Regulation Of Microtubule Formation Research Articles

Design of novel lead molecules against RhoG protein as cancer target – a computational study

Cancer is a class of diseases characterized by uncontrolled cell growth. Every year more than 2 million people are affected by the disease. Rho family proteins are actively involved in cytoskeleton regulation. Over-expression of Rho family proteins show oncogenic activity and promote cancer progression. In the present work RhoG protein is considered as novel target of cancer. It is a member of Rho family and Rac subfamily protein, which plays pivotal role in regulation of microtubule formation, cell migration and contributes in cancer progression. In order to understand the binding interaction between RhoG protein and the DH domain of Ephexin-4 protein, the 3D structure of RhoG was evaluated and Molecular Dynamic Simulations was performed to stabilize the structure. The 3D structure of RhoG protein was validated and active site identified using standard computational protocols. Protein–protein docking of RhoG with Ephexin-4 was done to understand binding interactions and the active site structure. Virtual screening was carried out with ligand databases against the active site of RhoG protein. The efficiency of virtual screening is analysed with enrichment factor and area under curve values. The binding free energy of docked complexes was calculated using prime MM-GBSA module. The SASA, FOSA, FISA, PISA and PSA values of ligands were carried out. New ligands with high docking score, glide energy and acceptable ADME properties were prioritized as potential inhibitors of RhoG protein.

Regulation of Microtubule Formation in Activated Mast Cells by Complexes of γ-Tubulin with Fyn and Syk Kinases

Aggregation of the high-affinity IgE receptors (FcepsilonRIs) on the surface of granulated mast cells initiates a chain of signaling events culminating in the release of allergy mediators. Although microtubules are involved in mast cell degranulation, the molecular mechanism that controls microtubule rearrangement after FcepsilonRI triggering is poorly understood. In this study, we show that the activation of bone marrow-derived mast cells (BMMCs) induced by FcepsilonRI aggregation or treatment with pervanadate leads to a rapid polymerization of microtubules. This polymerization was not dependent on the presence of Lyn kinase as determined by experiments with BMMCs isolated from Lyn-negative mice. One of the key regulators of microtubule polymerization is gamma-tubulin. Immunoprecipitation experiments revealed that gamma-tubulin from activated cells formed complexes with Fyn and Syk protein tyrosine kinases and several tyrosine phosphorylated proteins from both wild-type and Lyn(-/-) BMMCs. Pretreatment of the cells with Src-family or Syk-family selective tyrosine kinase inhibitors, PP2 or piceatannol, respectively, inhibited the formation of microtubules and reduced the amount of tyrosine phosphorylated proteins in gamma-tubulin complexes, suggesting that Src and Syk family kinases are involved in the initial stages of microtubule formation. This notion was corroborated by pull-down experiments in which gamma-tubulin complex bounds to the recombinant Src homology 2 and Src homology 3 domains of Fyn kinase. We propose that Fyn and Syk kinases are involved in the regulation of binding properties of gamma-tubulin and/or its associated proteins, and thus modulate the microtubule nucleation in activated mast cells.

A novel centrosome protein involved in regulation of microtubule formation

KIAA0092 is a former hypothetical protein possessing two coiled-coil segments. Upon over-expression in mammalian cells, KIAA0092 localizes and stabilizes microtubules (MTs) forming a remarkably stable ‘basket-like’ structure. Its C-terminal-half mutant retains the ability to stabilize MTs, whereas, the N-terminal-half localizes to centrosomes and appears to be involved in homo-multimerization. No basket structure but random aggregation was observed in cells pre-treated by nocodazole, implying that KIAA0092 hinders MT dynamics through inhibition of depolymerization. In in vitro tubulin polymerization studies, 1.25 uM recombinant KIAA0092 initiates polymerization of tubulin [12.5 uM] resulting in remarkably stable MTs, which tolerate prolonged storage at room temperature. In addition to MT formation, excess KIAA0092 leads to a 2-6 fold increase in OD350 as compared to MTs alone. Electron microscopy revealed that this increase is caused by aggregation of MTs mediated by KIAA0092. Localization of endogenous KIAA0092 at centrosomes was detected by two newly generated antibodies. Low-level expression of exogenous KIAA0092, insufficient to produce the basket structure and localized only to centrosomes, decreased the presence of mitotic cells, perhaps due to hindrance of endogenous protein function. Centrosomes have been shown to play a crucial role in MT regulation during mitosis. Knowing that KIAA0092 nucleates and enhances MT formation and that endogenous KIAA0092 is located at centrosomes, we propose that KIAA0092 plays a significant role in regulation of MT dynamics and mitosis during the cell cycle. (Supported by NIH PO1 HL 48807)

