Fig. 3.

MLK3 is required for ERK activation and the proliferation of NF2 tumor cells. Merlin suppresses MLK3 activation of JNK and mitogen activation of ERK. IB, immunoblot. (A) ERK activation in HEI 193 human NF2 schwannoma cells requires MLK3. Human HEI 193 cells were treated with human- or mouse-specific MLK3 siRNA (hsMLK3 or mmMLK3, respectively). Cells were serum-starved (0.5% serum) and then treated with 20% FCS. WCE were immunoblotted, as indicated, with anti-phospho-ERK (p-ERK) and total ERK (to monitor ERK activation), as well as anti-MLK3. (B) MLK3 is required for HEI 193 cell proliferation. HEI 193 cells were treated with hsMLK3 or mmMLK3 and serum-starved as in A. Cells (2.5 × 10⁴) were plated onto triplicate wells and treated with vehicle or serum (20%) as indicated. (Upper) Cell proliferation was quantitated, by hemocytometer, at the indicated times (bars indicate mean ± SD, n = 3). (Lower) In parallel, cell extracts were probed with anti-MLK3 to monitor MLK3 RNAi. For each immunoblot (either anti-MLK3 or anti-ERK), samples from the different sets of cells were run on the same gel. Different cell sample immunoblots are shown separately to improve legibility. (C) Merlin suppresses MLK3 activation of coexpressed JNK. HEK 293 cells were transfected with Myc-merlin, FLAG-MLK3, or GST-JNK as indicated. WCE were probed with the cognate Abs as indicated. JNK was isolated on glutathione agarose and assayed in vitro for phosphorylation of c-Jun (1–135). Autophosphorylation of the GST-JNK, as well as phosphorylation of the c-Jun, is apparent in the figure (³²P-JNK or c-Jun, respectively). KA, kinase assay. (D) Merlin suppresses mitogen activation of ERK. RT4 NF2.17 rat schwannoma cells expressing a TetON NF2 cDNA construct were treated with doxycycline (Dox) to induce merlin expression. Cells then were serum-starved and treated with serum as in B. WCE were assayed for in situ ERK activation as in A.