Myristoylation of the G alpha i2 polypeptide, a G protein alpha subunit, is required for its signaling and transformation functions.

Abstract

GTPase-inhibiting mutations of the a subunit (a12) of the G protein, Gj2, result in constitutive activation of a12 signal transduction functions. GTPase-inhibited a j2 mutant polypeptides, referred to as gip2 oncoproteins, have glutamine-205 mutated to leucine (a12Q205L). Expression of the a12Q205L polypeptide inhibits adenylyl cyclase stimulation, constitutively activates p42 mitogen-activated protein kinase, and transforms Rat la fibroblasts. The aI2 polypeptides are N-terminal-myristoylated, but the function of myristoylation is unclear in ae2 signal transduction. We have tested the requirement for myristoylation on the ability of the at2Q205L mutant polypeptide to constitutively regulate signal pathways and cell transformation. When expressed in Rat la cells, the nonmyristoylated ai2Q205L polypeptide is membrane associated but is unable to regulate adenylyl cyclase or p42 mitogen-activated protein kinase and does not induce cellular transformation. We conclude that myristoylation is absolutely necessary for ai2Q205L signal transduction and regulation of effector enzymes in the cell.Protein N-myristoylation involves the covalent attachment of a 14-carbon myristoyl fatty acid to the N-terminal glycine of a limited number of cellular proteins (1, 2). Among this group of proteins, cotranslational N-myristoylation appears to be important in protein localization and biological activity (2). Significant attention has been recently focused on the role of protein N-myristoylation because of its requirement for the fibroblast transforming ability of cellular oncoproteins such as v-Src and v-Abl (3-5). A subset of G protein a subunit family members, namely ail, aa2, ai3, a0, and a,, are N-terminal-myristoylated (6-8).It has been proposed that N-terminal amino acid residues and myristoylation of specific a subunits is involved in their membrane association (7)(8)(9)(10) would be similar to the proposed role of N-terminal myristoylation of v-Src and its interaction with cellular proteins required for Src signal transduction and cellular transformation by v-Src (3-6, 16). Until recently, it was difficult to approach the question of whether myristoylation was important in G protein a subunitmediated signal transduction. It has now been shown that GTPase-inhibiting mutations constitutively activate G protein a subunit-mediated signal transduction functions (17,18). One such mutation changes the glutamine-205 codon to a leucine codon in the ai2 gene (ai2Q205L). The GTPaseinhibited ai2Q205L mutant polypeptide has been found to constitutively inhibit adenylyl cyclase stimulation in several cell types (19,20). In addition, ai2Q205L is a tissue-selective oncogene referred to as gip2 (21), capable of causing transformation of Rat la fibroblasts (22, 23). The activated signal transducing ability of the gip2 oncoprotein also results in the constitutive stimulation of the p42 mitogen-activated protein kinase [p42 MAP kinase (24)]. Thus, the activated nature of the gip2 oncogene product provides the necessary dominant respons...

Regulation of G proteins by covalent modification

“…Interaction with adenylyl cyclase directly versus indirectly through membrane binding was not evaluated, however. Consistent with these data, constitutively active nonmyristoylated Ga i did not regulate adenylyl cyclase when expressed in Rat 1a cells, despite association with membrane, although the contributions of the loss of palmitoylation and potential mistargeting were not assessed (Gallego et al, 1992). Nonpalmitoylated mutants of Ga q (C9,10A or C9,10S) have a reduced ability to activate phospholipase-b in vitro, however Ga q that was palmitoylationde®cient as a result of treatment with an esterase was not aected, suggesting a role for the cysteine residue itself (Hepler et al, 1996).…”

“…The importance of these modi®cations to cell growth is highlighted in small part by the ability of mutations that prevent fatty acid acylation of certain Ga subunits to nullify the transforming activity of these subunits. Mutation of a constitutively active form of Ga i2 to prevent Nmyristoylation, for example, renders the subunit unable to transform Rat 1a ®broblasts (Gallego et al, 1992). A mutation preventing palmitoylation of a similarly active form of Ga 12 in NIH3T3 cells also prevents transforming activity (Jones and Gutkind, 1998).…”

Pheromone action regulates G-protein alpha-subunit myristoylation in the yeast Saccharomyces cerevisiae.

“…On the basis of their apparent size, the unidentified mammalian proteins of 42-48 kDa for which myristoylation is potentiated by a variety of extracellular stimuli (29,30) could be Ga subunits. In this regard, it is noteworthy that myristoylation seems necessary for the tumorigenic potential of mutationally activated inhibitory Gi2a subunit (65). Likewise, N-myristoylation of pp6Ov-src is required for its plasma-membrane association and oncogenicity (15).…”

Regulation of G-protein alpha i2 subunit expression by oxidized low-density lipoprotein.

“…Since a12 is associated with the plasma membrane, any changes in the physical dynamics of the membrane caused by cholesterol or phospholipids could potentially dissociate ci2 from the membrane thereby rendering it more susceptible to degradation (31). Furthermore, proper function and attachment of a12 to the plasma membrane requires NH2 terminal myristoylation, a process which may be altered by the effects of oxidized, but not native LDL (32,33). It remains to be determined whether reduced myristate incorporation and subsequent ai2 accumulation in the cytoplasm are observed in endothelial cells -treated with oxidized LDL.…”