Differential Subcellular Distribution and Translocation of Seven 14-3-3 Isoforms in Response to EGF and During the Cell Cycle.

Multiple isoforms of 14-3-3 proteins exist in different organisms. In mammalian cells, 14-3-3 protein has seven isoforms (α/ß, ε, η, γ, σ, θ/τ, and δ/ζ), with α and δ representing the phosphorylated versions of ß and ζ, respectively. While the existence of multiple isoforms may represent one more level of regulation in 14-3-3 signaling, our knowledge regarding the isoform-specific functions of 14-3-3 proteins is very limited. Determination of the subcellular localization of the different 14-3-3 isoforms could give us important clues of their specific functions. In this study, by using indirect immunofluorescence, subcellular fractionation, and immunoblotting, we studied the subcellular localization of the total 14-3-3 protein and each of the seven 14-3-3 isoforms; their redistribution throughout the cell cycle; and their translocation in response to EGF in Cos-7 cells. We showed that 14-3-3 proteins are broadly distributed throughout the cell and associated with many subcellular structures/organelles, including the plasma membrane (PM), mitochondria, ER, nucleus, microtubules, and actin fibers. This broad distribution underlines the multiple functions identified for 14-3-3 proteins. The different isoforms of 14-3-3 proteins have distinctive subcellular localizations, which suggest their distinctive cellular functions. Most notably, 14-3-3ƞ is almost exclusively localized to the mitochondria, 14-3-3γ is only localized to the nucleus, and 14-3-3σ strongly and specifically associated with the centrosome during mitosis. We also examined the subcellular localization of the seven 14-3-3 isoforms in other cells, including HEK-293, MDA-MB-231, and MCF-7 cells, which largely confirmed our findings with Cos-7 cells.

The novel p.Ser263Phe mutation in the human high-affinity choline transporter 1 (CHT1/SLC5A7) causes a lethal form of fetal akinesia syndrome.

A subset of a larger and heterogeneous class of disorders, the congenital myasthenic syndromes (CMS) are caused by pathogenic variants in genes encoding proteins that support the integrity and function of the neuromuscular junction (NMJ). A central component of the NMJ is the sodium-dependent high-affinity choline transporter 1 (CHT1), a solute carrier protein (gene symbol SLC5A7), responsible for the reuptake of choline into nerve termini has recently been implicated as one of several autosomal recessive causes of CMS. We report the identification and functional characterization of a novel pathogenic variant in SLC5A7, c.788C>T (p.Ser263Phe) in an El Salvadorian family with a lethal form of a congenital myasthenic syndrome characterized by fetal akinesia. This study expands the clinical phenotype and insight into a form of fetal akinesia related to CHT1 defects and proposes a genotype-phenotype correlation for the lethal form of SLC5A7-related disorder with potential implications for genetic counseling.

Interaction between Rho GTPases and 14-3-3 Proteins.

The Rho GTPase family accounts for as many as 20 members. Among them, the archetypes RhoA, Rac1, and Cdc42 have been the most well-characterized. Like all members of the small GTPases superfamily, Rho proteins act as molecular switches to control cellular processes by cycling between active, GTP-bound and inactive, GDP-bound states. The 14-3-3 family proteins comprise seven isoforms. They exist as dimers (homo- or hetero-dimer) in cells. They function by binding to Ser/Thr phosphorylated intracellular proteins, which alters the conformation, activity, and subcellular localization of their binding partners. Both 14-3-3 proteins and Rho GTPases regulate cell cytoskeleton remodeling and cell migration, which suggests a possible interaction between the signaling pathways regulated by these two groups of proteins. Indeed, more and more emerging evidence indicates the mutual regulation of these two signaling pathways. There have been many documented reviews of 14-3-3 protein and Rac1 separately, but there is no review regarding the interaction and mutual regulation of these two groups of proteins. Thus, in this article we thoroughly review all the reported interactions between the signaling pathways regulated by 14-3-3 proteins and Rho GTPases (mostly Rac1).

Rac1 S71 Mediates the Interaction between Rac1 and 14-3-3 Proteins.

Both 14-3-3 proteins (14-3-3s) and Rho proteins regulate cytoskeleton remodeling and cell migration, which suggests a possible interaction between the signaling pathways regulated by these two groups of proteins. Indeed, more and more emerging evidence indicates the mutual regulation of these two signaling pathways. However, all of the data regarding the interaction between Rac1 signaling pathways and 14-3-3 signaling pathways are through either the upstream regulators or downstream substrates. It is not clear if Rac1 could interact with 14-3-3s directly. It is interesting to notice that the Rac1 sequence 68RPLSYP73 is likely a 14-3-3 protein binding motif following the phosphorylation of S71 by Akt. Thus, we hypothesize that Rac1 directly interacts with 14-3-3s. We tested this hypothesis in this research. By using mutagenesis, co-immunoprecipitation (co-IP), Rac1 activity assay, immunoblotting, and indirect immunofluorescence, we demonstrate that 14-3-3s interact with Rac1. This interaction is mediated by Rac1 S71 in both phosphorylation-dependent and -independent manners, but the phosphorylation-dependent interaction is much stronger. Epidermal growth factor (EGF) strongly stimulates the phosphorylation of Rac1 S71 and the interaction between 14-3-3s and Rac1. Mutating S71 to A completely abolishes both phosphorylation-dependent and -independent interactions between 14-3-3s and Rac1. The interaction between 14-3-3s and Rac1 mostly serve to regulate the activity and subcellular localization of Rac1. Among the seven 14-3-3 isoforms, 14-3-3η, -σ, and -θ showed interactions with Rac1 in both Cos-7 and HEK 293 cells. 14-3-3γ also binds to Rac1 in HEK 293 cells, but not in Cos-7 cells. We conclude that 14-3-3s interact with Rac1. This interaction is mediated by Rac1 S71 in both phosphorylation-dependent and -independent manners. The interaction between 14-3-3 and Rac1 mostly serves to regulate the activity and subcellular localization of Rac1. Among the seven 14-3-3 isoforms, 14-3-3η, -γ, -σ, and -θ interact with Rac1.

An In Vitro Kinase Assay to Assess Rac1 Phosphorylation by ERK.

Recent findings suggest that phosphorylation might further contribute to the tight regulation of Rho GTPases. Interestingly, sequence analysis of Rac1 shows that T108 within the 106PNTP109 motif of Rac1 is likely an ERK phosphorylation site and Rac1 also has an ERK docking site 183KKRKRKCLLL192 (D-site) at the C-terminus. Protein phosphorylation could be assayed by many different methods. Here, we describe an in vitro kinase assay we used to assess Rac1 phosphorylation by ERK. Rac1 phosphorylation is detected based on the transfer of a radiolabeled phosphate from ATP to Rac1 by the phosphotransferase activity of the kinase EKR. This in vitro kinase assay uses commercially available purified active ERK. Substrate Rac1 was generated and purified as a glutathione S-transferase (GST) fusion protein. [γ-32P]ATP is used to radiolabel Rac1. Phosphorylation of Rac1 is viewed by autoradiography.