FIG 3.

T4519 is a secreted serine/threonine protein kinase. (A) SDS-PAGE (left) and Western blot (right) of purified recombinant 6His-tagged T4519. (B and C) In vitro kinase assays. Full-length (B) or truncated (C) purified rT4519 proteins (2 μg) or active ERK immunoprecipitated from LPS-stimulated THP-1 cells was incubated with the substrate (MBP; 5 μg) for 1 h. (D) Recombinant proteins (2 μg) were incubated with the substrate (MBP; 5 μg) for 1 h in the presence or absence of ATP (10 mM) or H7-dihydrochloride (1 μM). Kinase activities were monitored by immunoblotting for phosphorylation of MBP and autophosphorylation of T4519. Results of one of two independent experiments are shown. (E) PMA-differentiated THP-1 cells were infected with strain Ty2 or a mutant as indicated and were subjected to a gentamicin protection assay. Some of the mutant strains expressed the empty TEM1 vector (pCX340) or a TEM1 fusion protein. Cells were cultured as described for Fig. 1 for 6 h, followed by loading with CCF2-AM (1 μM), and were analyzed by confocal microscopy after excitation at 410 nm. Emission was captured at 510 nm (green) and 450 nm (blue). Results of one of two independent experiments are shown.