Myotonic dystrophy protein kinase domains mediate localization, oligomerization, novel catalytic activity, and autoinhibition

Human myotonic dystrophy protein kinase (DMPK) is a member of a novel class of multidomain protein kinases that regulate cell size and shape in a variety of organisms. However, little is currently known about the general properties of DMPK including domain function, substrate specificity, and potential mechanisms of regulation. Two forms of the kinase are expressed in muscle, DMPK-1 and DMPK-2. We demonstrate that the larger DMPK-1 form (the primary translation product) is proteolytically cleaved near the carboxy terminus to generate the smaller DMPK-2 form. We further demonstrate that the coiled-coil domain is required for DMPK oligomerization; coiled-coil mediated oligomerization also correlated with enhanced catalytic activity. DMPK was found to exhibit a novel catalytic activity similar to, but distinct from, related protein kinases such as protein kinase C and A, and the Rho kinases. We observed that recombinant DMPK-1 exhibits low activity, whereas the activity of carboxy-terminally truncated DMPK is increased approximately 3-fold. The inhibitory activity of the full-length kinase was mapped to what appears to be a pseudosubstrate autoinhibitory domain at the extreme carboxy terminus of DMPK. To date, endogenous activators of DMPK are unknown; however, we observed that DMPK purified from cells exposed to the G protein activator GTP-gamma-S exhibited an approximately 2-fold increase in activity. These results suggest a general model of DMPK regulation with two main regulatory branches: short-term activation of the kinase in response to G protein second messengers and long-term activation as a result of proteolysis.     

Implications of a poroelastic cytoplasm for the dynamics of animal cell shape

Two views have dominated recent discussions of the physical basis of cell shape change during migration and division of animal cells: the cytoplasm can be modeled as a viscoelastic continuum, and the forces that change its shape are generated only by actin polymerization and actomyosin contractility in the cell cortex. Here, we question both views: we suggest that the cytoplasm is better described as poroelastic, and that hydrodynamic forces may be generally important for its shape dynamics. In the poroelastic view, the cytoplasm consists of a porous, elastic solid (cytoskeleton, organelles, ribosomes) penetrated by an interstitial fluid (cytosol) that moves through the pores in response to pressure gradients. If the pore size is small (30-60nm), as has been observed in some cells, pressure does not globally equilibrate on time and length scales relevant to cell motility. Pressure differences across the plasma membrane drive blebbing, and potentially other type of protrusive motility. In the poroelastic view, these pressures can be higher in one part of a cell than another, and can thus cause local shape change. Local pressure transients could be generated by actomyosin contractility, or by local activation of osmogenic ion transporters in the plasma membrane. We propose that local activation of Na(+)/H(+) antiporters (NHE1) at the front of migrating cells promotes local swelling there to help drive protrusive motility, acting in combination with actin polymerization. Local shrinking at the equator of dividing cells may similarly help drive invagination during cytokinesis, acting in combination with actomyosin contractility. Testing these hypotheses is not easy, as water is a difficult analyte to track, and will require a joint effort of the cytoskeleton and ion physiology communities.     

Expression of typical osteoclast markers by PBMCs after PEG-induced fusion as a model for studying osteoclast differentiation

Bone is a metabolically active organ subjected to continuous remodeling process that involves resorption by osteoclast and subsequent formation by osteoblasts. Osteoclast involvement in this physiological event is regulated by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor κB ligand (RANKL). Fusion of mono-nuclear pre-osteoclasts is a critical event for osteoclast differentiation and for bone resorption. Here we show that PBMCs can be successfully fused with polyethylenglicol (PEG) in order to generated viable osteoclast-like cells that exhibit tartrate-resistant acid phosphatase (TRAP) and bone resorptive activities. PEG-fused PBMCs expressed additional markers compatible with osteoclastogenic differentiation such as carbonic anhydrase II (CAII), calcitonin receptor (CR), cathepsin K (Cat K), vacuolar ATPase (V-ATPase) subunit C1 (V-ATPase), integrin β3, RANK and cell surface aminopeptidase N/CD13. Actin redistribution in PEG-fused cells was found to be affected by cell cycle synchronization at G0/G1 or G2/M phases. PEG-induced fusion also led to expression of tyrosine kinases c-Src and Syk in their phosphorylated state. Scanning electron microscopy images showed morphological features typical of osteoclast-like cells. The results here shown allow concluding that PEG-induced fusion of PBMCs provides a suitable model system for understanding the mechanisms involved in osteoclastogenesis and for assaying new therapeutic strategies.     

