Nuclear Localization of Endogenous RGK Proteins and Modulation of Cell Shape Remodeling by Regulated Nuclear Transport

Abstract

The members of the RGK small GTP‐binding protein family, Kir/Gem, Rad, Rem and Rem2, are multifunctional proteins that regulate voltage‐gated calcium channel activity and cell shape remodeling. Calmodulin (CaM) or CaM 14‐3‐3 are regulators of RGK functions and their association defines the subcellular localization of RGK proteins. Abolition of CaM association results in the accumulation of RGK proteins in the nucleus, whereas 14‐3‐3 binding maintains them in the cytoplasm. Kir/Gem possesses nuclear localization signals (NLS) that mediate nuclear accumulation through an importin α5‐dependent pathway (see Mahalakshmi RN, Nagashima K, Ng MY, Inagaki N, Hunziker W, Béguin P. Nuclear transport of Kir/Gem requires specific signals and importin α5 and is regulated by Calmodulin and predicted service phosphorylations. Traffic 2007; doi: 10.1111/j.1600‐0854.2007.00598.x). Because the extent of nuclear localization depends on the RGK protein and the cell type, the mechanism and regulation of nuclear transport may differ. Here, we extend our analysis to the other RGK members and show that Rem also binds importin α5, whereas Rad associates with importins α3, α5 and β through three conserved NLS. Predicted phosphorylation of a serine residue within the bipartite NLS affects, as observed for Kir/Gem, nuclear accumulation of Rem, but not that of Rad or Rem2. We also identify an additional regulatory phosphorylation for all RGK proteins that prevents binding of 14‐3‐3 and thereby interferes with their cytosolic relocalization by 14‐3‐3. Functionally, nuclear localization of RGK proteins contributes to the suppression of RGK‐mediated cell shape remodeling. Importantly, we show that endogenous RGK proteins are localized predominantly in the nucleus of individual cells of the brain cortex ‘in situ’ as well as in primary hippocampal cells, indicating that transport between the nucleus and their site of action in the cytoplasm (i.e., cytoskeleton, endoplasmic reticulum or plasma membrane) is of physiological relevance for the regulation of RGK protein 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.…”

“…Additional studies will be necessary to determine whether PIP lipid binding is required for RGK activity, although the recent analysis of Rem and Rem2 C-terminal truncation mutants suggests a strong correlation between PIP-lipid interaction, plasma membrane localization, and Rem/Rem2-dependent Ca 2+ channel regulation ( [53] and unpublished data). 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].…”

“…Phosphorylation of the Gem C-terminus, and other RGK proteins, may also regulate subcellular distribution [41,44,46,47], with 14-3-3 binding modulating nuclear localization in heterologous expression systems [41,43,44,47]. Therefore, nucleocytoplasmic shuttling of RGK proteins may represent a distinct mechanism for controlling RGK-dependent cell shape remodeling, and this process appears to be dynamically regulated by calmodulin and 14-3-3 binding [41,43,44,46,47,61]. Whether these regulatory processes are correlated with RGKmediated Rho/ROK-cytoskeleton reorganization remains to be clarified.…”