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Regulated and constitutive activity by CDC25Mm (GRF), a Ras-specific exchange factor
H Cen1, A G Papageorge, W C Vass
1Laboratory of Cellular Oncology, National Cancer Institute, Bethesda, Maryland 20892.
Molecular and Cellular Biology
|December 1, 1993
Summary
CDC25Mm, also known as GRF, activates Ras protein, a key regulator of cell growth. This study identifies four GRF variants that induce cell transformation and increase GTP-bound Ras, with specific variants showing serum-dependent regulation.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Serum stimulation increases the active GTP-bound state of Ras protein.
- CDC25Mm (GRF) is a mammalian homolog of yeast CDC25, a Ras-specific exchange factor.
- Four cDNA types (I-IV) of CDC25Mm were identified, differing primarily in their N-terminal sequences.
Purpose of the Study:
- To investigate the function of different CDC25Mm/GRF cDNA types in cell transformation and Ras activation.
- To determine the role of N-terminal sequences and cellular localization in GRF activity.
- To elucidate the mechanism of serum-dependent Ras activation by GRF.
Main Methods:
- Cloning and expression of four CDC25Mm/GRF cDNA types in retroviral vectors.
- Morphological analysis and GTP-Ras level measurements in NIH 3T3 cells.
- In vitro guanine nucleotide exchange assays.
- Transfection with mutant ras genes and analysis of Ras-GTP levels in different cellular fractions.
Main Results:
- All four GRF types induced NIH 3T3 cell transformation and increased basal GTP-Ras levels.
- Type IV GRF, brain-specific, showed serum-dependent enhancement of GTP-Ras.
- GRF proteins were found in both cytosolic and membrane fractions and stimulated guanine nucleotide exchange.
- Expression of GRF increased basal GTP-bound Ras, particularly membrane-associated Ras, and serum-dependent activation required membrane association of both Ras and GRF.
Conclusions:
- CDC25Mm/GRF induces cell transformation through its C-terminus's guanine nucleotide exchange activity on Ras.
- N-terminal sequences of GRF are linked to serum-dependent modulation of GTP-Ras.
- Serum-dependent Ras activation by GRF likely necessitates membrane localization of both Ras and the exchange factor.