Loss of Hsp70-Hsp40 Chaperone Activity Causes Abnormal Nuclear Distribution and Aberrant Microtubule Formation in M-phase of Saccharomyces cerevisiae

The 70-kDa heat shock proteins, hsp70, are highly conserved among both prokaryotes and eukaryotes, and function as chaperones in diverse cellular processes. To elucidate the function of the yeast cytosolic hsp70 Ssa1p in vivo, we characterized a Saccharomyces cerevisiae ssa1 temperature-sensitive mutant (ssa1-134). After shifting to the restrictive temperature (37 degreesC), ssa1-134 mutant cells showed abnormal distribution of nuclei and accumulated as large-budded cells with a 2 N DNA content. We observed more prominent mutant phenotypes using nocodazole-synchronized cells: when cells were incubated at the restrictive temperature following nocodazole treatment, viability was rapidly lost and abnormal arrays of bent microtubules were formed. Chemical cross-linking and immunoprecipitation analyses revealed that the interaction of mutant Ssa1p with Ydj1p (cytosolic DnaJ homologue in yeast) was much weaker compared with wild-type Ssa1p. These results suggest that Ssa1p and Ydj1p chaperone activities play important roles in the regulation of microtubule formation in M phase. In support of this idea, a ydj1 null mutant at the restrictive temperature was found to exhibit more prominent phenotypes than ssa1-134. Furthermore, both ssa1-134 and ydj1 null mutant cells exhibited greater sensitivity to anti-microtubule drugs. Finally, the observation that SSA1 and YDJ1 interact genetically with a gamma-tubulin, TUB4, supports the idea that they play a role in the regulation of microtubule formation.

G protein beta subunit is closely associated with microtubules.

Previously, we have identified the association of G protein beta subunit (Gbeta) with mitotic spindles in various mammalian cells. Since microtubules are the main component of mitotic spindles, here we have isolated bovine brain microtubules and purified Gbeta subunit to identify the close association of Gbeta subunit with purified brain microtubules and have shown the direct incorporation of Gbeta subunit into the microtubules both in vitro and in vivo. It was found that: (1) microtubular fraction isolated from bovine brain contained Gbeta subunit, (2) coimmunoprecipitation demonstrated that Gbeta subunit could be coprecipitated with tubulin, (3) addition of purified Gbeta subunit into cytosolic extract for microtubule assembly caused direct incorporation of Gbeta subunit into assembled microtubules and increased the association of microtubule-associated proteins with microtubules, and (4) incubation of exogenous Gbeta subunit with detergent-permeabilized cells resulted in direct incorporation of Gbeta subunit into microtubule fibers and depolymerized tubulin molecules. We conclude that G protein beta subunit is closely associated with microtubules and may play an important role in the regulation of microtubule formation in addition to its regulatory role in cellular signal transduction.

P-81 - The role of brain protein phosphatases 1 and 2A in the regulation of microtubule formation

P-81 - The role of brain protein phosphatases 1 and 2A in the regulation of microtubule formation

Ca2+, Calmodulin‐Dependent Regulation of Microtubule Formation via Phosphorylation of Microtubule‐Associated Protein 2, τ Factor, and Tubulin, and Comparison with the Cyclic AMP‐Dependent Phosphorylation

Isolated microtubule-associated protein 2 (MAP2), tau factor, and tubulin were phosphorylated by a purified Ca2+, calmodulin-dependent protein kinase (640K enzyme) from rat brain. The phosphorylation of MAP2 and tau factor separately induced the inhibition of microtubule assembly, in accordance with the degree. Tubulin phosphorylation by the 640K enzyme induced the inhibition of microtubule assembly, whereas the effect of tubulin phosphorylation by the catalytic subunit was undetectable. The effects of tubulin and MAPs phosphorylation on microtubule assembly were greater than that of either tubulin or MAPs phosphorylation. Because MAP2, tau factor, and tubulin were also phosphorylated by the catalytic subunit of type-II cyclic AMP-dependent protein kinase from rat brain, the kinetic properties and phosphorylation sites were compared. The amount of phosphate incorporated into each microtubule protein was three to five times higher by the 640K enzyme than by the catalytic subunit. The Km values of the 640K enzyme for microtubule proteins were four to 24 times lower than those of the catalytic subunit. The peptide mapping analysis showed that the 640K enzyme and the catalytic subunit incorporated phosphate into different sites on MAP2, tau factor, and tubulin. Investigation of phosphoamino acids revealed that only the seryl residue was phosphorylated by the catalytic subunit, whereas both seryl and threonyl residues were phosphorylated by the 640K enzyme. These data suggest that the Ca2+, calmodulin system via phosphorylation of MAP2, tau factor, and tubulin by the 640K enzyme is more effective than the cyclic AMP system on the regulation of microtubule assembly.

