockade of SRC kinases or of actin polymerization prevented clustering of activated integrins as well as F-actin accumulation. FACS analysis showed that SRC inhibitors modified neither basal nor manganese-induced KIM127 binding. An SRC-dependent outside-in signalling initiated by β2 integrins was also required for adhesion triggered by interleukin-8. These results confirm the hypothesis that an SRC-dependent outside-in signalling triggered by high affinity and ligand binding is necessary to stabilize β2-integrin-mediated adhesion. Allowing clustering of activated integrins, SRC might link the high-affinity with the high-avidity state. Proline-rich tyrosine kinase-2 appears to be involved in this process.

scholarly journals β2 Integrin-dependent tyrosine phosphorylation of proline-rich tyrosine kinase 2 in platelet-activating factor-activated eosinophils

2000 ◽  
Vol 49 (4) ◽  
pp. 245-252
Author(s):  
Hiroshi Ohashi ◽  
Masao Takei ◽  
Hirohito Kita ◽  
Gerald J Gleich ◽  
Isao Serizawa ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Platelet Activating Factor ◽  
Β2 Integrin ◽  
Tyrosine Kinase 2

scholarly journals Regulation of the formation of osteoclastic actin rings by proline-rich tyrosine kinase 2 interacting with gelsolin

2003 ◽  
Vol 160 (4) ◽  
pp. 565-575 ◽  
Author(s):  
Qiang Wang ◽  
Yi Xie ◽  
Quan-Sheng Du ◽  
Xiao-Jun Wu ◽  
Xu Feng ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Tyrosine Kinase ◽  
Actin Polymerization ◽  
Actin Binding ◽  
Cell Periphery ◽  
Cytoskeletal Organization ◽  
Capping Protein ◽  
Tyrosine Kinase 2 ◽  
Multiple Cells ◽  
Actin Rings

Osteoclast activation is important for bone remodeling and is altered in multiple bone disorders. This process requires cell adhesion and extensive actin cytoskeletal reorganization. Proline-rich tyrosine kinase 2 (PYK2), a major cell adhesion–activated tyrosine kinase in osteoclasts, plays an important role in regulating this event. The mechanisms by which PYK2 regulates actin cytoskeletal organization and osteoclastic activation remain largely unknown. In this paper, we provide evidence that PYK2 directly interacts with gelsolin, an actin binding, severing, and capping protein essential for osteoclastic actin cytoskeletal organization. The interaction is mediated via the focal adhesion–targeting domain of PYK2 and an LD motif in gelsolin's COOH terminus. PYK2 phosphorylates gelsolin at tyrosine residues and regulates gelsolin bioactivity, including decreasing gelsolin binding to actin monomer and increasing gelsolin binding to phosphatidylinositol lipids. In addition, PYK2 increases actin polymerization at the fibroblastic cell periphery. Finally, PYK2 interacts with gelsolin in osteoclasts, where PYK2 activation is required for the formation of actin rings. Together, our results suggest that PYK2 is a regulator of gelsolin, revealing a novel PYK2–gelsolin pathway in regulating actin cytoskeletal organization in multiple cells, including osteoclasts.

scholarly journals Inhibition of Osteoclast Function by Adenovirus Expressing Antisense Protein-tyrosine Kinase 2

2000 ◽  
Vol 276 (10) ◽  
pp. 7484-7492 ◽  
Author(s):  
Le T. Duong ◽  
Ichiro Nakamura ◽  
Päivi T. Lakkakorpi ◽  
Lorraine Lipfert ◽  
Andrew J. Bett ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Protein Tyrosine Kinase ◽  
Protein Tyrosine ◽  
Tyrosine Kinase 2 ◽  
Osteoclast Function ◽  
Protein Tyrosine Kinase 2

scholarly journals Transfection of Syk protein tyrosine kinase reconstitutes high affinity IgE receptor-mediated degranulation in a Syk-negative variant of rat basophilic leukemia RBL-2H3 cells.

