Overexpression of kinase suppressor of Ras upregulates the high-molecular-weight tropomyosin isoforms in ras-transformed NIH 3T3 fibroblasts.
The down-regulation of the high-molecular-weight isoforms of tropomyosin (TM) is considered to be an essential event in cellular transformation. In ras-transformed fibroblasts, the suppression of TM is dependent on the activity of the Raf-1 kinase; however, the requirement for other downstream effectors of Ras, such as MEK and ERK, is less clear. In this study, we have utilized the mitogen-activated protein kinase scaffolding protein Kinase Suppressor of Ras (KSR) to further investigate the regulation of TM and to clarify the importance of MEK/ERK signaling in this process. Here, we report that overexpression of wild-type KSR1 in ras-transformed fibroblasts restores TM expression and induces cell flattening and stress fiber formation. Moreover, we find that the transcriptional activity of a TM-alpha promoter is decreased in ras-transformed cells and that the restoration of TM by KSR1 coincides with increased transcription from this promoter. Although ERK activity was suppressed in cells overexpressing KSR1, ERK inhibition alone was insufficient to upregulate TM expression. The KSR1-mediated effects on stress fiber formation and TM transcription required the activity of the ROCK kinase, because these effects could be suppressed by the ROCK inhibitor, Y27632. Overexpression of KSR1 did not directly regulate ROCK activity, but did permit the recoupling of ROCK to the actin polymerization machinery. Finally, all of the KSR1-induced effects were mediated by the C-terminal domain of KSR1 and were dependent on the KSR-MEK interaction.
Janssen
Richard A J
RA
Regulation of Cell Growth Laboratory, National Cancer Institute-Frederick, Frederick, Maryland 21702, USA. r.janssen@ncmls.kun.nl
Kim
Phillia N
PN
Mier
James W
JW
Morrison
Deborah K
DK
D016475:3T3 Cells; D000256:Adenoviridae / Q000235:genetics; D000577:Amides / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002461:Cell Line, Transformed; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D020033:Protein Isoforms; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors; D017434:Protein Structure, Tertiary; D011725:Pyridines / Q000494:pharmacology; D014158:Transcription, Genetic; D014335:Tropomyosin / Q000737:chemistry* / Q000378:metabolism; D015854:Up-Regulation; D018631:ras Proteins / Q000378:metabolism*; D054460:rho-Associated Kinases
D000577:Amides; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins; D020033:Protein Isoforms; D011725:Pyridines; D014335:Tropomyosin; C108830:Y 27632; D049452:Green Fluorescent Proteins; D011494:Protein Kinases; C097199:KSR-1 protein kinase; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D018631:ras Proteins