TAOK2 is an ER-localized kinase that catalyzes the dynamic tethering of ER to microtubules.
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ABSTRACT:
SUBMITTER: Nourbakhsh K
PROVIDER: S-EPMC8699727 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Centrosomes undergo dramatic changes in composition and activity during cell cycle progression. Yet mechanisms involved in recruiting centrosomal proteins are poorly understood. Nek2 is a cell cycle-regulated protein kinase required for regulation of centrosome structure at the G2/M transition. Here, we have addressed the processes involved in trafficking of Nek2 to the centrosome of human adult cells. We find that Nek2 exists in small, highly dynamic cytoplasmic particles that move to and from the centrosome. Many of these particles align along microtubules and a motif was identified in the Nek2 C-terminal noncatalytic domain that allows both microtubule binding and centrosome localization. FRAP experiments reveal that 70% of centrosomal Nek2 is rapidly turned over (t(1/2) approximately 3 s). Microtubules facilitate Nek2 trafficking to the centrosome but only over long distances. Cytoplasmic Nek2 particles colocalize in part with PCM-1 containing centriolar satellites and depletion of PCM-1 interferes with centrosomal recruitment of Nek2 and its substrate C-Nap1. Finally, we show that proteasomal degradation is necessary to allow rapid recruitment of new Nek2 molecules to the centrosome. Together, these data highlight multiple processes involved in regulating the abundance of Nek2 kinase at the centrosome including microtubule binding, the centriolar satellite component PCM-1, and localized protein degradation.
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Project description:Genes implicated in translation control have been associated with autism spectrum disorders (ASDs). However, some important genetic causes of autism, including the 16p11.2 microdeletion, bear no obvious connection to translation. Here, we use proteomics, genetics, and translation assays in cultured cells and mouse brain to reveal altered translation mediated by loss of the kinase TAOK2 in 16p11.2 deletion models. We show that TAOK2 associates with the translational machinery and functions as a translational brake by phosphorylating eukaryotic elongation factor 2 (eEF2). Previously, all signal-mediated regulation of translation elongation via eEF2 phosphorylation was believed to be mediated by a single kinase, eEF2K. However, we show that TAOK2 can directly phosphorylate eEF2 on the same regulatory site, but functions independently of eEF2K signaling. Collectively, our results reveal an eEF2K-independent signaling pathway for control of translation elongation and suggest altered translation as a molecular component in the etiology of some forms of ASD.
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Project description:Abnormalities in dendritic spines are manifestations of several neurodevelopmental and psychiatric diseases. TAOK2 is one of the genes in the 16p11.2 locus, copy number variations of which are associated with autism and schizophrenia. Here, we show that the kinase activity of the serine/threonine kinase encoded by TAOK2 is required for spine maturation. TAOK2 depletion results in unstable dendritic protrusions, mislocalized shaft-synapses, and loss of compartmentalization of NMDA receptor-mediated calcium influx. Using chemical-genetics and mass spectrometry, we identified several TAOK2 phosphorylation targets. We show that TAOK2 directly phosphorylates the cytoskeletal GTPase Septin7, at an evolutionary conserved residue. This phosphorylation induces translocation of Septin7 to the spine, where it associates with and stabilizes the scaffolding protein PSD95, promoting dendritic spine maturation. This study provides a mechanistic basis for postsynaptic stability and compartmentalization via TAOK2-Sept7 signaling, with implications toward understanding the potential role of TAOK2 in neurological deficits associated with the 16p11.2 region.
