Secondary antibodies for Western blots were donkey anti-mouse HRP and goat anti-rabbit HRP (both from Santa Cruz Biotechnology)

Secondary antibodies for Western blots were donkey anti-mouse HRP and goat anti-rabbit HRP (both from Santa Cruz Biotechnology). NMR analyses indicated that it can insert into the F2 subdomain. We suggest that this insertion determines the topology of the K2 FERM domain, and its deletion may affect the positioning of the membrane-binding functions of the F2 subdomain and the integrin-binding properties of its F3 subdomain. Free C-terminal peptide can still bind to K2 and displace the endogenous K2 NGI-1 C terminus but may not restore the conformation needed for integrin co-activation. Our findings indicate that the extreme C terminus of K2 is essential for integrin co-activation and highlight the importance of an atypical architecture of the K2 FERM domain in regulating integrin activation. or Atlantic salmon. In a lower organism, fermitin 1, transcript variant A (Fit1), the K2 homolog was still 69% identical and 89% conserved compared with the human K2 C-terminal sequence (Fig. 1and and supplemental Fig. S1). We then combined the two sets of mutations that each partially inhibited co-activator activity, K2Y673A and K2679 (referred to subsequently as the double mutant); this combination failed to support co-activator activity of K2 (Fig. 1and 0.001). 0.001). Intermediate flow cytometry data (dot plots and histograms) are shown in supplemental Fig. S2. The importance of the C-terminal region was also noted with another cell line and with a different target integrin. K2 overexpression in RAW 264.7 macrophage-like cells leads to activation of 1 1 integrins as monitored with 9EG7, an mAb specific for the activated conformation NGI-1 of these integrins (41). As originally reported by Moser (22) for kindlin-3/1 integrin, we found that 1 integrin activation by K2 did not require expression of exogenous talin in these cells. The intermediate histograms are shown in supplemental Fig. S2and quantified in Fig. 2are shown in supplemental Fig. S4. The EGFP fluorescence of cells transfected with the chimera, FL K3, and FL K2 were similar, indicating that all constructs were expressed at similar levels. Second, HEL cells were transfected with FL K3 and with a K3 truncated of its last 20 amino acids. When HEL cells were transfected with truncated K3 and stimulated with PMA, their spreading on fibrinogen was not affected; it was comparable with that of EGFP-transfected cells (data not shown). Open in a separate window Figure 4. The C-terminal segment of kindlin-3 is dispensable for K3 functions. (in Fig. 5shows that similar amounts of FL K2 and K2666 were loaded on the gels as detected with an anti-EGFP. Thus, the C-terminal deletion did not prevent co-activation by precluding interaction of K2 with the integrin cytoplasmic tail. In a recent publication (20), we localized an ILK binding site to K2(339C358). K2666 and the K2 double mutant retained their capacities to immunoprecipitate ILK (Fig. 5demonstrates that similar amounts of GST-tagged proteins were subjected to immunoprecipitation. Open in a separate window Figure 5. Effects of the C-terminal segment of kindlin-2 on its association with known binding partners. shows an equal amount of EGFP-tagged K2 in immunoprecipitates. 3 levels in total lysates (shows Coomassie BlueCstained GST and GSTCK2 constructs used for pulldown assays. Although less K2 double mutant was present, it still immunoprecipitated a similar amount of ILK. K2666 also retained the capacity to pull down actin, which binds to K2 via a site in F0 (not shown) (18). Thus, the C-terminal deletion did not lead to NGI-1 global loss of K2 binding functions. A vector for PSGL-1 was modified to replace its natural intracellular region with several K2 C-terminal extensions. We had previously used this approach to express various 3 CT Rabbit Polyclonal to FOXD3 mutants (10, 36); these 3 CT mutants that bound to talin-H or K2 competed with IIb3 in IIb3-CHO cells and inhibited spreading of the cells on fibrinogen, whereas mutants that did not bind either of.