Distribution and subcellular localization of calmodulin in adult and developing brain tissue

The distribution and subcellular localization of calmodulin in adult and developing cerebellum was studied in rats by immunocytochemistry. Calmodulin immunoreactivity was found both in neurons and in glial cells. Within neurons the staining was particularly intense in the cell nucleus and in dendrites, the cytoplasm of the cell body was more lightly stained than the nucleus, and light immunoreactivity was observed in axons. Electron microscopic analysis confirmed the association of calmodulin with the nuclear chromatin, while the nucleolus remained unstained. The reaction product was also found overlying the membranes of several organelles, in postsynaptic densities and decorating both dendritic and axonal microtubules. In developing Purkinje cells, calmodulin immunoreactivity was found as early as 5 days after birth. During the initial phases of dendritic development (5–10 days post-natal), the reaction product was associated with the organelles of the apical cone, while little or no staining was observed in the elongating dendrites or in the cell nucleus. Later in development, calmodulin was found in primary and secondary dendrites, and by 20 days after birth immunoreactivity appeared in the cell nucleus, and in the postsynaptic densities of immature spines located in dendrites. The presence of calmodulin in the apical cone suggests the possibility that this protein may participate in the regulation of microtubule formation during the initial stages of dendritic development. Its presence in dendrites at later stages (during the period of synaptogenesis) may indicate that it also participates in the formation of synapses between the parallel fibres and dendritic spines.

Binding of ATP to tubulin.

Microtubules are present in all eukaryotic cells and participate in a variety of cellular processes1. From recent studies it has become clear that nucleotides have a central role in the assembly of tubulin into microtubules2–5. The tubulin dimer binds two moles of guanine nucleotide: one (at the E-site) which can exchange with exogenous nucleotide and one (at the N-site) which does not exchange6. E-site GTP is hydrolysed during the assembly process4,7,8. Microtubule assembly can be induced by ATP through a contaminating nucleoside diphosphokinase activity that regenerates GTP following its hydrolysis—this is associated with tubulin polymerization4,9,10. Recent reports suggest that ATP has other effects on the assembly kinetics11–13, and we have shown14,15, using tubulin preparations lacking the nucleoside diphosphokinase activity, that ATP may play a critical part in the regulation of microtubule formation by binding to tubulin at a site which is distinct from the N- and E-sites. Kinetic data suggest a model in which ATP can act at the level of nucleation to stimulate assembly15. We now report the first direct evidence for the binding of ATP to tubulin and show that the corresponding dissociation constant is in a range of physiological significance.

Binding sites for calcium on tubulin.

Calcium ions can inhibit the in vitro assembly of microtubules and, therefore, may play a role in the regulation of microtubule formation in vivo. In order to test the validity of this hypothesis; the interaction between calcium and pruified brain microtubular protein has been investigated by standard binding assays. We have detected and characterized two classes of binding sites for calcium on tubulin, the major component of cytoplasmic microtubules. There is a single high-affinity site per tubulin molecule, characterized by a dissociation constant of 3.2 X 10(-6) M. That site is inhibited by magnesium (k1 = 5 X 10(-5) M) and potassium chloride. There are approximately 16 low-affinity sites which have a dissociation constant of 2.8 X 10(-4) M, and which are also inhibited by potassium chloride. Binding at the low-affinity sites is slightly enhanced by low magnesium concentrations. Both classes of sites are distinguishable from the colchicine binding site, and are apparently also distinct from the vinblastine and guanine nucleotide sites. The characteristics of the calcium binding activity of tubulin are similar to those found for the calcium-binding proteins of sarcoplasmic reticulum. The results are consistent with a physiological role for calcium in the regulation of microtubule assembly.