The Saccharomyces cerevisiae SDC25 C-domain gene product overcomes the dominant inhibitory activity of Ha-Ras Asn-17.

The carboxy-terminal part of the Saccharomyces cerevisiae SDC25 gene product (SDC25 C domain) can elicit activation of mammalian Ras proteins. Specifically, SDC25 C domain functions as an exchange factor for cellular Ras proteins in CHO cells. In this study, we used the dominant inhibitory Ha-Ras Asn-17 mutant and SDC25 C domain to further investigate the interaction between cellular Ras proteins and their putative endogenous guanine nucleotide-releasing factors. Transcription from the polyomavirus thymidine kinase gene (Py tk) promoter is strongly inhibited by the expression of Ha-Ras Asn-17 in NIH 3T3 cells. Coexpression of SDC25 C domain overcomes the negative effect of the Ras mutant on the Py tk promoter. On the other hand, transactivation of the Ras-responsive element of the Py tk promoter induced by SDC25 C domain is lost upon coexpression of increasing amounts of Ha-Ras Asn-17. In addition, coexpression of SDC25 C domain overcomes the inhibition of proliferation of NIH 3T3 cells caused by Ha-Ras Asn-17. These results are consistent with the idea that the Ha-Ras Asn-17 mutant functions by titrating an upstream activator of cellular Ras proteins.

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The COOH-terminal domain of the Rap1A (Krev-1) protein is isoprenylated and supports transformation by an H-Ras:Rap1A chimeric protein

Molecular and Cellular Biology ◽

10.1128/mcb.11.3.1523-1530.1991 ◽

1991 ◽

Vol 11 (3) ◽

pp. 1523-1530

Author(s):

J E Buss ◽

L A Quilliam ◽

K Kato ◽

P J Casey ◽

P A Solski ◽

...

Keyword(s):

Biological Activities ◽

Chimeric Protein ◽

Cell System ◽

Full Length ◽

Ras Proteins ◽

3T3 Cells ◽

Farnesyl Transferase ◽

Oncogenic Ras ◽

Nih 3T3 ◽

Nih 3T3 Cells

Although the Rap1A protein resembles the oncogenic Ras proteins both structurally and biochemically, Rap1A exhibits no oncogenic properties. Rather, overexpression of Rap1A can reverse Ras-induced transformation of NIH 3T3 cells. Because the greatest divergence in amino acid sequence between Ras and Rap1A occurs at the COOH terminus, the role of this domain in the opposing biological activities of these proteins was examined. COOH-terminal processing and membrane association of Rap1A were studied by constructing and expressing a chimeric protein (composed of residues 1 to 110 of an H-Ras activated by a Leu-61 mutation attached to residues 111 to 184 of Rap1A) in NIH 3T3 cells and a full-length human Rap1A protein in a baculovirus-Sf9 insect cell system. Both the chimeric protein and the full-length protein were synthesized as a 23-kDa cytosolic precursor that rapidly bound to membranes and was converted into a 22-kDa form that incorporated label derived from [3H]mevalonate. The mature 22-kDa form also contained a COOH-terminal methyl group. Full-length Rap1A, expressed in insect cells, was modified by a C20 (geranylgeranyl) isoprenoid. In contrast, H-Ras, expressed in either Sf9 insect or NIH 3T3 mouse cells contained a C15 (farnesyl) group. This suggests that the Rap1A COOH terminus is modified by a prenyl transferase that is distinct from the farnesyl transferase that modifies Ras proteins. Nevertheless, in NIH 3T3 cells the chimeric Ras:Rap1A protein retained the transforming activity conferred by the NH2-terminal Ras61L domain. This demonstrates that the modifications and localization signals of the COOH terminus of Rap1A can support the interactions between H-Ras and membranes that are required for transformation.

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Soluble form of the ST2 gene product exhibits growth promoting activity in NIH-3T3 cells

Biochemistry and Biophysics Reports ◽

10.1016/j.bbrep.2015.11.020 ◽

2016 ◽

Vol 5 ◽

pp. 8-15 ◽

Cited By ~ 1

Author(s):

Shin-ichi Tominaga ◽

Satoshi Ohta ◽

Kenji Tago

Keyword(s):

Gene Product ◽

Soluble Form ◽

3T3 Cells ◽

Growth Promoting ◽

Nih 3T3 ◽

Nih 3T3 Cells

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The COOH-terminal domain of the Rap1A (Krev-1) protein is isoprenylated and supports transformation by an H-Ras:Rap1A chimeric protein.

Molecular and Cellular Biology ◽

10.1128/mcb.11.3.1523 ◽

1991 ◽

Vol 11 (3) ◽

pp. 1523-1530 ◽

Cited By ~ 72

Author(s):

J E Buss ◽

L A Quilliam ◽

K Kato ◽

P J Casey ◽

P A Solski ◽

...

Keyword(s):

Biological Activities ◽

Chimeric Protein ◽

Cell System ◽

Full Length ◽

Ras Proteins ◽

3T3 Cells ◽

Farnesyl Transferase ◽

Oncogenic Ras ◽

Nih 3T3 ◽

Nih 3T3 Cells

Although the Rap1A protein resembles the oncogenic Ras proteins both structurally and biochemically, Rap1A exhibits no oncogenic properties. Rather, overexpression of Rap1A can reverse Ras-induced transformation of NIH 3T3 cells. Because the greatest divergence in amino acid sequence between Ras and Rap1A occurs at the COOH terminus, the role of this domain in the opposing biological activities of these proteins was examined. COOH-terminal processing and membrane association of Rap1A were studied by constructing and expressing a chimeric protein (composed of residues 1 to 110 of an H-Ras activated by a Leu-61 mutation attached to residues 111 to 184 of Rap1A) in NIH 3T3 cells and a full-length human Rap1A protein in a baculovirus-Sf9 insect cell system. Both the chimeric protein and the full-length protein were synthesized as a 23-kDa cytosolic precursor that rapidly bound to membranes and was converted into a 22-kDa form that incorporated label derived from [3H]mevalonate. The mature 22-kDa form also contained a COOH-terminal methyl group. Full-length Rap1A, expressed in insect cells, was modified by a C20 (geranylgeranyl) isoprenoid. In contrast, H-Ras, expressed in either Sf9 insect or NIH 3T3 mouse cells contained a C15 (farnesyl) group. This suggests that the Rap1A COOH terminus is modified by a prenyl transferase that is distinct from the farnesyl transferase that modifies Ras proteins. Nevertheless, in NIH 3T3 cells the chimeric Ras:Rap1A protein retained the transforming activity conferred by the NH2-terminal Ras61L domain. This demonstrates that the modifications and localization signals of the COOH terminus of Rap1A can support the interactions between H-Ras and membranes that are required for transformation.

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