Structure-function relationships in the stem cell's mechanical world B: emergent anisotropy of the cytoskeleton correlates to volume and shape changing stress exposure

In the preceding study (Part A), we showed that prescribed seeding conditions as well as seeding density can be used to subject multipotent stem cells (MSCs) to volume changing stresses and that changes in volume of the cell are associated with changes in shape, but not volume, of the cell nucleus. In the current study, we aim to control the mechanical milieu of live cells using these prescribed seeding conditions concomitant to delivery of shape changing stresses via fluid flow, while observing adaptation of the cytoskeleton, a major cellular transducer that modulates cell shape, stiffness and remodeling. We hypothesize that the spatiotemporal organization of tubulin and actin elements of the cytoskeleton changes in response to volume and shape changing stresses emulating those during development, prior to the first beating of the heart or twitching of muscle. Our approach was to quantify the change over baseline in spatiotemporal distribution of actin and tubulin in live C3H/10T1/2 model stem cells subjected to volume changing stresses induced by seeding at density as well as low magnitude, short duration, shape changing (shear) stresses induced by fluid flow (0.5 or 1.0 dyne/cm2 for 30/60/90 minutes). Upon exposure to fluid flow, both tubulin thickness (height) and concentration (fluorescence intensity) change significantly over baseline, as a function of proximity to neighboring cells (density) and the substrate (apical-basal height). Given our recently published studies showing amplification of stress gradients (flow velocity) with increasing distance to nearest neighbors and the substrate, i.e. with decreasing density and toward the apical side of the cell, tubulin adaptation appears to depend significantly on the magnitude of the stress to which the cell is exposed locally. In contrast, adaptation of actin to the changing mechanical milieu is more global, exhibiting less significant differences attributable to nearest neighbors or boundaries than differences attributable to magnitude of the stress to which the cell is exposed globally (0.5 versus 1.0 dyne/cm2). Furthermore, changes in the actin cytoskeletal distribution correlate positively with one pre-mesenchymal condensation marker (Msx2) and negatively with early markers of chondrogenesis (ColIIaI alone, indicative of pre-hypertrophic chondrogenesis) and osteogenesis (Runx2). Changes in the tubulin cytoskeletal distribution correlate positively with a marker of pericondensation (Sox9 alone), negatively with chondrogenesis (ColIIaI) and positively with adipogenesis (Ppar-gamma 2). Taken as a whole, exposure of MSCs to volume and shape changing stresses results in emergent anisotropy of cytoskeletal architecture (structure), which relate to emergent cell fate (function).     

A mitotic role for a novel fission yeast protein kinase dsk1 with cell cycle stage dependent phosphorylation and localization.

The fission yeast dsk1+ gene, a multicopy suppressor for cold-sensitive dis1 mutants, encodes a novel 61-kd protein kinase. It is a phosphoprotein, and phosphoserine is the major phosphorylated amino acid. Hyperphosphorylation of dsk1 causes a mobility shift, resulting in two dsk1-specific protein bands. The phosphorylation pattern is strikingly altered when cell cycle progression is delayed or arrested. The slowly migrating phosphorylated form is prominent in mitotically arrested cells, and the fast migrating form is enriched in interphase-arrested cells. dsk1 is a protein kinase. It auto-phosphorylates as well as phosphorylates myelin basic protein (MBP). Phosphotyrosine as well as phosphoserine/threonine were found in autophosphorylation, but no tyrosine phosphorylation occurs when MBP was used as the substrate. The dsk1 immunoprecipitates from mitotically arrested cells have a several-fold higher kinase activity than that from wild type. The haploid gene disruptant is viable, indicating that the dsk1+ gene is non-essential for viability. High dosage of dsk1+, however, strongly delays the G2/M progression. Immunofluorescence microscopy using anti-dsk1 antibody shows that localization pattern of dsk1 protein strikingly alters depending on cell cycle stages. In G2-arrested cells, dsk1 locates in the cytoplasm, whereas in mitotically arrested cells, nuclear stain is intense. In wild-type cells, nuclear stain is seen only in mitotic cells. Hence dsk1 protein may play an important role in mitotic control by altering cellular location, degree of phosphorylation and kinase activity. We discuss possible roles of dsk1 kinase as an add-on regulator in mitosis.     