1996 ◽  
Vol 184 (1) ◽  
pp. 71-79 ◽  
Author(s):  
J Zhang ◽  
E H Berenstein ◽  
R L Evans ◽  
R P Siraganian
Keyword(s):  
Tyrosine Kinase ◽  
Histamine Release ◽  
Phospholipase C ◽  
Tyrosine Phosphorylation ◽  
Protein Tyrosine Kinase ◽  
Immunoglobulin E ◽  
Critical Role ◽  
Protein Tyrosine ◽  
High Affinity ◽  
Phospholipase C Gamma

Aggregation of the high affinity receptor for immunoglobulin E (Fc epsilon RI) on mast cells results in rapid tyrosine phosphorylation and activation of Syk, a cytoplasmic protein tyrosine kinase. To examine the role of Syk in the Fc epsilon RI signaling pathway, we identified a variant of RBL-2H3 cells that has no detectable Syk by immunoblotting and by in vitro kinase reactions. In these Syk-deficient TB1A2 cells, aggregation of Fc epsilon RI induced no histamine release and no detectable increase in total cellular protein tyrosine phosphorylation. However, stimulation of these cells with the calcium ionophore did induce degranulation. Fc epsilon RI aggregation induced tyrosine phosphorylation of the beta and gamma subunits of the receptor, but no increase in the tyrosine phosphorylation of phospholipase C-gamma 1 and phospholipase C-gamma 2 and no detectable increase in intracellular free Ca2+ concentration. By transfection, cloned lines were established with stable expression of Syk. In these reconstituted cells, Fc epsilon RI aggregation induced tyrosine phosphorylation of phospholipase C-gamma 1 and phospholipase C-gamma 2, an increase in intracellular free Ca2+ and histamine release. These results demonstrate that Syk plays a critical role in the early Fc epsilon RI-mediated signaling events. It further demonstrates that Syk activation occurs downstream of receptor phosphorylation, but upstream of most of the Fc epsilon RI-mediated protein tyrosine phosphorylations.

scholarly journals Src-family kinases mediate an outside-in signal necessary for β2 integrins to achieve full activation and sustain firm adhesion of polymorphonuclear leucocytes tethered on E-selectin

2006 ◽  
Vol 396 (1) ◽  
pp. 89-98 ◽  
Author(s):  
Licia Totani ◽  
Antonio Piccoli ◽  
Stefano Manarini ◽  
Lorenzo Federico ◽  
Romina Pecce ◽  
...  
Keyword(s):  
Kinase Inhibitors ◽  
Chinese Hamster Ovary Cells ◽  
Src Family Kinases ◽  
Cell Suspensions ◽  
Polymorphonuclear Cells ◽  
Polymorphonuclear Leucocytes ◽  
Dependent Manner ◽  
Src Family Kinase ◽  
Β2 Integrin ◽  
Β2 Integrins

In cell suspensions subjected to high-shear rotatory motion, human PMN (polymorphonuclear cells) adhered to E-selectin-expressing CHO (Chinese-hamster ovary) cells (CHO-E), and formed homotypic aggregates when challenged by E-selectin–IgG fusion protein, by a mechanism that involved β2 integrins. Both heterotypic and homotypic PMN adhesion was accompanied by tyrosine phosphorylation of a 110 kDa protein (P110). This event was prevented by blocking anti-(β2 integrin) antibodies and by inhibitors of Src-family kinases, suggesting that it was part of an ‘outside-in’ signalling that was initiated by integrin engagement. Interestingly, Src-family kinase inhibitors prevented β2-integrin-mediated (i) homotypic PMN adhesion triggered by E-selectin–IgG, (ii) heterotypic CHO-E/PMN adhesion in mixed-cell suspensions, and (iii) firm adhesion of PMN to CHO-E monolayers under physiological flow. Similarly to PMN treated with Src-family kinase inhibitors, PMN from hck−/−fgr−/− and hck−/−fgr−/−lyn−/− mice showed significant impairment of β2-integrin-mediated adhesion to CHO-E. Moreover, the expression of β2 integrin activation epitopes at the sites of cell–cell contact in CHO-E/PMN conjugates was abolished by Src-family kinase inhibitors. One component of P110 was identified as the FAK (focal adhesion kinase) Pyk2 (proline-rich tyrosine kinase 2), which was phosphorylated in a β2 integrin- and Src-family-kinase-dependent manner. Thus, Src-family kinases, and perhaps Pyk2, mediate a signal necessary for β2 integrin function in PMN tethered by E-selectin.

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