Membrane-targeting potentiates guanine nucleotide exchange factor CDC25 and SOS1 activation of Ras transforming activity

Lawrence Quilliam, Shayne Y. Huff, Kelly M. Rabun, Wen Wei, Weonmee Park, Daniel Broek, Channing J. Der

Research output: Contribution to journalArticle

123 Citations (Scopus)

Abstract

Growth factor-triggered activation of Ras proteins is believed to be mediated by guanine nucleotide exchange factors (CDC25/GRF and SOS1/2) that promote formation of the active Ras GTP-bound state. Although the mechanism(s) of guanine nucleotide exchange factor regulation is unclear, recent studies suggest that translocation of SOS1 to the plasma membrane, where Ras is located, might be responsible for Ras activation. To evaluate this model, we generated constructs that encode the catalytic domains of human CDC25 or mouse SOS1, either alone (designated cCDC25 and cSOS1, respectively) or terminating in the carboxyl-terminal CAAX membrane-targeting sequence from K-Ras4B (designated cCDC25-CAAX and cSOS1-CAAX, respectively; in CAAX, C is Cys, A is an aliphatic amino acid, and X is Ser or Met). We then compared the transforming potential of cCDC25 and cSOS1 with their membrane-targeted counterparts. We observed that addition of the Ras plasma membrane-targeting sequence to the catalytic domains of CDC25 and SOS1 greatly enhanced their focus-forming activity (10- to 50-fold) in NIH 3T3 transfection assays. Similarly, we observed that the membrane-targeted versions showed a 5- to 10-fold enhanced ability to induce transcriptional activation from the Ets/AP-1 Ras-responsive element. Furthermore, whereas cells that stably expressed cCDC25 or cSOS1 exhibited the same morphologies as untransformed NIH 3T3 cells, cells expressing cCDC25-CAAX or cSOS1-CAAX displayed transformed morphologies that were indistinguishable from the elongated and refractile morphology of oncogenic Ras-transformed cells. Thus, these results suggest that membrane translocation alone is sufficient to potentiate guanine nucleotide exchange factor activation of Ras.

Original languageEnglish (US)
Pages (from-to)8512-8516
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume91
Issue number18
StatePublished - Aug 30 1994
Externally publishedYes

Fingerprint

Guanine Nucleotide Exchange Factors
Membranes
Guanine Nucleotide-Releasing Factor 2
Catalytic Domain
ras Guanine Nucleotide Exchange Factors
Cell Membrane
ras Proteins
NIH 3T3 Cells
Transcription Factor AP-1
Guanosine Triphosphate
Transcriptional Activation
Transfection
Intercellular Signaling Peptides and Proteins
Fatty Acids
Amino Acids

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

Membrane-targeting potentiates guanine nucleotide exchange factor CDC25 and SOS1 activation of Ras transforming activity. / Quilliam, Lawrence; Huff, Shayne Y.; Rabun, Kelly M.; Wei, Wen; Park, Weonmee; Broek, Daniel; Der, Channing J.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 91, No. 18, 30.08.1994, p. 8512-8516.

Research output: Contribution to journalArticle

Quilliam, Lawrence ; Huff, Shayne Y. ; Rabun, Kelly M. ; Wei, Wen ; Park, Weonmee ; Broek, Daniel ; Der, Channing J. / Membrane-targeting potentiates guanine nucleotide exchange factor CDC25 and SOS1 activation of Ras transforming activity. In: Proceedings of the National Academy of Sciences of the United States of America. 1994 ; Vol. 91, No. 18. pp. 8512-8516.
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abstract = "Growth factor-triggered activation of Ras proteins is believed to be mediated by guanine nucleotide exchange factors (CDC25/GRF and SOS1/2) that promote formation of the active Ras GTP-bound state. Although the mechanism(s) of guanine nucleotide exchange factor regulation is unclear, recent studies suggest that translocation of SOS1 to the plasma membrane, where Ras is located, might be responsible for Ras activation. To evaluate this model, we generated constructs that encode the catalytic domains of human CDC25 or mouse SOS1, either alone (designated cCDC25 and cSOS1, respectively) or terminating in the carboxyl-terminal CAAX membrane-targeting sequence from K-Ras4B (designated cCDC25-CAAX and cSOS1-CAAX, respectively; in CAAX, C is Cys, A is an aliphatic amino acid, and X is Ser or Met). We then compared the transforming potential of cCDC25 and cSOS1 with their membrane-targeted counterparts. We observed that addition of the Ras plasma membrane-targeting sequence to the catalytic domains of CDC25 and SOS1 greatly enhanced their focus-forming activity (10- to 50-fold) in NIH 3T3 transfection assays. Similarly, we observed that the membrane-targeted versions showed a 5- to 10-fold enhanced ability to induce transcriptional activation from the Ets/AP-1 Ras-responsive element. Furthermore, whereas cells that stably expressed cCDC25 or cSOS1 exhibited the same morphologies as untransformed NIH 3T3 cells, cells expressing cCDC25-CAAX or cSOS1-CAAX displayed transformed morphologies that were indistinguishable from the elongated and refractile morphology of oncogenic Ras-transformed cells. Thus, these results suggest that membrane translocation alone is sufficient to potentiate guanine nucleotide exchange factor activation of Ras.",
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