HIV-1 Vif protein mediates the degradation of APOBEC3G in fission yeast when over-expressed using codon optimization

Interaction between the HIV-1 Vif protein and the cellular host APOBEC3G protein is a promising target for inhibition of HIV-1 replication. Considering that human cells are a very complicated environment for the study of protein interactions, the goal of this study was to check whether fission yeast could be used as a model cell for studying the Vif-APOBEC3G interaction. Vif and APOBEC3G were expressed in fusion with GFP protein in the S. pombe SP223 strain. Subcellular localizations of Vif and APOBEC3G were observed with fluorescent microscopy. Codon optimization was used to over express the Vif protein in S. pombe cells. The degradation of APOBEC3G mediated by Vif was tested through expressing Vif and GFP-APOBEC3G proteins in the same cell. Western Blot analysis was used to measure the corresponding protein levels under different experimental conditions. The results showed that the Vif protein was predominantly localized in the nucleus of S.pombe cells, APOBEC3G was localized in the cytoplasm and concentrated at punctate bodies that were often in close proximity to the nucleus but were not necessarily restricted from other regions in the cytoplasm. Vif protein expression levels were increased significantly by using codon optimization and APOBEC3G was degraded when Vif was over-expressed in the same S. pombe cells. These results indicate that fission yeast is a good model for studying the interaction between the Vif and APOBEC3G proteins.     

Antigen receptor-induced cell cycle arrest in WEHI-231 B lymphoma cells depends on the duration of signaling before the G1 phase restriction point.

Stimulation of antigen receptors on WEHI-231 B lymphoma cells with anti-receptor antibodies (anti-immunoglobulin M [IgM]) causes irreversible growth arrest. This may be a model for antigen-induced tolerance to self components in the immune system. Antigen receptor stimulation also causes inositol phospholipid hydrolysis, producing diacylglycerol, which activates protein kinase C, and inositol 1,4,5-trisphosphate, which causes release of calcium from intracellular stores. To better understand the nature of the antigen receptor-induced growth arrest of WEHI-231 cells, we have examined the basis for it. WEHI-231 cells in various phases of the cell cycle were isolated by centrifugal elutriation, and their response was evaluated following treatment with either anti-IgM or pharmacologic agents that raise intracellular free calcium levels and activate protein kinase C. Treatment with anti-IgM or the pharmacologic agents did not lengthen the cell cycle. Instead, growth inhibition was solely the result of arrest in the G1 phase. The efficiency of G1 arrest increased with the length of time during which the cells received signaling before reaching the G1 phase arrest point. Maximum efficiency of arrest was achieved after approximately one cell cycle of receptor signaling. These results imply that anti-IgM causes G1 arrest of WEHI-231 cells by slowly affecting components required for S phase progression, rather than by rapidly inhibiting such components or by rapidly activating a suicide mechanism. Antigen receptor stimulation was twice as effective as stimulation via the mimicking reagents phorbol dibutyrate and ionomycin. Thus, although the phosphoinositide second messengers diacylglycerol and calcium probably play roles in mediating the effects of anti-IgM on WEHI-231 cells, other second messengers may also be involved.     

Nuclear diacylglycerol kinases: emerging downstream regulators in cell signaling networks

There exists an active lipid metabolism in the nucleus, which is regulated differentially from the lipid metabolism taking place elsewhere in the cell. Evidence has been accumulated that nuclear lipid metabolism is closely involved in a variety of cell responses, including proliferation, differentiation, and apoptosis. A fundamental lipid second messenger which is generated in the nucleus is diacylglycerol, that is mainly known for its role as an activator of some protein kinase C isoforms. Diacylglycerol kinases attenuate diacylglycerol signaling by converting this lipid to phosphatidic acid, which also has signaling functions. Ten mammalian diacylglycerol kinase isoforms have been cloned so far, and some of them are found also in the nucleus, either as resident proteins or after migration from cytoplasm in response to various agonists. Experiments using cultured cells have demonstrated that nuclear diacylglycerol kinases have prominent roles in cell cycle regulation and differentiation. In this review, the emerging roles played by diacylglycerol kinases in the nucleus, such as the control of G1/S phase transition, are discussed.     

