LIM proteins: association with the actin cytoskeleton

The LIM domain is an evolutionary conserved double-zinc finger motif found in a variety of proteins exhibiting diverse biological roles. LIM domains have been observed to act as modular protein-binding interfaces mediating protein-protein interactions in the cytoplasm and the nucleus. Interaction of LIM domains with specific protein partners is now known to influence its subcellular localization and activity; however, no single binding motif has been identified as a common target for LIM domains. Several LIM domain-containing proteins associated with the actin cytoskeleton have been identified, playing a role in signal transduction and organization of the actin filaments during various cellular processes.     

Actin binding and proline rich motifs of CR16 play redundant role in growth of vrp1Delta cells

CR16, (Glucocorticoid-regulated) belongs to the verprolin family of proteins which are characterized by the presence of a V domain (verprolin) at the N-terminal. Expression of CR16 suppressed the growth and endocytosis defect of vrp1Delta strain without correcting the actin patch polarization defect. The V domain of CR16 is critical for suppression of the growth defect of vrp1Delta strain but not for localisation to cortical actin patches. Mutations in the actin binding motif alone did not abolish the activity of CR16 but the mutations in combination with deletion of N-terminal proline rich motif abolished the ability of CR16 to suppress the growth defect. This suggests that the V domain of CR16 has two functionally redundant motifs and either one of these motifs is sufficient for suppressing the growth defect of vrp1Delta strain. This is in contrast to the observation that both WIP and WIRE require the actin binding motif for their activity.     

A nuclear localization domain in the hnRNP A1 protein

The heterogeneous nuclear RNP (hnRNP) A1 protein is one of the major pre-mRNA/mRNA binding proteins in eukaryotic cells and one of the most abundant proteins in the nucleus. It is localized to the nucleoplasm and it also shuttles between the nucleus and the cytoplasm. The amino acid sequence of A1 contains two RNP motif RNA-binding domains (RBDs) at the amino terminus and a glycine-rich domain at the carboxyl terminus. This configuration, designated 2x RBD-Gly, is representative of perhaps the largest family of hnRNP proteins. Unlike most nuclear proteins characterized so far, A1 (and most 2x RBD-Gly proteins) does not contain a recognizable nuclear localization signal (NLS). We have found that a segment of ca. 40 amino acids near the carboxyl end of the protein (designated M9) is necessary and sufficient for nuclear localization; attaching this segment to the bacterial protein beta- galactosidase or to pyruvate kinase completely localized these otherwise cytoplasmic proteins to the nucleus. The RBDs and another RNA binding motif found in the glycine-rich domain, the RGG box, are not required for A1 nuclear localization. M9 is a novel type of nuclear localization domain as it does not contain sequences similar to classical basic-type NLS. Interestingly, sequences similar to M9 are found in other nuclear RNA-binding proteins including hnRNP A2.     

Phosphorylation-dependent regulation of unique nuclear and nucleolar localization signals of LIM kinase 2 in endothelial cells

LIM kinases (LIMKs) regulate actin dynamics through cofilin phosphorylation and also have a function in the nucleus. Recently we have shown that LIMK2 shuttles between cytoplasm and nucleus in endothelial cells and that nuclear import is inhibited by protein kinase C-mediated phosphorylation of Ser-283. Here we aimed to identify the structural features of LIMK2 responsible for nuclear import. We found that the kinase domain of LIMK2 is localized exclusively in the nucleus and, in contrast to the kinase domain of LIMK1, it accumulated in the nucleolus. Through site-directed mutagenesis, we identified the basic amino acid-rich motif KKRTLRKNDRKKR (amino acids 491-503) as the functional nuclear and nucleolar localization signal of LIMK2. After fusing this motif to enhanced green fluorescent protein, the fusion protein localized exclusively in the nucleus and nucleolus. Mutagenesis studies showed that phosphorylation of Thr-494, a putative protein kinase C phosphorylation site identified within the nuclear localization signal, inhibits nuclear import of the enhanced green fluorescent protein-PDZ kinase domain of LIMK2. After inhibiting nuclear export with leptomycin B, phosphorylation of either Ser-283 or Thr-494 reduced the nuclear import of LIMK2. Phosphorylation of both Ser-283 and Thr-494 sites inhibited nuclear import completely. Our findings identify a unique basic amino acid-rich motif (amino acids 491-503) in LIMK2 which is not present in LIMK1 that serves to target the protein not only to the nucleus but also to the nucleolus. Phosphorylation of Thr-494 within this motif negatively regulates nuclear import of LIMK2.     

