The Cytoplasmic Domain of the Platelet Glycoprotein Ibα Is Phosphorylated at Serine 609

Bodnar, Richard J.; Gu, Minghong; Li, Zhenyu; Englund, Graham D.; Du, Xiaoping

doi:10.1074/jbc.274.47.33474

Cited by 67 publications

(68 citation statements)

References 42 publications

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“…Antibodies-To generate anti-peptide antibodies, synthetic peptides conjugated to keyhole limpet hemocyanin were used to immunize rabbits (24). The antibody pY759 was generated using the peptide CTpYRGT with a linker cystein and a 5-residue sequence corresponding to tyrosine-phosphorylated ␤ 3 C terminus.…”

Section: Methodsmentioning

confidence: 99%

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

Flevaris

Stojanović

et al. 2006

Journal of Biological Chemistry

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Outside-in signaling of ␤ 3 integrins induces and requires phosphorylation at tyrosine 747 (Tyr 747 ) and tyrosine 759 (Tyr 759 ) of the ␤ 3 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 ␤ 3 cytoplasmic domain. Here we show that ␤ 3 tyrosine phosphorylation inhibits calpain cleavage. Mutating both tyrosines to phenylalanine sensitizes ␤ 3 to calpain cleavage. Furthermore, phosphorylation at Tyr 747 and Tyr 759 of ␤ 3 in the focal adhesion sites and the leading edge of spreading platelets was differentially regulated. Selective dephosphorylation of Tyr 759 is associated with calpain cleavage at Tyr 759 . Thus, one mechanism by which tyrosine phosphorylation promotes integrin signaling and cell spreading is its inhibition of calpain cleavage of the ␤ 3 cytoplasmic domain.Integrins mediate cell adhesion and transduce signals that are critical in the dynamic regulation of cell adhesion, spreading, migration, and proliferation (1, 2). Integrin signaling is a two-way process exemplified by inside-out and outside-in signaling of the platelet integrin, ␣ IIb ␤ 3 . Inside-out signaling is believed to be transduced by talin binding to the cytoplasmic domain of ␣ IIb ␤ 3 (3-7), and consequent conformational changes (5, 6), which propagate to the ligand binding domain of ␣ IIb ␤ 3 , activating ligand binding function (8,9). Ligand binding to ␣ IIb ␤ 3 not only forms adhesive bond but also induces outside-in signaling, leading to cell spreading, secretion, stabilization of platelet adhesion, and amplification of platelet aggregation (10, 11).The cytoplasmic domain of ␤ 3 is critical in bidirectional signaling (12-15). Inside-out signaling requires the membrane proximal region and the two NXXY motifs in the ␤ 3 cytoplasmic domain (3)(4)(5)(6)(15)(16)(17)(18)(19). Outside-in signaling requires the intact cytoplasmic domain of ␤ 3 (15) and also requires tyrosine phosphorylation in NXXY motifs (20,21). However, the mechanism responsible for the role of tyrosine phosphorylation of ␤ 3 in outside-in signaling is unclear. On the other hand, the cytoplasmic domain of ␤ 3 is cleaved by the calcium-dependent proteases (calpain) at sites flanking two NXXY motifs, preferentially at the C-terminal side of Tyr 759 (15,22,23). A consequence of calpain cleavage of ␤ 3 at Tyr 759 is the inhibition of ␤ 3 -dependent cell spreading, which is an outside-in signaling event (15). In studying the relationship between these two seemingly unrelated ␤ 3 modifications that regulate the function of the cytoplasmic domain of ␤ 3 , we found that tyrosine phosphorylation in ␤ 3 cytoplasmic domain inhibits cleavage of ␤ 3 by calpain. Since calpain cleavage negatively regulates outside-in signaling-mediated cell spreading, our finding provides a mechanism by which tyrosine phosphorylation of ␤ 3 promotes integrin outside-in signaling. EXPERIMENTAL PROCEDURESPeptides-Peptides were synthesized by Protein Chemistry Laboratory, Universit...

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Section: Methodsmentioning

confidence: 99%

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

Flevaris

Stojanović

et al. 2006

Journal of Biological Chemistry

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“…. ) (12,15). Immediately N-terminal to the 14-3-3x-binding site of GPIbb is a positively-charged, membrane-proximal sequence that binds calmodulin (24).…”

Section: Gpib-ix-vmentioning

confidence: 99%

Platelet Interactions in Thrombosis

et al. 2004

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SummaryPatho/physiological platelet aggregate (thrombus) formation is initiated by engagement of platelet surface receptors, glycoprotein (GP)Ib-IX-V and GPVI that bind von Willebrand factor or collagen. Although beneficial in response to vascular injury by preventing blood loss (haemostasis), platelet aggregation in a sclerotic coronary artery or other diseased blood vessel (thrombosis) can cause thrombotic diseases like heart attack and stroke. At the molecular level, ligand interactions with GPIb-IX-V or GPVI trigger signalling responses, including elevation of cytosolic Ca 2 + , dissociation of calmodulin from their cytoplasmic domains, cytoskeletal actin-filament rearrangements, activation of src-family kinases or PI 3-kinase, and 'inside-out' activation of the integrin, aIIbb3 (GPIIb-IIIa), that binds von Willebrand factor or fibrinogen and mediates platelet aggregation. Furthermore, emerging evidence supports a topographical coassociation of these receptors of the leucine-rich repeat family (GPIb-IX-V) and immunoglobulin superfamily (GPVI) in an adhesive cluster or 'adhesosome'. This arrangement may underlie common mechanisms of initiating thrombus formation in haemostasis or thrombotic disease. IUBMB Life, 56: 13-18, 2004

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“…Consensus mode I and mode II motifs and the reported C-terminal mode III binding sequences are shown. S/T: phosphorylated serine or threonine required for 14-3-3 binding, : aromatic residue, Reference#1: (Bodnar et al, 1999) , #2: (Sliva et al, 2000), #3 and #4: (O'Kelly et al, 2002):, #5 and #6: , #7: (Rong et al, 2007), #8: (Wurtele et al, 2003), #9: (Ganguly et al, 2005), #10: (Fujita et al, 2003). (Yaffe et al, 1997) and by random C-terminal peptide selection in a cell-based genetic screen .…”

Section: -3-3 Proteins and 14-3-3 Binding Sitesmentioning

confidence: 99%