The effect of ADF/cofilin and profilin on the dynamics of monomeric actin

The main goal of the work was to uncover the dynamical changes in actin induced by the binding of cofilin and profilin. The change in the structure and flexibility of the small domain and its function in the thermodynamic stability of the actin monomer were examined with fluorescence spectroscopy and differential scanning calorimetry (DSC). The structure around the C-terminus of actin is slightly affected by the presence of cofilin and profilin. Temperature dependent fluorescence resonance energy transfer measurements indicated that both actin binding proteins decreased the flexibility of the protein matrix between the subdomains 1 and 2. Time resolved anisotropy decay measurements supported the idea that cofilin and profilin changed similarly the dynamics around the fluorescently labeled Cys-374 and Lys-61 residues in subdomains 1 and 2, respectively. DSC experiments indicated that the thermodynamic stability of actin increased by cofilin and decreased in the presence of profilin. Based on the information obtained it is possible to conclude that while the small domain of actin acts uniformly in the presence of cofilin and profilin the overall stability of actin changes differently in the presence of the studied actin binding proteins. The results support the idea that the small domain of actin behaves as a rigid unit during the opening and closing of the nucleotide binding pocket in the presence of profilin and cofilin as well. The structural arrangement of the nucleotide binding cleft mainly influences the global stability of actin while the dynamics of the different segments can change autonomously.     

Actin Dynamics Regulated by the Balance of Neuronal Wiskott-Aldrich Syndrome Protein (N-WASP) and Cofilin Activities Determines the Biphasic Response of Glucose-induced Insulin Secretion

Actin dynamics in pancreatic β-cells is involved in insulin secretion. However, the molecular mechanisms of the regulation of actin dynamics by intracellular signals in pancreatic β-cells and its role in phasic insulin secretion are largely unknown. In this study, we elucidate the regulation of actin dynamics by neuronal Wiskott-Aldrich syndrome protein (N-WASP) and cofilin in pancreatic β-cells and demonstrate its role in glucose-induced insulin secretion (GIIS). N-WASP, which promotes actin polymerization through activation of the actin nucleation factor Arp2/3 complex, was found to be activated by glucose stimulation in insulin-secreting clonal pancreatic β-cells (MIN6-K8 β-cells). Introduction of a dominant-negative mutant of N-WASP, which lacks G-actin and Arp2/3 complex-binding region VCA, into MIN6-K8 β-cells or knockdown of N-WASP suppressed GIIS, especially the second phase. We also found that cofilin, which severs F-actin in its dephosphorylated (active) form, is converted to the phosphorylated (inactive) form by glucose stimulation in MIN6-K8 β-cells, thereby promoting F-actin remodeling. In addition, the dominant-negative mutant of cofilin, which inhibits activation of endogenous cofilin, or knockdown of cofilin reduced the second phase of GIIS. However, the first phase of GIIS occurs in the G-actin predominant state, in which cofilin activity predominates over N-WASP activity. Thus, actin dynamics regulated by the balance of N-WASP and cofilin activities determines the biphasic response of GIIS.     

O-GlcNAcylation of Cofilin Promotes Breast Cancer Cell Invasion

O-GlcNAcylation is a post-translational modification that regulates a broad range of nuclear and cytoplasmic proteins and is emerging as a key regulator of various biological processes. Previous studies have shown that increased levels of global O-GlcNAcylation and O-GlcNAc transferase (OGT) are linked to the incidence of metastasis in breast cancer patients, but the molecular basis behind this is not fully known. In this study, we have determined that the actin-binding protein cofilin is O-GlcNAcylated by OGT and mainly, if not completely, mediates OGT modulation of cell mobility. O-GlcNAcylation at Ser-108 of cofilin is required for its proper localization in invadopodia at the leading edge of breast cancer cells during three-dimensional cell invasion. Loss of O-GlcNAcylation of cofilin leads to destabilization of invadopodia and impairs cell invasion, although the actin-severing activity or lamellipodial localization is not affected. Our study provides insights into the mechanism of post-translational modification in fine-tuning the regulation of cofilin activity and suggests its important implications in cancer metastasis.     

Mammalian and Malaria Parasite Cyclase-associated Proteins Catalyze Nucleotide Exchange on G-actin through a Conserved Mechanism

Cyclase-associated proteins (CAPs) are among the most highly conserved regulators of actin dynamics, being present in organisms from mammals to apicomplexan parasites. Yeast, plant, and mammalian CAPs are large multidomain proteins, which catalyze nucleotide exchange on actin monomers from ADP to ATP and recycle actin monomers from actin-depolymerizing factor (ADF)/cofilin for new rounds of filament assembly. However, the mechanism by which CAPs promote nucleotide exchange is not known. Furthermore, how apicomplexan CAPs, which lack many domains present in yeast and mammalian CAPs, contribute to actin dynamics is not understood. We show that, like yeast Srv2/CAP, mouse CAP1 interacts with ADF/cofilin and ADP-G-actin through its N-terminal α-helical and C-terminal β-strand domains, respectively. However, in the variation to yeast Srv2/CAP, mouse CAP1 has two adjacent profilin-binding sites, and it interacts with ATP-actin monomers with high affinity through its WH2 domain. Importantly, we revealed that the C-terminal β-sheet domain of mouse CAP1 is essential and sufficient for catalyzing nucleotide exchange on actin monomers, although the adjacent WH2 domain is not required for this function. Supporting these data, we show that the malaria parasite Plasmodium falciparum CAP, which is entirely composed of the β-sheet domain, efficiently promotes nucleotide exchange on actin monomers. Collectively, this study provides evidence that catalyzing nucleotide exchange on actin monomers via the β-sheet domain is the most highly conserved function of CAPs from mammals to apicomplexan parasites. Other functions, including interactions with profilin and ADF/cofilin, evolved in more complex organisms to adjust the specific role of CAPs in actin dynamics.     