Ca2+ and intracellular signalling in plant cells: a role in phytochrome transduction.

The role of Ca2+ as a mediator and its participation in intracellular signaling in relation to phytochrome regulatory action are considered. Using the literature data, the non-random distribution of Ca2+ in the plant cell cytoplasm, the mechanisms of Ca2+ homeostasis and the function of putative messengers of Ca2+ are described. On the basis of the data obtained at the author's laboratory, a dual effect of phytochrome on the cytosolic Ca2+ concentration is demonstrated, i.e. phytochrome controls Ca2+ permeability of plasma membrane and induces transient changes in [Ca2+]cyt. Evidence is presented that the endoplasmic reticulum and vacuole are intracellular Ca2+ stores in the plant cell sensitive to phytochrome regulatory action. It is also demonstrated that phytochrome controls the metabolism of a key phosphoinositide in plant cell membranes. It stimulates the hydrolysis of phosphatidyl 4,5-bisphosphate, a precursor of inositol 1,4,5-trisphosphate, the potent mobilizer of Ca2+ in cytoplasm. Finally, the data on the close interactions between Ca2+ and other secondary messengers in the plant cell cytoplasm are presented. Using the transgenic tobacco expressing an apoprotein of Ca(2+)-sensitive photoprotein aequorin, evidence was obtained that cyclic mononucleotides (cGMP, cAMP) control the Ca2+ concentration in the cytoplasm testifying a crosstalk among Ca2+ and cyclic mononucleotides as messengers.     

Cholesterol is superior to 7-ketocholesterol or 7 alpha-hydroxycholesterol as an allosteric activator for acyl-coenzyme A:cholesterol acyltransferase 1

We compared the abilities of cholesterol versus various oxysterols as substrate and/or as activator for the enzyme acyl-coenzyme A:cholesterol acyltransferase (ACAT), by monitoring the activity of purified human ACAT1 in response to sterols solubilized in mixed micelles or in reconstituted vesicles. The results showed that 5 alpha,6 alpha-epoxycholesterol and 7 alpha-hydroxycholesterol are comparable with cholesterol as the favored substrates, whereas 7-ketocholesterol, 7 beta-hydroxycholesterol, 5 beta,6 beta-epoxycholesterol, and 24(S),25-epoxycholesterol are very poor substrates for the enzyme. We then tested the ability of 7-ketocholesterol as an activator when cholesterol was measured as the substrate, and vice versa. When cholesterol was measured as the substrate, the addition of 7-ketocholesterol could not activate the enzyme. In contrast, when 7-ketocholesterol was measured as the substrate, the addition of cholesterol significantly activated the enzyme and changed the shape of the substrate saturation curve from sigmoidal to essentially hyperbolic. Additional results show that, as an activator, cholesterol is much better than all the oxysterols tested. These results suggest that ACAT1 contains two types of sterol binding sites; the structural requirement for the ACAT activator site is more stringent than it is for the ACAT substrate site. Upon activation by cholesterol, ACAT1 becomes promiscuous toward various sterols as its substrate.     

Diffusion of the second messengers in the cytoplasm acts as a variability suppressor of the single photon response in vertebrate phototransduction

The single photon response in vertebrate phototransduction is highly reproducible despite a number of random components of the activation cascade, including the random activation site, the random walk of an activated receptor, and its quenching in a random number of steps. Here we use a previously generated and tested spatiotemporal mathematical and computational model to identify possible mechanisms of variability reduction. The model permits one to separate the process into modules, and to analyze their impact separately. We show that the activation cascade is responsible for generation of variability, whereas diffusion of the second messengers is responsible for its suppression. Randomness of the activation site contributes at early times to the coefficient of variation of the photoresponse, whereas the Brownian path of a photoisomerized rhodopsin (Rh*) has a negligible effect. The major driver of variability is the turnoff mechanism of Rh*, which occurs essentially within the first 2-4 phosphorylated states of Rh*. Theoretically increasing the number of steps to quenching does not significantly decrease the corresponding coefficient of variation of the effector, in agreement with the biochemical limitations on the phosphorylated states of the receptor. Diffusion of the second messengers in the cytosol acts as a suppressor of the variability generated by the activation cascade. Calcium feedback has a negligible regulatory effect on the photocurrent variability. A comparative variability analysis has been conducted for the phototransduction in mouse and salamander, including a study of the effects of their anatomical differences such as incisures and photoreceptors geometry on variability generation and suppression.     

