Query: GSK-1 inhibitor kinesin-5 Eg5

title: Structure of Microtubule Trapped Human Kinesin 5 and Its Mechanism of Inhibition Revealed Using Cryoelectron Microscopy
abstract: Kinesin 5 motors are vital mitotic spindle components, and disruption of their function perturbs cell division. We investigated the molecular mechanism of the human kinesin 5 inhibitor GSK 1, which allosterically promotes tight microtubule binding. GSK 1 inhibits monomeric human kinesin 5 ATPase and microtubule gliding activities, and promotes the motor's microtubule stabilization activity. Using cryoelectron microscopy, we determined the 3D structure of the microtubule bound motor GSK 1 at 3.8 A overall resolution. The structure reveals that GSK 1 stabilizes the microtubule binding surface of the motor in an ATP like conformation, while destabilizing regions of the motor around the empty nucleotide binding pocket. Density corresponding to GSK 1 is located between helix a4 and helix a6 in the motor domain at its interface with the microtubule. Using a combination of difference mapping and protein ligand docking, we characterized the kinesin 5 GSK 1 interaction and further validated this binding site using mutagenesis. This work opens up new avenues of investigation of kinesin inhibition and spindle perturbation.

title: Kinesin 5 Eg5 is essential for spindle assembly and chromosome alignment of mouse spermatocytes
abstract: Microtubule organization is essential for bipolar spindle assembly and chromosome segregation, which contribute to genome stability. Kinesin 5 Eg5 is known to be a crucial regulator in centrosome separation and spindle assembly in mammalian somatic cells, however, the functions and mechanisms of Eg5 in male meiotic cell division remain largely unknown.  In this study, we have found that Eg5 proteins are expressed in mouse spermatogonia, spermatocytes and spermatids. After Eg5 inhibition by specific inhibitors Monastrol, STLC and Dimethylenastron, the meiotic spindles of dividing spermatocytes show spindle collapse and the defects in bipolar spindle formation. We demonstrate that Eg5 regulates spindle bipolarity and the maintenance of meiotic spindles in meiosis. Eg5 inhibition leads to monopolar spindles, spindle abnormalities and chromosome misalignment in cultured GC 2 spd cells. Furthermore, Eg5 inhibition results in the decrease of the spermatids and the abnormalities in mature sperms.  Our results have revealed an important role of kinesin 5 Eg5 in male meiosis and the maintenance of male fertility. We demonstrate that Eg5 is crucial for bipolar spindle assembly and chromosome alignment in dividing spermatocytes. Our data provide insights into the functions of Eg5 in meiotic spindle assembly of dividing spermatocytes.

title: Src family kinase phosphorylation of the motor domain of the human kinesin 5, Eg5
abstract: Spindle formation in mammalian cells requires precise spatial and temporal regulation of the kinesin 5, Eg5, which generates outward force to establish spindle bipolarity. Our results demonstrate that Eg5 is phosphorylated in cultured cells by Src family kinases (SFKs) at three sites in the motor head: Y125, Y211, and Y231. Mutation of these sites diminishes motor activity in vitro, and replacement of endogenous Eg5 with phosphomimetic Y211 in LLC Pk1 cells results in monopolar spindles, consistent with loss of Eg5 activity. Cells treated with SFK inhibitors show defects in spindle formation, similar to those in cells expressing the nonphosphorylatable Y211 mutant, and distinct from inhibition of other mitotic kinases. We propose that this phosphoregulatory mechanism tunes Eg5 enzymatic activity for optimal spindle morphology.

