Figure 2.

Cell cycle stimulation of MLK3's phosphorylation state and kinase activity in the absence of JNK activation. HeLa cells were synchronized at G₁/S by a double-thymidine block (0) and then released into media lacking thymidine. Samples were collected at subsequent times for 14 h and analyzed as described. (A) Flow cytometric quantification of DNA content per cell (x-axis, propidium iodide stain) vs. cell number (y-axis). Distribution of DNA content of cells from an asynchronous culture is also shown (Asynch). Twenty micrograms of each sample was separated by SDS-PAGE and subjected to immunoblot analysis using anti-MLK3 (B) or antiphosphorylated histone H3 (D). (C) Full-length immunoblot of MLK3 from 0- and 10-h samples. Molecular weight markers are indicated on the left. (E) Anticyclin B1 immune precipitation/kinase assay analysis (100 μg/sample) using histone H1 as substrate. (F) Immunoblot of MLK3 in G1/S (0 h) and G2/M (10 h) samples that were incubated with λ-phosphatase in the presence or absence of the phosphatase inhibitor, EDTA, or were left untreated. (G, upper left panel) MLK3 kinase activity toward myelin basic protein (MBP) was determined for G₁/S (0 h) and G₂/M (9 h) cell extracts after immunoprecipitation with anti-MLK3 (I) or with nonimmune (N) antibodies. (G, lower left panel) Average values of MLK3 immune precipitation/kinase assays from six different synchronous cultures collected at G₁/S (0 h) and G2/M (9, 10, 11 or 12 h, depending on culture). Shown is the average fold stimulation of activity from G2/M samples (2.4 times) relative to G₁/S (set at 1) ± the SD (0.67). (G, right panels) Immunoblots of MLK3 and actin in G₁/S and G₂/M samples before (Tot) and after (Post IP) immunoprecipitation with anti-MLK3 (I) or with nonimmune (N) antibodies. (H) Immunoblot of samples from (A) reacted with anti-JNK antibodies. Twenty micrograms of a sample collected from an asynchronous HeLa culture treated with anisomycin for 20 min was also included (Anis). (I) Anti-JNK immune precipitation/kinase assay analysis (100 μg/sample) of the samples in (H) using GST-N-Jun as substrate. As part of the JNK assays, the anisomycin-treated sample also was subjected to immune precipitation with the anti-JNK antibody (I) or with a nonimmune control antibody (NI) followed by incubation in kinase assay buffer. Products of the kinase reactions (E and H) were separated by SDS-PAGE and substrate incorporation of ³²P was detected by phosphorimager exposure.