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Fig. 3. (A) An in vitro kinase assay using purified GST, GST-GEF-H1 (763-921) and GST-Maguin-like protein as substrates for PAK4. The negative control autophosphorylation reaction is the reaction mix without substrate. A concentration gradient of reaction substrate is indicated by the triangles. (B) In vivo phosphorylation of GEF-H1 by PAK4 is demonstrated in western blots of co-transfected lysates probed with phosphospecific antisera (lower panel). Anti-HA serum detects the level of GEF-H1 in these lysates (upper panel). Increased phosphorylation is seen in the presence of PAK4 and PAK4 S474E (lanes 2-3 compared with lane 1). Basal level activity is further reduced by kinase-inactive PAK4 K350,351A. (C) PAK4 alleles, including wild-type, kinase domain and H19,22L (CRIB mutant), co-expressed with GEF-H1 induce phosphorylation of GEF-H1 (lanes 3-5) whereas Cdc42 and vector retain basal activity (lanes 1-2), middle panel. Upper and lower panels are probed with tag-specific serum to detect the levels of GEF-H1 and PAK4 alleles present in these lysates.





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