title: SRC Phosphorylation Regulates the Human Kinesin 5, Eg5, and Disrupts the Binding of Eg5 Inhibitors
abstract: The human kinesin 5 motor, Eg5, is required to establish and maintain the mitotic spindle. We show that Src kinase binds to a unique motif in the microtubule binding interface of the Eg5 enzymatic head domain and phosphorylates three specific tyrosine residues in endogenous Eg5. These tyrosines are located near the nucleotide pocket and the functionally critical Loop 5 region within the Eg5 head. We have also found that phosphomimetic Eg5 motor proteins have altered motility characteristics relative to wild type and non phosphorylatable mutant proteins. Furthermore, cells expressing phosphomimetic Eg5 motors have increased spindle polarity defects. These results implicate Eg5 as a potential direct mitotic target of tyrosine kinases, most likely Src family kinases. Phosphomimetic motors also have greatly reduced affinity for the Eg5 inhibitor S trityl L cysteine (STLC) In cells with high Src activity, including many types of cancers, the same mechanism may provide rapid resistance to therapy with Eg5 inhibitors.

title: Src Kinase regulates the Human Kinesin 5, Eg5, by Phosphorylating Tyrosines in the Eg5 Motor Domain
abstract: The human kinesin 5, Eg5, is required to establish and maintain the mitotic spindle. using in silico, in vitro and cell culture methods, we show that Src kinase phosphorylates specific tyrosine residues in Eg5. These residues are located near the nucleotide pocket and the functionally critical L5 loop. Phosphomimetic and non phosphorylatable Eg5 mutant proteins have diminished ATPase activity and microtubule sliding relative to wild type Eg5. We also report that phosphomimetic proteins have greatly reduced affinity for the Eg5 inhibitor S trityl L cysteine. This finding suggests that Src phosphorylation of Eg5 may provide cells a non mutagenesis dependent strategy to evolve resistance to anti mitotic Eg5 inhibitors. In this case, combination treatment targeting both SFKs and Eg5 may inhibit mitosis more effectively than anti Eg5 treatment alone. Ultimately, Src phosphorylation of Eg5 represents a novel regulatory mechanism for a human kinesin, and links the chemical and physical processes that cause mitosis.

title: Terpendole E and its Derivative Inhibit STLC and GSK 1 Resistant Eg5
abstract: Terpendole E is first natural product found to inhibit mitotic kinesin Eg5, but its inhibitory mechanism remains to be revealed. Here, we report the effects of terpendole E and 11ketopaspaline (a new natural terpendole E analogue) on the Eg5 microtubule interaction and in several Eg5 mutants. 11 Ketopaspaline is a shunt product from terpendole E, and it shows potent inhibitory activity against the microtubule stimulated ATPase activity of Eg5. Unlike other Eg5 inhibitors, such as S trityl L cysteine (STLC) and GSK 1, both terpendole E and 11 ketopaspaline only partially inhibited Eg5 microtubule interaction. Furthermore, terpendole E and 11 ketopaspaline inhibited several Eg5 mutants that are resistant to STLC (Eg5D130A, Eg5L214A) or GSK 1 (Eg5I299F, Eg5A356T) but with the same extent of inhibition against wild type Eg5. Because Eg5D130A and Eg5L214A show cross resistance to most known Eg5 inhibitors, which bind the L5 loop, these results suggest that terpendole E and its analogues have a different binding site and/or inhibitory mechanism to those for L5 loop binding type Eg5 inhibitors.