Alzheimer's disease pathogenesis: The role of disturbed sleep in attenuated brain plasticity and neurodegenerative processes

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by cognitive impairments. The classical symptoms of the disease include gradual deterioration of memory and language. Epidemiological studies indicate that around 25–40% of AD patients have sleep-wake cycle disturbances. Importantly, a series of studies suggested that the relationship between AD and sleep disturbance may be complex and bidirectional. Indeed, accumulation of the extracellular neuronal protein amyloid-beta (Aβ) leads to altered sleep-wake behavior in both mice and humans. At the same time, disturbances of the normal sleep-wake cycle may facilitate AD pathogenesis. This paper will review the mechanisms underlying this potential interrelated connection including locus coeruleus damage, reductions in orexin neurotransmission, alterations in melatonin levels, and elevated cytokine levels. In addition, we will also highlight how both the development of AD and sleep disturbances lead to changes in intracellular signaling pathways involved in regulating neuronal plasticity and connectivity, particularly extremes in cofilin phosphorylation. Finally, current pharmacological and nonpharmacological therapeutic approaches will be discussed.     

Identification of differentially expressed genes like cofilin2 in growing collateral arteries

Arteriogenesis, the growth of pre-existing collateral arteries, can be induced in rabbits by occlusion of the femoral artery. In order to analyze the differential gene expression in arteriogenesis, cDNA of collateral arteries 24h after femoral occlusion or sham operation was subjected to suppression subtractive hybridization (SSH). We demonstrated an upregulation of the U6 snRNA binding protein Lsm5, cytochrome b, an expressed sequence tag, and the actin-depolymerizing factor cofilin2 mRNA in collateral arteries 24h after femoral ligation. For cofilin2, we also detected an increase in the protein level and a localization predominantly in smooth muscle cells of collaterals. Simultaneously with the upregulation of cofilin2 we found a downregulation of the alpha-smooth muscle actin mRNA in growing collateral arteries. In summary, our data showed an augmented expression level of genes contributing to different fundamental processes of arteriogenesis.     

Cofilin phosphorylation and actin polymerization by NRK/NESK,a member of the germinal center kinase family

Nck-interacting kinase (NIK)-related kinase (NRK)/NIK-like embryo-specific kinase (NESK) is a protein kinase that belongs to the germinal center kinase family, and activates the c-Jun N-terminal kinase (JNK) signaling pathway. In this study, we examined the effect of NRK/NESK on actin cytoskeletal organization. Overexpression of NRK/NESK in COS7 cells induced accumulation of polymerized actin at the perinuclear. Phosphorylation of cofilin, an actin-depolymerizing factor, was increased in NRK/NESK-expressing HEK 293T cells. In addition, in vitro phosphorylation of cofilin was observed on NRK/NESK immunoprecipitates from HEK 293T cells expressing the kinase domain of NRK/NESK. The cofilin phosphorylation occurred at the serine residue of position 3 (Ser-3). Since the phosphorylation at Ser-3 inactivates the actin-depolymerizing activity of cofilin, these results suggest that NRK/NESK induces actin polymerization through cofilin phosphorylation. The cofilin phosphorylation did not appear to be mediated through activation of LIM-kinasel, a cofilin-phosphorylating kinase, or through the activation of JNK. Thus, cofilin is likely to be a direct substrate of NRK/NESK. NRK/NESK is predominantly expressed in skeletal muscle during the late stages of mouse embryogenesis. Thus, NRK/NESK may be involved in the regulation of actin cytoskeletal organization in skeletal muscle cells through cofilin phosphorylation.     

Proteomic profiling and identification of cofilin responding to oxidative stress in vascular smooth muscle

We used 2-DE and MALDI-TOF/TOF to identify proteins of vascular smooth muscle cells whose expression was or was not altered by exposure to 500 microM H2O2 for 30 min. We detected more than 800 proteins on silver-stained gels of whole protein extracts from rat aortic smooth muscle strips. Of these proteins, 135 clearly unaffected and 19 having levels altered by exposure to H2O2 were identified. Protein characterization revealed that the most prominent vascular smooth muscle proteins were those with antioxidant, cytoskeletal structure, or muscle contraction. In addition, cofilin, an isoform of the actin depolymerizing factor family, shifted to its basic site on the 2-DE gel as a result of H2O2 treatment. In Western blot analysis of proteins from A7r5 aortic smooth muscle cells, the phosphorylation, but not the expression, of cofilin was decreased by H2O2 in a dose-dependent manner. The H2O2-induced dephosphorylation of cofilin and apoptosis was inhibited by Na3VO4, an inhibitor of protein tyrosine phosphatase (PTP). These results suggest that cofilin is one of the proteins regulated by H2O2 treatment in vascular smooth muscle, and has an important role in the induction of vascular apoptosis through PTP-dependent mechanisms.     

Extracellular potassium deprivation reversibly dephosphorylates cofilin

Cofilin is essential for actin filament remodeling in various cell types. We previously reported that ouabain leads to dephosphorylation of cofilin, resulting in cytoskeletal reorganization and cell volume change. We report here that the extracellular [K(+)](e) deprivation causes dephosphorylation of cofilin with disruption of cytoskeletal structures and cell shrinkage. These changes can be reversed by time-dependent restoration of [K(+)](e). Thus, these results suggest that the acute shift of [K(+)](e) triggers a reversible change of cofilin phosphorylation which may be an essential event in the regulation of cytoskeletal architecture and cell volume.