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 extracellular signals, many of which activate extracellular signal-regulated kinase (ERK)/mitogen-activated protein (MAP) kinases. Components of the MAP kinase pathways also cause oncogenic transformation in their constitutively active forms. Moreover, expression of activated ras 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 kinases, called MEKs (MAP kinase/ERK kinase). MEK1 is activated by phosphorylation at Ser218 and Ser222 by Raf. Mutation of these two sites to acidic residues, specifically [Asp218], [Asp218, Asp222], and [Glu218, Glu222], 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, independent of ERK activity. The transformed cell lines show increased expression of matrix metalloproteinases 2 and 9 and cathepsin L, proteinases that have been implicated in the metastatic process. We tested NIH3T3 cells transfected with the [Asp218] or [Asp218, Asp222] for metastatic potential after i.v. injection into athymic mice. Parental 3T3 cells formed no tumors grossly or histologically. However, all MEK1 mutant transformants formed macroscopic metastases. Thus, like activated Ras, MEK1 can confer both tumorigenic and metastatic potential upon NIH3T3 cells. These results refine the mechanism through which ras could confer tumorigenic and metastatic potential (ie., the critical determinants of tumorigenic and metastatic potential are downstream of MEK1).
Welch
D R
DR
Jake Gittlen Cancer Research Institute, The Pennsylvania State University College of Medicine, Hershey 17033-2390, USA.
Sakamaki
T
T
Pioquinto
R
R
Leonard
T O
TO
Goldberg
S F
SF
Hon
Q
Q
Erikson
R L
RL
Rieber
M
M
Rieber
M S
MS
Hicks
D J
DJ
Bonventre
J V
JV
Alessandrini
A
A
D016475:3T3 Cells; D000818:Animals; D002448:Cell Adhesion; D002461:Cell Line, Transformed; D002471:Cell Transformation, Neoplastic; D002999:Clone Cells / Q000166:cytology; D005260:Female; D008175:Lung Neoplasms / Q000201:enzymology / Q000556:secondary; D048369:MAP Kinase Kinase 1; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D051379:Mice; D008819:Mice, Nude; D020929:Mitogen-Activated Protein Kinase Kinases / Q000235:genetics; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D009362:Neoplasm Metastasis; D009374:Neoplasms, Experimental / Q000209:etiology / Q000473:pathology; D017346:Protein Serine-Threonine Kinases / Q000235:genetics; D014162:Transfection
D017346:Protein Serine-Threonine Kinases; D020928:Mitogen-Activated Protein Kinases; D048369:MAP Kinase Kinase 1; C482127:Map2k1 protein, mouse; D020929:Mitogen-Activated Protein Kinase Kinases; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9