title: Inhibition of the Motor Protein Eg5/Kinesin 5 in Amyloid b Mediated Impairment of Hippocampal Long Term Potentiation and Dendritic Spine Loss
abstract: Alzheimer's disease (AD) is characterized by neurofibrillary tangles, amyloid plaques, and neurodegeneration. However, this pathology is preceded by increased soluble amyloid beta (Ab) 1 42 oligomers that interfere with the glutamatergic synaptic plasticity required for learning and memory, including N methyl d aspartate receptor (NMDAR) dependent long term potentiation (LTP) In particular, soluble Ab(1 42) acutely inhibits LTP and chronically causes synapse loss. Many mechanisms have been proposed for Ab induced synaptic dysfunction, but we recently found that Ab(1 42) inhibits the microtubule motor protein Eg5/kinesin 5. Here we compared the impacts of Ab(1 42) and monastrol, a small molecule Eg5 inhibitor, on LTP in hippocampal slices and synapse loss in neuronal cultures. Acute (20 minute) treatment with monastrol, like Ab, completely inhibited LTP at doses >100 nM. In addition, 1 nM Ab(1 42) or 50 nM monastrol inhibited LTP #x223c;50% and when applied together caused complete LTP inhibition. At concentrations that impaired LTP, neither Ab(1 42) nor monastrol inhibited NMDAR synaptic responses until #x223c;60 minutes, when only #x223c;25% inhibition was seen for monastrol, indicating that NMDAR inhibition was not responsible for LTP inhibition by either agent when applied for only 20 minutes. Finally, 48 hours of treatment with either 0.5 1.0 mM Ab(1 42) or 1 5 mM monastrol reduced the dendritic spine/synapse density in hippocampal cultures up to a maximum of #x223c;40% and when applied together at maximal concentrations, no additional spine loss resulted. Thus, monastrol can mimic and in some cases occlude the impact of Ab on LTP and synapse loss, suggesting that Ab induces acute and chronic synaptic dysfunction in part through inhibiting Eg5.

title: Kinesin 5 inhibitor resistance is driven by kinesin 12
abstract: The kinesin 5 Eg5 is essential for mitotic progression, and the lethal effects of Eg5 inhibitors make these inhibitors attractive candidates for chemotherapy drugs. Sturgill et al. show that kinesin 12 and a nonmotile Eg5 mutant form an alternative spindle assembly pathway that provides resistance to Eg5 inhibitors.

title: Alzheimer amyloid beta inhibition of Eg5/kinesin 5 reduces neurotrophin and/or transmitter receptor function
abstract: The mechanism by which amyloid beta (Ab) causes neuronal dysfunction and/or death in Alzheimer's disease (AD) is unclear. Previously, we showed that Ab inhibits several microtubule dependent kinesin motors essential for mitosis and also present in mature neurons. Here, we show that inhibition of kinesin 5 (Eg5) by Ab blocks neuronal function by reducing transport of neurotrophin and neurotransmitter receptors to the cell surface. Specifically, cell surface NGF/NTR(p75) and NMDA receptors decline in cells treated with Ab or the kinesin 5 inhibitor monastrol, or expressing APP. Ab and monastrol also inhibit NGF dependent neurite outgrowth from PC12 cells and glutamate dependent Ca+ entry into primary neurons. Like Ab, monastrol inhibits long term potentiation, a cellular model of NMDA dependent learning and memory, and kinesin 5 activity is absent from APP/PS transgenic mice brain or neurons treated with Ab. These data imply that cognitive deficits in AD may derive in part from inhibition of neuronal Eg5 by Ab, resulting in impaired neuronal function and/or survival through receptor mislocalization. Preventing inhibition of Eg5 or other motors by Ab may represent a novel approach to AD therapy.

title: Kinesin 5 promotes microtubule nucleation and assembly by stabilizing a lattice competent conformation of tubulin
abstract: Besides sliding apart antiparallel microtubules during spindle elongation, the mitotic kinesin 5, Eg5 promotes microtubule polymerization, emphasizing its importance in mitotic spindle length control. Here, we characterize the Eg5 microtubule polymerase mechanism by assessing motor induced changes in the longitudinal and lateral tubulin tubulin bonds that form the microtubule lattice. Isolated Eg5 motor domains promote microtubule nucleation, growth and stability. Eg5 binds preferentially to microtubules over free tubulin, and colchicine like inhibitors that stabilize the bent conformation of tubulin allosterically inhibit Eg5 binding, consistent with a model in which Eg5 induces a curved to straight transition in tubulin. Domain swap experiments establish that the family specific Loop11, which resides near the nucleotide sensing Switch II domain, is necessary and sufficient for the polymerase activity of Eg5. Thus, we propose a microtubule polymerase mechanism in which Eg5 at the plus end promotes a curved to straight transition in tubulin that enhances lateral bond formation and thereby promotes microtubule growth and stability.