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Results: 4

1.
FIGURE 4.

FIGURE 4. From: Tyrosine Phosphorylation of Integrin ?3 Regulates Kindlin-2 Binding and Integrin Activation.

Phosphorylation of Tyr795 in the β1 peptide inhibits kindlin-2 binding. A, the amino acid sequence of the β1 CT is shown. B, the inhibitory effects of the indicated β1 peptides on kindlin-2 and β3 CT interaction were evaluated in pulldown assays, and kindlin-2 binding was evaluated by Western blotting. IB, immunoblot; β1C-pep-phos, Tyr795-phosphorylated β1 CT C-terminal peptide.

Kamila Bledzka, et al. J Biol Chem. 2010 October 1;285(40):30370-30374.
2.
FIGURE 2.

FIGURE 2. From: Tyrosine Phosphorylation of Integrin ?3 Regulates Kindlin-2 Binding and Integrin Activation.

Phosphorylation of the NxxY759 motif in the β3 CT peptides disables its ability to block interaction of kindlin-2 and the β3 CT. A, the amino acid sequences of the β3 CT C-terminal peptide containing the NITY759 motif and a modified peptide with Tyr759 phosphorylation (β3C-pep-phos) are shown. B, kindlin-2 and β3 CT interaction was evaluated by pulldown assays in the presence of the indicated peptides. IB, immunoblot. C, kindlin-2 or a mixture of kindlin-2 and the indicated peptide at a 1:100 molar ratio was passed over an SA5 biosensor chip coated with biotinylated β3 CT peptides, and the binding curves were recorded over time. To block nonspecific binding of the peptide to the chips, 0.1% BSA was included in the running buffer.

Kamila Bledzka, et al. J Biol Chem. 2010 October 1;285(40):30370-30374.
3.
FIGURE 3.

FIGURE 3. From: Tyrosine Phosphorylation of Integrin ?3 Regulates Kindlin-2 Binding and Integrin Activation.

Phosphorylation of the NxxY759 motif in the β3 CT peptides dampens direct binding to kindlin-2. Biotinylated β3 CT peptides with or without modification at Tyr759 are displayed (A). Phos, phosphorylated. The WT biotin-β3 peptide (B), its phosphorylated form (C), and other mutants (D) were immobilized on the streptavidin sensor chips. Various concentrations of kindlin-2 protein were injected over the chips in HEPES buffer (0.1% BSA) at a flow rate of 25 μl/min. The binding signals were recorded, and the results are expressed in RU. The RU values of the chips prior to introduction of kindlin-2 were similar (∼200 RU), indicating that the coating densities of the peptides were similar.

Kamila Bledzka, et al. J Biol Chem. 2010 October 1;285(40):30370-30374.
4.
FIGURE 1.

FIGURE 1. From: Tyrosine Phosphorylation of Integrin ?3 Regulates Kindlin-2 Binding and Integrin Activation.

Phosphomimetic mutation of the NxxY759 motif in the β3 CT perturbs kindlin-2 binding and suppresses kindlin-2-mediated integrin αIIbβ3 activation. A, kindlin-2 and talin-H can interact with the integrin β3 CT. The kindlin-2-binding site is localized in the membrane-distal NxxY motif, NITY759, but talin-H binding depends on the membrane-proximal NxxY motif, NPLY747. B, GST-fused β3 CT, with or without the indicated mutations, was used in pulldown assays to precipitate kindlin-2 from cell lysates. The bound kindlin-2 was measured by Western blotting. IB, immunoblot. C, a representative FACS histogram developed with the activation-specific PAC1 mAb is shown together with the median fluorescence intensities (MFI) demonstrating that total αIIbβ3 expression, measured with a mAb unaffected by the activation status of the receptor, is unaffected. D, wild-type αIIbβ3 and its mutants were coexpressed with talin-H and kindlin-2 or their empty vectors in CHO cells by transient transfection, and the extent of αIIbβ3 activation was analyzed by PAC1 binding of the DsRed and EGFP double-positive cells, correcting for any differences in total αIIbβ3 expression. Results are means ± S.D. (n = 3). **, p < 0.01.

Kamila Bledzka, et al. J Biol Chem. 2010 October 1;285(40):30370-30374.

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