Figure 4.

Effect of pITAM and integrin β₃ tail on Syk wildtype activity. A, kinetic measurement of Basal Syk in the absence of peptide (open diamonds), in presence of 30 μm integrin β₃ cytoplasmic tail peptide (diamonds), 10 μm pITAM peptide (triangles), and a combination of both (open circles). The activity is expressed in terms of normalized product formation in time (min); basal Syk was used at concentration of 17 nm. Error bars correspond to the standard deviation calculated from six different experiments. B, ANOVA of the 5-min time point in the experiment shown in panel A. The data indicate that the presence of soluble pITAM activates the protein reducing the lag-phase of basal Syk. p value for basal Syk versus Syk + pITAM and basal Syk versus Syk + pITAM + integrin were both <0.0001. Comparing the basal Syk versus Syk + integrin and the Syk + pITAM versus Syk + integrin + pITAM with the ANOVA test resulted in nonsignificant (ns) p values thus showing that the soluble integrin β₃ cytoplasmic tail peptide had no effect. C, kinetic measurement of active Syk in the absence of peptide (open diamonds), in the presence of 30 μm integrin β₃ cytoplasmic tail peptide (diamonds), 10 μm pITAM peptide (triangles), and a combination of both (open circles). The activity is expressed in terms of normalized product formation in time (min). Active Syk was used at 17 nm. D, the ANOVA of the 5-min time point in panel C; it resulted in a nonsignificant (ns) p value suggesting that the pre-activated Syk is not modulated by the presence of either of two peptides. E and F, Lineweaver-Burk plots showing the competitive inhibition toward SOX peptide (E) and uncompetitive inhibition toward ATP (F) by the integrin β₃ cytoplasmic tail. In each case 1/v_i was plotted against 1/[SOX] (E) or 1/[ATP] (F). The experiments were carried out using active Syk at 4 nm.