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Project description:Platelet endothelial adhesion molecule-1 (PECAM-1) is a part of intercellular junctions and triggers intracellular signaling cascades upon homophilic binding. The intracellular domain of PECAM-1 is tyrosine phosphorylated upon homophilic engagement. However, it remains unclear which tyrosine kinase phosphorylates PECAM-1. We sought to isolate tyrosine kinases responsible for PECAM-1 phosphorylation and identified Fer as a candidate, based on expression cloning. Fer kinase specifically phosphorylated PECAM-1 at the immunoreceptor tyrosine-based inhibitory motif. Notably, Fer induced tyrosine phosphorylation of SHP-2, which is known to bind to the immunoreceptor tyrosine-based inhibitory motif of PECAM-1, and Fer also induced tyrosine phosphorylation of Gab1 (Grb2-associated binder-1). Engagement-dependent PECAM-1 phosphorylation was inhibited by the overexpression of a kinase-inactive mutant of Fer, suggesting that Fer is responsible for the tyrosine phosphorylation upon PECAM-1 engagement. Furthermore, by using green fluorescent protein-tagged Fer and a time-lapse fluorescent microscope, we found that Fer localized at microtubules in polarized and motile vascular endothelial cells. Fer was dynamically associated with growing microtubules in the direction of cell-cell contacts, where p120catenin, which is known to associate with Fer, colocalized with PECAM-1. These results suggest that Fer localized on microtubules may play an important role in phosphorylation of PECAM-1, possibly through its association with p120catenin at nascent cell-cell contacts.
| S-EPMC196549 | biostudies-literature
Project description:When cells with a mesenchymal type of motility come into contact with an adhesive substrate they adhere and start spreading by the formation of lamellipodia. Using a label-free approach and virtual synchronization approach we analyzed spreading in fibroblasts and cancer cells. In all cell lines spreading is a non-linear process undergoing isotropic or anisotropic modes with first fast (5-20?min) and then slow (30-120?min) phases. In the first 10 min cell area increases 2-4 times, while the absolute rate of initial spreading decreases 2-8 times. Fast spreading depends on actin polymerization and dynamic microtubules. Inhibition of microtubule growth was sufficient for a slowdown of initial spreading. Inhibition of myosin II in the presence of stable microtubules restored fast spreading. Inhibition of actin polymerization or complete depolymerization of microtubules slowed down fast spreading. However, in these cases inhibition of myosin II only partially restored spreading kinetics. We conclude that rapid growth of microtubules towards cell margins at the first stage of cell spreading temporarily inhibits phosphorylation of myosin II and is essential for the fast isotropic spreading. Comparison of the fibroblasts with cancer cells shows that fast spreading in different cell types shares similar kinetics and mechanisms, and strongly depends on dynamic microtubules.
| S-EPMC6310885 | biostudies-other
Project description:Mitochondria form close physical associations with the endoplasmic reticulum (ER) that regulate a number of physiological functions. One mechanism by which regions of ER are recruited to mitochondria involves binding of the ER protein VAPB to the mitochondrial protein PTPIP51, which act as scaffolds to tether the two organelles. Here, we show that the VAPB-PTPIP51 tethers regulate autophagy. We demonstrate that overexpression of VAPB or PTPIP51 to tighten ER-mitochondria contacts impairs, whereas small interfering RNA (siRNA)-mediated loss of VAPB or PTPIP51 to loosen contacts stimulates, autophagosome formation. Moreover, we show that expression of a synthetic linker protein that artificially tethers ER and mitochondria also reduces autophagosome formation, and that this artificial tether rescues the effects of siRNA loss of VAPB or PTPIP51 on autophagy. Thus, these effects of VAPB and PTPIP51 manipulation on autophagy are a consequence of their ER-mitochondria tethering function. Interestingly, we discovered that tightening of ER-mitochondria contacts by overexpression of VAPB or PTPIP51 impairs rapamycin- and torin 1-induced, but not starvation-induced, autophagy. This suggests that the regulation of autophagy by ER-mitochondria signaling is at least partly dependent upon the nature of the autophagic stimulus. Finally, we demonstrate that the mechanism by which the VAPB-PTPIP51 tethers regulate autophagy involves their role in mediating delivery of Ca2+ to mitochondria from ER stores. Thus, our findings reveal a new molecular mechanism for regulating autophagy.
