Deletion of the COOH terminus converts the ST5 p70 protein from an inhibitor of RAS signaling to an activator with transforming activity in NIH-3T3 cells.
Expression of the human protein ST5-p70 correlates with reduced tumorigenic phenotype in mammalian cells, reverts their transformed phenotype, and restores their contact-dependent growth. Furthermore, expression of p70 in COS-7 cells suppresses activation of mitogen activated protein kinase MAPK/ERK2 by the largest ST5 product, p126, in response to epidermal growth factor stimulation. Here we show that deletions of the COOH-terminal region of p70 transform NIH3T3 cells and induce their anchorage-independent growth. Analysis of signaling leading to MAPK/ERK2 stimulation revealed that in COS-7 cells, expression of either p70-DeltaC1 or p70-DeltaC2 markedly enhanced ERK2 activity in a growth factor-independent manner. Whereas wild-type p70 slightly inhibited ERK2 activation by RAS and MEK2, co-expression or p70-DeltaC1 or p70-DeltaC2 with either protein stimulated ERK2 cooperatively. This activity was completely blocked by the dominant negative mutants RAS17N or MEKAA, suggesting that p70 functions upstream of RAS. Unlike wild-type p70, expression of p70-DeltaC1 or p70-DeltaC2 mutant did not interfere with the ability of ST5-p126 to stimulate ERK2. Taken together, the data suggest that the COOH-terminal tail, residues 489-609, contains some of the critical determinants for the function of p70. Loss of this region converts the protein from an inhibitor to a constitutive activator of the RAS-ERK2 pathway.
Majidi
M
M
Department of Cellular Pathology, Armed Forces Institute of Pathology, Washington, D. C. 20306-6000, USA. majidi@afip.osd.mil
Gutkind
J S
JS
Lichy
J H
JH
D016475:3T3 Cells; D000818:Animals; D019556:COS Cells; D002455:Cell Division / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics*; D004789:Enzyme Activation / Q000235:genetics; D006801:Humans; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics; D009154:Mutation; D017384:Sequence Deletion / Q000235:genetics*; D015398:Signal Transduction / Q000235:genetics*; D014162:Transfection; D014170:Transformation, Genetic; D025521:Tumor Suppressor Proteins; D018631:ras Proteins / Q000037:antagonists & inhibitors*
C077272:DENND2B protein, human; D004268:DNA-Binding Proteins; C486188:St5 protein, mouse; D025521:Tumor Suppressor Proteins; D019950:Mitogen-Activated Protein Kinase 1; D020928:Mitogen-Activated Protein Kinases; D018631:ras Proteins