Interaction of the Ras-related protein associated with diabetes Rad and the putative tumor metastasis suppressor NM23 provides a novel mechanism of GTPase regulation

Abstract

Rad is the prototypic member of a new class of Ras-related GTPases. Purification of the GTPase-activating protein (GAP) for Rad revealed nm23, a putative tumor metastasis suppressor and a development gene in Drosophila. Antibodies against nm23 depleted Rad-GAP activity from human skeletal muscle cytosol, and bacterially expressed nm23 reconstituted the activity. The GAP activity of nm23 was specific for Rad, was absent with the S105N putative dominant negative mutant of Rad, and was reduced with mutations of nm23. In the presence of ATP, GDP⅐Rad was also reconverted to GTP⅐Rad by the nucleoside diphosphate (NDP) kinase activity of nm23. Simultaneously, Rad regulated nm23 by enhancing its NDP kinase activity and decreasing its autophosphorylation. Melanoma cells transfected with wild-type Rad, but not the S105N-Rad, showed enhanced DNA synthesis in response to serum; this effect was lost with coexpression of nm23. Thus, the interaction of nm23 and Rad provides a potential novel mechanism for bidirectional, bimolecular regulation in which nm23 stimulates both GTP hydrolysis and GTP loading of Rad whereas Rad regulates activity of nm23. This interaction may play important roles in the effects of Rad on glucose metabolism and the effects of nm23 on tumor metastasis and developmental regulation. Ras-related GTP-binding proteins are a superfamily consisting of many members that play important roles in cell proliferation and differentiation (1), intracellular vesicular trafficking (2), cytoskeletal rearrangement (3), cell cycle regulation (4), and glucose transport into cells (5, 6). These GTPases cycle between GTP-bound (active) and GDP-bound states (inactive) in a controlled manner, stimulated by interaction with GTPaseactivating proteins (GAPs) present in the cytoplasm of the cells (7,8). Conversely, the reloading of the GTPases is made possible by the action of guanine nucleotide exchange factors (GEFs), which facilitates the exchange of free GTP for G protein-bound GDP (7), or is slowed by the presence of GDP dissociation inhibitors (9).Rad is the prototype of a new subfamily of Ras-related GTPases, which are of molecular mass 33-35 kDa and lack typical prenylation motifs at the C terminus. It was initially identified by subtraction cloning and is overexpressed in skeletal muscle of a subset of humans with type 2 diabetes (10). Overexpression of Rad in 3T3-L1 adipocytes and C2C12 myocytes causes a marked reduction in glucose uptake in response to insulin stimulation, suggesting that it contributes to the insulin resistance of diabetes (6). Rad also interacts with calmodulin and calmodulin kinase II (11) and ␤-tropomyosin (12). These interactions are enhanced by an increase in the calcium influx and favor the GDP-bound form of Rad. The other two members of Rad family are Gem͞Kir and Rem. Gem͞Kir was identified in activated T lymphocytes (13) and v-abl transformed pre-B cells (14). Rem was cloned as a PCR product by using primers derived from conserved regions of Rad and Gem͞Kir, is expressed in heart, lung, and oth...

Direct Inhibition of the Interaction between α-Interaction Domain and β-Interaction Domain of Voltage-dependent Ca2+ Channels by Gem

“…NM23, which was identified as GTPase-activating protein and guanine nucleotide exchange factor (GEF) for Rad, acts as Gem-GEF in vitro (42). It has been shown that NM23 inhibits Rad-induced growth and tumorigenicity of breast cancer in breast tissues (43).…”

The RGK family of GTP-binding proteins: Regulators of voltage-dependent calcium channels and cytoskeleton remodeling

“…While these proteins have not yet been identified, the in vitro GTP/GDP state of Rad has been found to be modified by the tumor suppressor nm23, which can either phosphorylate GDP or dephosphoryate GTP while the nucleotide is bound to Rad [14]. It remains to be determined whether nm23 functions as an in vivo regulator of Rad, or regulates the actions of other RGK proteins.…”

Arabidopsis nucleoside diphosphate kinase-2 as a plant GTPase activating protein

“…NDPK is capable of maintaining nucleotide levels in mammals, implying NDPK may be a critical component of G protein-mediated signaling (2)(3)(4)(5)(6). Mammalian NDPK is a multifunctional enzyme, involved in the regulation of growth and development (7)(8)(9).…”