Diacylglycerol kinase zeta is associated with chromatin, but dissociates from condensed chromatin during mitotic phase in NIH3T3 cells

Diacylglycerol kinase (DGK) converts diacylglycerol (DG) to phosphatidic acid, both of which act as second messengers to mediate a variety of cellular mechanisms. Therefore, DGK contributes to the regulation of these messengers in cellular signal transduction. Of DGK isozymes cloned, DGKzeta is characterized by a nuclear localization signal that overlaps with a sequence similar to the myristoylated alanine-rich C-kinase substrate. Previous studies showed that nuclear DG is differentially regulated from plasma membrane DG and that the nuclear DG levels fluctuate in correlation with cell cycle progression, suggesting the importance of nuclear DG in cell cycle control. In this connection, DGKzeta has been shown to localize to the nucleus in fully differentiated cells, such as neurons and lung cells, although it remains elusive how DGK behaves during the cell cycle in proliferating cells. Here we demonstrate that DGKzeta localizes to the nucleus during interphase including G1, S, and G2 phases and is associated with chromatin although it dissociates from condensed chromatin during mitotic phase in NIH3T3 cells. Furthermore, this localization pattern is also observed in proliferating spermatogonia in the testis. These results suggest a reversible association of DGKzeta with histone or its related proteins in cell cycle, plausibly dependent on their post-translational modifications.     

Nuclear protein kinase activities during the cell cycle of HeLa S3 cells

To ascertain the activity and substrate specificity of nuclear protein kinases during various stages of the cell cycle of HeLa S3 cells, a nuclear phospho-protein-enriched sample was extracted from synchronised cells and assayed in vitro in the presence of homologous substrates. The nuclear protein kinases increased in activity during S and G2 phase to a level that was twice that of kinases from early S phase cells. The activity was reduced during mitosis but increased again in G1 phase. When the phosphoproteins were separated into five fractions by cellulose-phosphate chromatography each fraction, though not homogenous, exhibited differences in activity. Variations in the activity of the protein kinase fractions were observed during the cell cycle, similar to those observed for the unfractionated kinases. Sodium dodecyl sulfate polyacrylamide gel electrophoretic analysis of the proteins phosphorylated by each of the five kinase fractions demonstrated a substrate specificity. The fractions also exhibited some cell cycle stage-specific preference for substrates; kinases from G1 cells phosphorylated mainly high molecular weight polypeptides, whereas lower molecular weight species were phosphorylated by kinases from the S, G2 and mitotic stages of the cell cycle. Inhibition of DNA and histone synthesis by cytosine arabinoside had no effect on the activity or substrate specificity of S phase kinases. Some kinase fractions phosphorylated histones as well as non-histone chromosomal proteins and this phosphorylation was also cell cycle stage dependent. The presence of histones in the in vitro assay influenced the ability of some fractions to phosphorylate particular non-histone polypeptides; non-histone proteins also appeared to affect the in vitro phosphorylation of histones.     

A pre-start checkpoint preventing mitosis in fission yeast acts independently of p34cdc2 tyrosine phosphorylation.

We have monitored the tyrosine (Y15) phosphorylated and dephosphorylated forms of p34cdc2 from Schizosaccharomyces pombe as cells proceed through the cell cycle. Y15 is dephosphorylated in G1 before start and becomes phosphorylated only after cells pass start and enter late G1. This transition is associated with a switch from one checkpoint which restrains mitosis in pre-start G1, by a mechanism independent from Y15 phosphorylation, to a second checkpoint acting post-start during late G1 and S phase operating through Y15 phosphorylation. The pre-start checkpoint may act by preventing formation of the p34cdc2/p56cdc13 complex. The complex between Y15-phosphorylated p34cdc2 and p56cdc13 accumulates during S phase and G2, but the level generated is not solely dependent on the amount of p34cdc2 and p56cdc13 present in the cell. The extent of p56cdc13 breakdown at the end of mitosis may be determined by the amount complexed with p34cdc2. We have also shown that an insoluble form of p34cdc2 is associated with the progression of the cell through late G1 into S phase.     

