14-3-3 and calmodulin control subcellular distribution of Kir/Gem and its regulation of cell shape and calcium channel activity

Abstract

Individual members of the RGK family of Ras-related GTPases, which comprise Rad, Gem/Kir, Rem and Rem2, have been implicated in important functions such as the regulation of voltage-gated calcium channel activity and remodeling of cell shape. The GTPase Kir/Gem inhibits the activity of calcium channels by interacting with the β-subunit and also regulates cytoskeleton dynamics by inhibiting the Rho-Rho kinase pathway. In addition, Kir/Gem interacts with 14-3-3 and calmodulin, but the significance of this interaction on Kir/Gem function is poorly understood. Here, we present a comprehensive analysis of the binding of 14-3-3 and calmodulin to Kir/Gem. We show that 14-3-3, in conjunction with calmodulin, regulates the subcellular distribution of Kir/Gem between the cytoplasm and the nucleus. In addition, 14-3-3 and calmodulin binding modulate Kir/Gem-mediated cell shape remodeling and downregulation of calcium channel activity. Competition experiments show that binding of 14-3-3, calmodulin and calcium channel β-subunits to Kir/Gem is mutually exclusive, providing a rationale for the observed regulatory effects of 14-3-3 and calmodulin on Kir/Gem localization and function.

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

“…Rad is an in vitro substrate for several kinases, including PKA, PKC, CaMKII, and casein kinase II [12], which phosphorylate several distinct serine residues within the protein. 14-3-3 proteins interact specifically with RGK proteins phosphorylated on N-and C-terminal serines [41][42][43][44][45] and 14-3-3 binding appears to modulate the subcellular localization of Rad, Rem, Rem2, and Gem/Kir proteins [41,43,44,46,47]. Phosphorylation of Rad by PKC or casein kinase II, on the other hand, reduces binding to CaM [12].…”

“…RGK proteins do not contain canonical lipid modification motifs [48], though there is enrichment of these proteins at the plasma membrane, and individual proteins have been shown to be localized to the cytosol, nucleus, and with both the actin and microtubule networks [4,5,18,41,43,44,[46][47][48][49][50][51][52][53][54]. The carboxyl terminus is well conserved (Fig.…”

“…The carboxyl terminus is well conserved (Fig. 1) and has been shown to play a critical role in the function of all RGK GTPases [49,55], regulating subcellular distribution [4,5,53], and directing protein-protein interactions [45,53,55], to control both Ca 2+ channel activity [49,53,55] and cytoskeletal reorganization [41][42][43][44]46,47,52]. This region contains a cysteine residue within a highly conserved domain termed the C-7 motif (Fig.…”

“…In addition to plasma membrane targeting through the C-terminus, three specific nuclear localization signals are well conserved in all RGK proteins [46,47]. Recent studies suggest that nuclear sequestration may play a role in regulating RGK GTPase function, and appears to be controlled in part through interactions with both calmodulin and 14-3-3 proteins (see sections 5 and 6) [41][42][43][44]46,47].…”

“…RGK proteins have been found to promote cell shape remodeling through the regulation of the actin [21,26,[41][42][43][44][58][59][60][61], and perhaps microtubule, cytoskeletons [29,51]. The identification of the microtubule-associated protein, kinesin-like protein (KIF9), as a Gem-binding partner suggests that microtubule localization may facilitate Gem-dependent activation of downstream effector proteins [51].…”