Fig 3.

PikA phosphorylates PI to generate PI3P. (A) Biochemical assays for the kinase activity of PikA. Purified His₆-PikA was incubated in reactions containing the indicated PI substrates, and the production of ADP was measured using the ADP-Glo Kinase Assay. RLU, relative luminescence units. (B) The His-Asn-Asp motif is critical for the kinase activity of PikA. His6-PikA or its mutants were incubated with PI or PI5P, and the activity was determined as described in A. (C) Determination of PikA-catalyzed production of a panel of PI substrates by TLC assays. His₆-PikA or its inactive mutant His₆-PikA_H171A was incubated with the indicated diC8-Bodipy-FL-PI species at 25°C for 14 h, and the products were separated by TLC. Images of the plates were acquired using the Goodsee-5 TLC imager. (D) Mutations in H171, D194 eliminate the monophosphorylation of PI catalyzed by PikA. Experiments were performed with the same procedure as described in panel C. (E) Dephosphorylation of PikA-produced products by PI phosphatases. The products produced by PikA were incubated with the indicated phosphatases at 37°C for 15 min, and the products were detected by TLC assays. (F) Suppression of PikA-induced cell rounding by MTM. HeLa cells were transfected to express the indicated protein combination for 14 h, and cell morphology was analyzed using a fluorescence microscope for image acquisition (left) and quantitation (right). Bar, 10 µm. Results (mean ± s.e.) shown were from three independent experiments, each done in triplicate. At least 300 cells were scored for each sample (right). ***, P < 0.001.