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Figure 11


Fig. 11. L-MLCK N-terminal sequences reduce the rate of cell spreading and bind the SV N-terminus. (A) Diagram of L-MLCK, S-MLCK and mutants. Catalytic core (dark gray); immunoglobulin-like domains (Ig, stippled); Asp-X-Lysine sequences (DXR, black); and location (*) of the kinase-inactivating (KD) point mutation are shown. (B) EGFP-tagged fusion proteins containing N-terminal L-MLCK sequences (Poperechnaya et al., 2000) reduce the rate of cell spreading. N-terminal sequences unique to L-MLCK without (Ig) or with (Ig+5DXRs) all five of the DXRXXL actin-binding motifs reduce the rate of spreading by ~50%; the 5DXR motifs alone slow cell spreading by ~30%. Histogram of the percentages of spread COS7 cells transfected for 16 hours with EGFP as a control or EGFP-tagged avian L-MLCK sequences, as indicated. Means±s.e.m.; 100 cells/experiment, n=6; *P<0.05; ***P<0.001 versus EGFP-only control. (C) The L-MLCK Ig domains, but not the 5DXRs, bind directly to SV1-174. GST-tagged SV1-174 (lanes 1-6) or GST only (lanes 7-12) were pre-bound to glutathione-agarose beads and incubated with purified recombinant 6xHis-tagged Ig domains (upper panel) or 6xHis-tagged 5DXRs (lower panel). Void volumes (V, lanes 1, 12), washes with the indicated concentrations of NaCl (lanes 2-5, 8-11) and glutathione eluates (E, lanes 6,7) were analyzed on immunoblots stained with anti-6xHis antibodies.





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