Na(+)/H(+) exchanger 1 directly binds to calcineurin A and activates downstream NFAT signaling, leading to cardiomyocyte hypertrophy

The calcineurin A (CaNA) subunit was identified as a novel binding partner of plasma membrane Na(+)/H(+) exchanger 1 (NHE1). CaN is a Ca(2+)-dependent phosphatase involved in many cellular functions, including cardiac hypertrophy. Direct binding of CaN to the (715)PVITID(720) sequence of NHE1, which resembles the consensus CaN-binding motif (PXIXIT), was observed. Overexpression of NHE1 promoted serum-induced CaN/nuclear factor of activated T cells (NFAT) signaling in fibroblasts, as indicated by enhancement of NFAT promoter activity and nuclear translocation, which was attenuated by NHE1 inhibitor. In neonatal rat cardiomyocytes, NHE1 stimulated hypertrophic gene expression and the NFAT pathway, which were inhibited by a CaN inhibitor, FK506. Importantly, CaN activity was strongly enhanced with increasing pH, so NHE1 may promote CaN/NFAT signaling via increased intracellular pH. Indeed, Na(+)/H(+) exchange activity was required for NHE1-dependent NFAT signaling. Moreover, interaction of CaN with NHE1 and clustering of NHE1 to lipid rafts were also required for this response. Based on these results, we propose that NHE1 activity may generate a localized membrane microdomain with higher pH, thereby sensitizing CaN to activation and promoting NFAT signaling. In cardiomyocytes, such signaling can be a pathway of NHE1-dependent hypertrophy.     

Nuclear ERM (ezrin, radixin, moesin) proteins: regulation by cell density and nuclear import

The highly conserved ERM (ezrin-radixin-moesin) family of proteins function as molecular linkers between the actin cytoskeleton and transmembrane receptors. We now provide unequivocal evidence that full-length endogenous ezrin and moesin also localise to the nucleus in two independent mammalian cell lines. All three ERM family members can localise to the nucleus upon exogenous expression of their GFP-tagged counterparts, suggesting a common family trend. Furthermore, Dmoesin, the Drosophila ERM homologue, is present in the nucleus of an insect cell line and can localise to the nucleus when exogenously expressed in MDCK cells. The nuclear localisation of endogenous ezrin and moesin is regulated by cell density and is resistant to detergent extraction, suggesting tight association with nuclear structures. Furthermore, phosphorylation in the actin-binding domain is not a prerequisite for nuclear localisation. We have identified a specific nuclear localisation sequence, which is conserved and functional in all ERM family members, implying specific regulated nuclear import. Although the precise nuclear function of the ERM proteins is unknown, these data provide further evidence that an increasing number of cytoskeletal components directly link the plasma membrane with nuclear events.     

Identification of a novel plant MAR DNA binding protein localized on chromosomal surfaces

We identified a novel nucleoplasm localized protein in Arabidopsis called AT-hook motif nuclear localized protein 1 (AHL1), which was isolated by visual screening of transformants using random GFP::cDNA fusions. AHL1 contains an AT-hook motif and unknown conserved PPC (plants and prokaryotes conserved) domain that includes a hydrophobic region. Approximately 30 paralogues were identified in the Arabidopsis genome. Proteins with PPC-like domains are found in Bacteria, Archaea and the plant kingdom, but in Bacteria and Archaea the PPC containing proteins of do not have an AT-hook motif. Thus, the PPC domain is evolutionary conserved and has a new function such as AT-rich DNA binding. AHL1 was mainly localized in the nucleoplasm, but little in the nucleolus and heterochromatic region, and was concentrated in the boundary region between euchromatin and heterochromatin. Biochemically, AHL1 was also found in the nuclear matrix fraction. In the M phase, AHL1 was localized on the chromosomal surface. The AT-hook motif was essential for matrix attachment region (MAR) binding, and the hydrophobic region of the PPC was indispensable for nuclear localization. Our results suggest that AHL1 is a novel plant MAR binding protein, which is related to the positioning of chromatin fibers in the nucleus by the presence of an AT-hook motif and PPC domain. In addition, AHL1 is located on the surface of chromosomes during mitosis.     

Angiotensin II stimulates α-skeletal actin expression in cadiomyocytes in vitro and in vivo in the absence of hypertension

Using a specific alpha-skeletal actin antibody, we have previously shown, that during hypertension-associated cardiac hypertrophy in the rat, the expression of alpha-skeletal actin in the myocardium is increased, but maintains focal distribution, compared to normotensive animals. In the present study, we have investigated whether alpha-skeletal actin expression can be induced in the absence of hypertension. For this purpose, we have examined transgenic mice overexpressing angiotensinogen exclusively in the heart. These animals are characterized by high cardiac angiotensin II levels and cardiac hypertrophy accompanied or not by high blood pressure depending on their genetic background, i.e. presence of one or two renin genes. Alpha-skeletal actin levels were highly increased in transgenic compared to wild-type myocardium independently of the number of renin genes, indicating that angiotensin II can stimulate alpha-skeletal actin expression in normotensive animals. Additional in vitro experiments using cultured mouse and rat cardiomyocytes showed that angiotension II not only increases alpha-skeletal actin expression but also induces an increase of its incorporation within II-bands compared to control cardiomyocytes. Angiotensin II increases also the expression of alpha-smooth muscle actin in sarcomeres of cardiomyocytes as well as in fibroblastic cells present within the culture.