Membrane movements and fluidity during rotational motility of a termite flagellate. A freeze-fracture study

Freeze-fracture electron microscopy was used to examine the structure of a region of plasma membrane that undergoes continual, unidirectional shear. Membrane shear arises from the continual clockwise rotation of one part (head) of a termite flagellate relative to the rest of the cell. Freeze-fracture replicas show that the lipid bilayer is continuous across the shear zone. Thus, the relative movements of adjacent membrane regions are visible evidence of membrane fluidity. The distribution and density of intramembrane particles within the membrane of the shear zone is not different from that in other regions of the cell membrane. Also, an additional membrane shear zone arises when body membrane becomes closely applied to the rotating axostyle as cells change shape in vitro. This suggests that the entire membrane is potentially as fluid as the membrane between head and body but that this fluidity is only expressed at certain locations for geometrical and/or mechanical reasons. Membrane movements may be explained solely by cell shape and proximity to rotating structures, although specific membrane-cytoskeletal connections cannot be ruled out. The membrane of this cell may thus be viewed as a fluid which adheres to the underlying cytoplasm/cytoskeleton and passively follows its movements.     

Architecture of tissue cells the structural basis which determines shape and locomotion of cells

Shape and locomotion of tissue cells depend on the interaction of elements of the cytoskeleton, adhesion to the substrate and an intracellular hydrostatic pressure. The existence of this pressure becomes obvious from increase in cell volume on cessation of contractile forces and from observations with ultrasound acoustic microscopy. Wherever such an internal pressure is established, it is involved in generation of shape and driving force of cell locomotion. Therefore each hypothesis on cell shape and locomotion must consider this property of a living cell. Apparently different types of locomotion depend on differences in substrate adhesion and/or cytoskeleton organisation.     

How Listeria exploits host cell actin to form its own cytoskeleton. II. Nucleation, actin filament polarity, filament assembly, and evidence for a pointed end capper

Processes such as cell locomotion and morphogenesis depend on both the generation of force by cytoskeletal elements and the response of the cell to the resulting mechanical loads. Many widely accepted theoretical models of processes involving cell shape change are based on untested hypotheses about the interaction of these two components of cell shape change. I have quantified the mechanical responses of cytoplasm to various chemical environments and mechanical loading regimes to understand better the mechanisms of cell shape change and to address the validity of these models. Measurements of cell mechanical properties were made with strands of cytoplasm submerged in media containing detergent to permeabilize the plasma membrane, thus allowing control over intracellular milieu. Experiments were performed with equipment that generated sinusoidally varying length changes of isolated strands of cytoplasm from Physarum polycephalum. Results indicate that stiffness, elasticity, and viscosity of cytoplasm all increase with increasing concentration of Ca2+, Mg2+, and ATP, and decrease with increasing magnitude and rate of deformation. These results specifically challenge assumptions underlying mathematical models of morphogenetic events such as epithelial folding and cell division, and further suggest that gelation may depend on both actin cross-linking and actin polymerization.     

Aurora-A and an interacting activator,the LIM protein Ajuba,are required for mitotic commitment in human cells

Aurora family kinases contribute to regulation of mitosis. Using RNA interference in synchronized HeLa cells, we now show that Aurora-A is required for mitotic entry. We found that initial activation of Aurora-A in late G2 phase of the cell cycle is essential for recruitment of the cyclin B1-Cdk1 complex to centrosomes, where it becomes activated and commits cells to mitosis. A two-hybrid screen identified the LIM protein Ajuba as an Aurora-A binding protein. Ajuba and Aurora-A interact in mitotic cells and become phosphorylated as they do so. In vitro analyses revealed that Ajuba induces the autophosphorylation and consequent activation of Aurora-A. Depletion of Ajuba prevented activation of Aurora-A at centrosomes in late G2 phase and inhibited mitotic entry. Overall, our data suggest that Ajuba is an essential activator of Aurora-A in mitotic commitment.