Phosphorylation of α-tubulin by protein kinase C stimulates microtubule dynamics in human breast cells (original) (raw)
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Intrinsic and Extrinsic Factors Affecting Microtubule Dynamics in Normal and Cancer Cells
Molecules
Microtubules (MTs), highly dynamic structures composed of α- and β-tubulin heterodimers, are involved in cell movement and intracellular traffic and are essential for cell division. Within the cell, MTs are not uniform as they can be composed of different tubulin isotypes that are post-translationally modified and interact with different microtubule-associated proteins (MAPs). These diverse intrinsic factors influence the dynamics of MTs. Extrinsic factors such as microtubule-targeting agents (MTAs) can also affect MT dynamics. MTAs can be divided into two main categories: microtubule-stabilizing agents (MSAs) and microtubule-destabilizing agents (MDAs). Thus, the MT skeleton is an important target for anticancer therapy. This review discusses factors that determine the microtubule dynamics in normal and cancer cells and describes microtubule–MTA interactions, highlighting the importance of tubulin isoform diversity and post-translational modifications in MTA responses and the conse...
Molecular Mechanism of Microtubules Dynamics and its Precise Regulation Inside Cells
Microtubules are tubulin polymers that use nucleoside triphosphate (GTP) hydrolysis for polymerization. Microtubules (MTs) are involved in diverse and dynamic cellular functions like cell shape maintenance, cell division, cell migration, and signalling. Microtubules display dynamic behaviour of Treadmilling and microtubule dynamics, these processes are precisely regulated by microtubule associated proteins. Inside the cells, soluble and polymeric fraction of tubulin is in equilibrium state that is regulated by microtubule polymerizing and depolymerizing proteins.
Journal of Biological Chemistry, 2005
Phosphoinositide-specific phospholipase C-␥1 (PLC-␥1) has two pleckstrin homology (PH) domains, an Nterminal domain and a split PH domain. Here we show that pull down of NIH3T3 cell extracts with PLC-␥1 PH domain-glutathione S-transferase fusion proteins, followed by matrix-assisted laser desorption ionizationtime of flight-mass spectrometry, identified -tubulin as a binding protein of both PLC-␥1 PH domains. Tubulin is a main component of microtubules and mitotic spindle fibers, which are composed of ␣and -tubulin heterodimers in all eukaryotic cells. PLC-␥1 and -tubulin colocalized in the perinuclear region in COS-7 cells and cotranslocated to the plasma membrane upon agonist stimulation. Membrane-targeted translocation of depolymerized tubulin by agonist stimulation was also supported by immunoprecipitation analyses. The phosphatidylinositol 4,5-bisphosphate (PIP 2 ) hydrolyzing activity of PLC-␥1 was substantially increased in the presence of purified tubulin in vitro, whereas the activity was not promoted by bovine serum albumin, suggesting that -tubulin activates PLC-␥1. Furthermore, indirect immunofluorescent microscopy showed that PLC-␥1 was highly concentrated in mitotic spindle fibers, suggesting that PLC-␥1 is involved in spindle fiber formation. The effect of PLC-␥1 in microtubule formation was assessed by overexpression and silencing PLC-␥1 in COS-7 cells, which resulted in altered microtubule dynamics in vivo. Cells overexpressing PLC-␥1 showed higher microtubule densities than controls, whereas PLC-␥1 silencing with small interfering RNAs led to decreased microtubule network densities as compared with control cells. Taken together, our results suggest that PLC-␥1 and -tubulin transmodulate each other, i.e. that PLC-␥1 modulates microtubule assembly by -tubulin, and -tubulin promotes PLC-␥1 activity.
Isolation of Functional Tubulin Dimers and of Tubulin-Associated Proteins from Mammalian Cells
Current biology : CB, 2016
The microtubule (MT) cytoskeleton forms a dynamic filamentous network that is essential for many processes, including mitosis, cell polarity and shape, neurite outgrowth and migration, and ciliogenesis [1, 2]. MTs are built up of α/β-tubulin heterodimers, and their dynamic behavior is in part regulated by tubulin-associated proteins (TAPs). Here we describe a novel system to study mammalian tubulins and TAPs. We co-expressed equimolar amounts of triple-tagged α-tubulin and β-tubulin using a 2A "self-cleaving" peptide and isolated functional fluorescent tubulin dimers from transfected HEK293T cells with a rapid two-step approach. We also produced two mutant tubulins that cause brain malformations in tubulinopathy patients [3]. We then applied a paired mass-spectrometry-based method to identify tubulin-binding proteins in HEK293T cells and describe both novel and known TAPs. We find that CKAP5 and the CLASPs, which are MT plus-end-tracking proteins with TOG(L)-domains [4], b...
Microtubule Regulation in Mitosis: Tubulin Phosphorylation by the Cyclin-dependent Kinase Cdk1
Molecular Biology of The Cell, 2005
The activation of the cyclin-dependent kinase Cdk1 at the transition from interphase to mitosis induces important changes in microtubule dynamics. Cdk1 phosphorylates a number of microtubule-or tubulin-binding proteins but, hitherto, tubulin itself has not been detected as a Cdk1 substrate. Here we show that Cdk1 phosphorylates -tubulin both in vitro and in vivo. Phosphorylation occurs on Ser172 of -tubulin, a site that is well conserved in evolution. Using a phosphopeptide antibody, we find that a fraction of the cell tubulin is phosphorylated during mitosis, and this tubulin phosphorylation is inhibited by the Cdk1 inhibitor roscovitine. In mitotic cells, phosphorylated tubulin is excluded from microtubules, being present in the soluble tubulin fraction.
Tubulin: Structure, Functions and Roles in Disease
Cells
Highly conserved α- and β-tubulin heterodimers assemble into dynamic microtubules and perform multiple important cellular functions such as structural support, pathway for transport and force generation in cell division. Tubulin exists in different forms of isotypes expressed by specific genes with spatially- and temporally-regulated expression levels. Some tubulin isotypes are differentially expressed in normal and neoplastic cells, providing a basis for cancer chemotherapy drug development. Moreover, specific tubulin isotypes are overexpressed and localized in the nuclei of cancer cells and/or show bioenergetic functions through the regulation of the permeability of mitochondrial ion channels. It has also become clear that tubulin isotypes are involved in multiple cellular functions without being incorporated into microtubule structures. Understanding the mutations of tubulin isotypes specifically expressed in tumors and their post-translational modifications might help to identif...