Transfection of constitutively active mitogen-activated protein/extracellular signal-regulated kinase kinase confers tumorigenic and metastatic potentials to NIH3T3 cells

Cellular growth and differentiation are controlled by multiple extracellula r signals, many of which activate extracellular signal-regulated kinase (ER K)/mitogen-activated protein (MAP) kinases, Components of the MAP kinase pa thways also cause oncogenic transformation in their constitutively active f orms. Moreover, expression of activated ms can confer metastatic potential upon some cells. Activation of MAP kinases requires phosphorylation of both Thr and Tyr in the catalytic domain by a family of dual-specificity kinase s, called MEKs (MAP kinase/ERK kinase). MEK1 is activated by phosphorylatio n at Ser(218) and Ser(222) by Raf. Mutation of these two sites to acidic re sidues, specifically [Asp(218)], [Asp(218), Asp(222)], and [Glu(218), Glu(2 22)], results in constitutively active MEK1. Using these mutant variants of MEK1, we showed previously that transfection of NIH/3T3 or Swiss 3T3 cells causes morphological transformation and increases growth on soft agar, ind ependent of ERR activity, The transformed cell fines show increased express ion of matrix metalloproteinases 2 and 9 and cathepsin L, proteinases that have been implicated in the metastatic process. We tested NIH3T3 cells tran sfected with the [Asp(218)] or [Asp(218), Asp(222)] for metastatic potentia l after i.v. injection into athymic mice. Parental 3T3 cells formed no tumo rs grossly or histologically. However, all MEK1 mutant transformants formed macroscopic metastases. Thus, like activated Ras, MEK1 can confer both tum origenic and metastatic potential upon NIH3T3 cells. These results refine t he mechanism through which ras could confer tumorigenic and metastatic pote ntial (i.e., the critical determinants of tumorigenic and metastatic potent ial are downstream of MEK1).