FIG. 4.

The MLK family is upstream of MKK4 and -7 and c-Jun in the apoptotic pathway. (a) A dominant-negative form of c-Jun suppresses death induced by overexpression of Rac1 V12 and Cdc42 V12 as well as by the MLK family. pCMS.EGFP as well as DNA-encoding MLK family members, Rac1 V12, and Cdc42 V12 (all in the pCMS.EGFP vector) were cotransfected with either pCMV vector as a control or the dominant-negative (d/n) form of c-Jun (pCMV-TAM67 c-Jun) as described in Materials and Methods. Three days after transfection, cell numbers were determined as described in Materials and Methods. Cell numbers in cultures transfected with pCMS.EGFP-pCDNA3 and pCMS.EGFP-pCMV were defined as 100% survival, and cell numbers in the other transfected cultures were normalized accordingly. (b) Dominant-negative (d/n) forms of MKK4 and MKK7 suppress death induced by MLK expression. pCMS.EGFP and constructs encoding different MLK family members were cotransfected with either pCDNA3 vector or dominant-negative MKK4 and/or dominant-negative MKK7 (both in pCDNA3) as described in Materials and Methods. In those cotransfected with both dominant-negative MKK4 and dominant-negative MKK7, the amounts of dominant-negative MKK4 and dominant-negative MKK7 DNA were equal and the total amounts of both together (1.25 μg/well) were equal to five times the amount of DNA (0.25 μg/well) with which they were cotransfected. Three days after transfection, cell numbers were counted as described in Materials and Methods. Numbers of cells transfected with pCMS.EGFP-pCDNA3 were defined as 100%, and survival in cultures with other transfections were normalized to these values. The values are the means from three independent experiments plus SEM.