FIG. 7.

MEKK3 inhibits cyclin D1 expression and G₁ progression via p38. (A) p38 activity is necessary for inhibition of cyclin D1 induction by MEKK3-ER. NIH 3T3 [MEKK3-ER] cells were kept without serum for 4 days, preincubated for 30 min with or without 10 μM SB203580 (SB), incubated in the presence of E2 for another 2 h, and then stimulated with 10% FCS in the presence or absence of SB203580 and/or E2 for 12 h. Equal amounts of protein (100 μg) were separated by SDS-PAGE and then sequentially immunoblotted with an anti-cyclin D1 antibody (cycD1), followed by an anti-estrogen receptor antibody to detect the stably expressed fusion protein (MEKK3-ER). (B) MEKK3-induced expression of MKP-1 is largely dependent on p38 activity. NIH 3T3 [MEKK3-ER] cells were rendered quiescent by being cultured for 2 days without serum and then were stimulated with E2 for various times, as indicated, without serum. Before addition of E2, some cells were preincubated with 10 μM SB203580 (SB) as well. MKP-1 expression was detected by Western analysis (100 μg/lane) with anti-Pac1 antibody, which cross-reacts with MKP-1. (C) Inhibition of G₁/S progression by wild-type MEKK3 but relief of inhibition in the presence of MKK6[KN]. NIH 3T3 cells were transfected with GFP expression plasmid alone or together with 0.1 μg of an expression plasmid expressing full-length MEKK3 (MEKK3-F), in the absence or presence of 0.3 μg of an expression vector expressing MKK6[KN], as indicated. After transfection, the cells were incubated overnight, serum starved for 30 h, and then stimulated with 10% FCS in the presence of BrdU. The bars represent percentages of BrdU-positive cells in the transfected, GFP-positive population (±mean standard deviation of duplicate sets). More than 100 cells were counted for each sample, and similar results were obtained in two independent experiments. (D) Wild-type MEKK3 and a constitutively active form of MKK6 inhibit G₁/S progression to similar extents. NIH 3T3 cells were transfected with 0.1 μg of GFP expression plasmid and 0.2 μg of an expression vector encoding MEKK3-F or MKK6[2E], a constitutively active form of MKK6. The cells were treated and evaluated as for panel C. In all experiments, total amounts of transfected DNA were kept constant with the addition of empty vectors as needed.