Figure 4. Partial inhibition of MAP4K4 kinase activity elicits transformation.

(A) Quantification of MAP4K4 in vitro kinase activity after MAP4K4 was tandem-affinity purified from cells expressing ST or GFP control. (B) Quantification of AI growth after increasing concentrations of the MAP4K4 inhibitor C29. (C) Quantification of AI growth after expression of MAP4K4 WT, MAP4K4 K54R mutant, ST, or GFP in HEK TER cells (Student’s t-test, *p<0.01, **p<0.001, ***p<0.0001, n.s. = not significant).

Figure 4—figure supplement 1. Changes in AI growth and proliferation with inhibition of MAP4K4 kinase activities.

(A) In vitro kinase assay of tandem-affinity purified MAP4K4 (Flag/HA) from HEK TER cells expressing either ST or GFP was performed and MBP was used as a substrate. HA and phospho-MBP were detected by immunoblotting. (B) In vitro MAP4K4 kinase assay from HEK TER cells using increasing concentrations of the MAP4K4 inhibitor (Compound 29). MBP was used as a substrate and phosphorylation determined by phospho-MBP immunoblotting (bottom). Quantification of relative kinase activity (top). (C) Proliferation of HEK TER cells was measured after exposure to compound 29 at the indicated concentrations. The Student’s t-test was performed based on absorbance values measured between 0 uM and 1 uM at five days. (D) Representative immunoblot showing MAP4K4 in vitro kinase assay using tandem-affinity purified wild-type (WT) or kinase-dead mutant (K54R) MAP4K4. (E) Mean MAP4K4 activity of WT relative to kinase-dead mutant (K54R) assessed using in vitro kinase assay (student’s t-test: n.s. = not significant, **p<0.001, ***p<0.0001).