ZO-1 regulates Erk, Smad1/5/8, Smad2, and RhoA activities to modulate self-renewal and differentiation of mouse embryonic stem cells.

ZO-1/Tjp1 is a cytosolic adaptor that links tight junction (TJ) transmembrane proteins to the actin cytoskeleton and has also been implicated in regulating cell proliferation and differentiation by interacting with transcriptional regulators and signaling proteins. To explore possible roles for ZO-1 in mouse embryonic stem cells (mESCs), we inactivated the ZO-1 locus by homologous recombination. The lack of ZO-1 was found to affect mESC self-renewal and differentiation in the presence of leukemia-inhibiting factor (LIF) and Bmp4 or following removal of the growth factors. Our data suggest that ZO-1 suppresses Stat3 and Smad1/5/8 activities and sustains extracellular-signal-regulated kinase (Erk) activity to promote mESC differentiation. Interestingly, Smad2, critical for human but not mESC self-renewal, was hyperactivated in ZO-1(-/-) mESCs and RhoA protein levels were concomitantly enhanced, suggesting attenuation of the noncanonical transforming growth factor ß (Tgfß)/Activin/Nodal pathway that mediates ubiquitination and degradation of RhoA via the TJ proteins Occludin, Par6, and Smurf1 and activation of the canonical Smad2-dependent pathway. Furthermore, Bmp4-induced differentiation of mESCs in the absence of LIF was suppressed in ZO-1(-/-) mESCs, but differentiation down the neural or cardiac lineages was not disturbed. These findings reveal novel roles for ZO-1 in mESC self-renewal, pluripotency, and differentiation by influencing several signaling networks that regulate these processes. Possible implications for the differing relevance of Smad2 in mESC and human ESC self-renewal and how ZO-1 may connect to the different pathways are discussed.

BARP suppresses voltage-gated calcium channel activity and Ca2+-evoked exocytosis.

Voltage-gated calcium channels (VGCCs) are key regulators of cell signaling and Ca(2+)-dependent release of neurotransmitters and hormones. Understanding the mechanisms that inactivate VGCCs to prevent intracellular Ca(2+) overload and govern their specific subcellular localization is of critical importance. We report the identification and functional characterization of VGCC ß-anchoring and -regulatory protein (BARP), a previously uncharacterized integral membrane glycoprotein expressed in neuroendocrine cells and neurons. BARP interacts via two cytosolic domains (I and II) with all Cavß subunit isoforms, affecting their subcellular localization and suppressing VGCC activity. Domain I interacts at the α1 interaction domain-binding pocket in Cavß and interferes with the association between Cavß and Cavα1. In the absence of domain I binding, BARP can form a ternary complex with Cavα1 and Cavß via domain II. BARP does not affect cell surface expression of Cavα1 but inhibits Ca(2+) channel activity at the plasma membrane, resulting in the inhibition of Ca(2+)-evoked exocytosis. Thus, BARP can modulate the localization of Cavß and its association with the Cavα1 subunit to negatively regulate VGCC activity.

Zona occludens-2 is critical for blood-testis barrier integrity and male fertility.

Tight junction integral membrane proteins such as claudins and occludin are tethered to the actin cytoskeleton by adaptor proteins, notably the closely related zonula occludens (ZO) proteins ZO-1, ZO-2, and ZO-3. All three ZO proteins have recently been inactivated in mice. Although ZO-3 knockout mice lack an obvious phenotype, animals deficient in ZO-1 or ZO-2 show early embryonic lethality. Here, we rescue the embryonic lethality of ZO-2 knockout mice by injecting ZO-2(-/-) embryonic stem (ES) cells into wild-type blastocysts to generate viable ZO-2 chimera. ZO-2(-/-) ES cells contribute extensively to different tissues of the chimera, consistent with an extraembryonic requirement for ZO-2 rather than a critical role in epiblast development. Adult chimera present a set of phenotypes in different organs. In particular, male ZO-2 chimeras show reduced fertility and pathological changes in the testis. Lanthanum tracer experiments show a compromised blood-testis barrier. Expression levels of ZO-1, ZO-3, claudin-11, and occludin are not apparently affected. ZO-1 and occludin still localize to the blood-testis barrier region, but claudin-11 is less well restricted and the localization of connexin-43 is perturbed. The critical role of ZO-2 for male fertility and blood-testis barrier integrity thus provides a first example for a nonredundant role of an individual ZO protein in adult mice.

Nuclear localization of endogenous RGK proteins and modulation of cell shape remodeling by regulated nuclear transport.

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 alpha5-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 alpha5 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 alpha5, whereas Rad associates with importins alpha3, alpha5 and beta 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.

Nuclear transport of Kir/Gem requires specific signals and importin alpha5 and is regulated by calmodulin and predicted serine phosphorylations.

Kir/Gem, together with Rad, Rem and Rem2, is a member of the RGK small GTP-binding protein family. These multifunctional proteins regulate voltage-gated calcium channel (VGCC) activity and cell-shape remodeling. Calmodulin and 14-3-3 binding modulate the functions of RGK proteins. Intriguingly, abolishing the binding of calmodulin or calmodulin and 14-3-3 results in nuclear accumulation of RGK proteins. Under certain conditions, the Ca(v)beta3-subunit of VGCCs can be translocated into the nucleus along with the RGK proteins, resulting in channel inactivation. The mechanism by which nuclear localization of RGK proteins is accomplished and regulated, however, is unknown. Here, we identify specific nuclear localization signals (NLS) in Kir/Gem that are both required and sufficient for nuclear transport. Importin alpha5 binds to Kir/Gem, and its depletion using RNA interference impairs nuclear translocation of this RGK protein. Calmodulin and predicted phosphorylations on serine residues within or in the vicinity of a C-terminal bipartite NLS regulate nuclear translocation by interfering with the association between importinalpha5 and Kir/Gem. These predicted phosphorylations, however, do not affect Kir/Gem-mediated calcium channel downregulation but rather, as shown in the accompanying paper (Mahalakshmi RN, Ng MY, Guo K, Qi Z, Hunziker W, Béguin P. Nuclear localization of endogenous RGK proteins and modulation of cell shape remodeling by regulated nuclear transport. Traffic 2007; doi:10.1111/j.1600-0854.2007.00599.x), interfere with cell-shape remodeling.

RGK small GTP-binding proteins interact with the nucleotide kinase domain of Ca2+-channel beta-subunits via an uncommon effector binding domain.

RGK proteins (Kir/Gem, Rad, Rem, and Rem2) form a small subfamily of the Ras superfamily. Despite a conserved GTP binding core domain, several differences suggest that structure, mechanism of action, and functional regulation differ from Ras. RGK proteins down-regulate voltage-gated calcium channel activity by binding in a GTP-dependent fashion to the Cavbeta subunits. Mutational analysis combined with homology modeling reveal a novel effector binding mechanism distinct from that of other Ras GTPases. In this model the Switch 1 region acts as an allosteric activator that facilitates electrostatic interactions between Arg-196 in Kir/Gem and Asp-194, -270, and -272 in the nucleotide-kinase (NK) domain of Cavbeta3 and wedging Val-223 and His-225 of Kir/Gem into a hydrophobic pocket in the NK domain. Kir/Gem interacts with a surface on the NK domain that is distinct from the groove where the voltage-gated calcium channel Cavalpha1 subunit binds. A complex composed of the RGK protein and the Cavbeta3 and Cav1.2 subunits could be revealed in vivo using coimmunoprecipitation experiments. Intriguingly, docking of the RGK protein to the NK domain of the Cavbeta subunit is reminiscent of the binding of GMP to guanylate kinase.