Abstract
In contrast to familiar role for Ras in proliferation, we and others previously suggested that Ras also mediates hypertrophy, the increase in cell mass characteristic of post-natal ventricular muscle. We showed that activated (G12R) and dominant-negative (S17N) Ha-Ras regulate "constitutive" and growth factor-responsive genes equivalently, in both cardiac myocytes and non-cardiac, Mv1Lu cells. Here, we attempt to delineate pathways by which Ras exerts this global effect. The E63K mutation, which impairs binding of guanine nucleotide releasing factor to Ras, alleviated suppression by S17N, consistent with sequestration of exchange factors as the mechanism for inhibition. To compare potential Ras effector proteins, we first engineered G12R/D38N, to abolish binding of Raf and phosphatidylinositol-3-kinase and established that this site was indispensable for augmenting gene expression. To distinguish between inhibition of Ras by Ras GTPase-activating protein (GAP) versus a potential effector function of GAP, we tested the effector domain substitution P34R: this mutation, which abolishes GAP binding, enhanced Ras-dependent transcription in Mv1Lu cells, yet interfered with Ras-dependent expression in ventricular myocytes. To examine the dichotomous role of Ras-GAP predicted from these P34R results, we transfected both cell types with full-length GAP, the C-terminal catalytic domain (cGAP), or N-terminal Src homology domains (nGAP). In Mv1Lu cells, cGAP markedly inhibited both reporter genes, whereas GAP and nGAP had little effect. Antithetically, in ventricular myocytes, GAP and nGAP activated gene expression, whereas cGAP was ineffective. Thus, Ras activates gene expression through differing effectors contingent on cell type, and an effector-like function of GAP predominates in ventricular muscle.
Highlights
Ras, the archetype of low molecular weight GTP-binding proteins, first was identified in mammalian cells through gainof-function mutations in human tumors, corresponding to the transforming genes of Harvey and Kirsten murine sarcoma viruses [1,2,3]
Repression of a Constitutive Promoter by S17N Ras Is Alleviated by Mutation of the Binding Site for Guanine Nucleotide Exchange Factors—To test the prediction that the E63K substitution, which impairs the binding of guanine nucleotide exchange factors to Ras [37], would at least partially reverse the inhibition of gene expression by dominant-negative Ras, we generated the S17N/E63K double mutation
The inference that Ras activity is required for efficient expression of both growth factor-inducible and nominally constitutive genes was the conclusion of our prior investigations, implicating Ras as a governor of global gene expression in both cardiac myocytes and mink lung epithelial cells [6]
Summary
The archetype of low molecular weight GTP-binding proteins, first was identified in mammalian cells through gainof-function mutations in human tumors, corresponding to the transforming genes of Harvey and Kirsten murine sarcoma viruses [1,2,3]. No indications were found for the hypothesis that TGF might signal through Ras. dominant-negative, wild-type, or activated Ras cDNAs regulated all seven reporter genes tested, including those driven by nominally constitutive viral control regions, the Rous sarcoma virus promoter, and minimal herpes simplex virus thymidine kinase (HSV tk) promoter, and even a TATA-less adeno-associated virus initiator element [6]. Dominant-negative, wild-type, or activated Ras cDNAs regulated all seven reporter genes tested, including those driven by nominally constitutive viral control regions, the Rous sarcoma virus promoter, and minimal herpes simplex virus thymidine kinase (HSV tk) promoter, and even a TATA-less adeno-associated virus initiator element [6] These broadly inclusive effects suggested the interpretation that Ras activity regulates an unexpectedly generalized or global set of genes or, conceivably, the basic transcriptional machinery [6]. Given the diversity of Ras effects and Ras-associated proteins, the extrapolation from transforming activity to transcriptional control is uncertain, in the novel context of Ras as a potential governor of global gene expression
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