| S-EPMC5300905 | biostudies-other
Project description:Although endolysosomal trafficking is well defined, how it is regulated and coordinates with cellular metabolism is unclear. To identify genes governing endolysosomal dynamics, we conducted a global fluorescence-based screen to reveal endomembrane effector genes. Screening implicated Phox (PX) domain-containing protein Mdm1 in endomembrane dynamics. Surprisingly, we demonstrate that Mdm1 is a novel interorganelle tethering protein that localizes to endoplasmic reticulum (ER)-vacuole/lysosome membrane contact sites (MCSs). We show that Mdm1 is ER anchored and contacts the vacuole surface in trans via its lipid-binding PX domain. Strikingly, overexpression of Mdm1 induced ER-vacuole hypertethering, underscoring its role as an interorganelle tether. We also show that Mdm1 and its paralogue Ydr179w-a (named Nvj3 in this study) localize to ER-vacuole MCSs independently of established tether Nvj1. Finally, we find that Mdm1 truncations analogous to neurological disease-associated SNX14 alleles fail to tether the ER and vacuole and perturb sphingolipid metabolism. Our work suggests that human Mdm1 homologues may play previously unappreciated roles in interorganelle communication and lipid metabolism.
| S-EPMC4539980 | biostudies-literature
Project description:BACKGROUND:Mitochondria form a dynamic tubular network within the cell. Proper mitochondria movement and distribution are critical for their localized function in cell metabolism, growth, and survival. In mammalian cells, mechanisms of mitochondria positioning appear dependent on the microtubule cytoskeleton, with kinesin or dynein motors carrying mitochondria as cargos and distributing them throughout the microtubule network. Interestingly, the timescale of microtubule dynamics occurs in seconds, and the timescale of mitochondria distribution occurs in minutes. How does the cell couple these two time constants? RESULTS:Fission yeast also relies on microtubules for mitochondria distribution. We report here a new microtubule-dependent but motor-independent mechanism for proper mitochondria positioning in fission yeast. We identify the protein mmb1p, which binds to mitochondria and microtubules. mmb1p attaches the tubular mitochondria to the microtubule lattice at multiple discrete interaction sites. mmb1 deletion causes mitochondria to aggregate, with the long-term consequence of defective mitochondria distribution and cell death. mmb1p decreases microtubule dynamicity. CONCLUSIONS:mmb1p is a new microtubule-mitochondria binding protein. We propose that mmb1p acts to couple long-term mitochondria distribution to short-term microtubule dynamics by attenuating microtubule dynamics, thus enhancing the mitochondria-microtubule interaction time.
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Project description:Communication between organelles is an important feature of all eukaryotic cells. To uncover components involved in mitochondria/endoplasmic reticulum (ER) junctions, we screened for mutants that could be complemented by a synthetic protein designed to artificially tether the two organelles. We identified the Mmm1/Mdm10/Mdm12/Mdm34 complex as a molecular tether between ER and mitochondria. The tethering complex was composed of proteins resident of both ER and mitochondria. With the use of genome-wide mapping of genetic interactions, we showed that the components of the tethering complex were functionally connected to phospholipid biosynthesis and calcium-signaling genes. In mutant cells, phospholipid biosynthesis was impaired. The tethering complex localized to discrete foci, suggesting that discrete sites of close apposition between ER and mitochondria facilitate interorganelle calcium and phospholipid exchange.
| S-EPMC2933203 | biostudies-literature
Project description:The fusion of yeast vacuolar membranes depends on the disassembly of cis-soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes and the subsequent reassembly of new SNARE complexes in trans. The disassembly of cis-SNARE complexes by Sec17/Sec18p releases the soluble SNARE Vam7p from vacuolar membranes. Consequently, Vam7p needs to be recruited to the membrane at future sites of fusion to allow the formation of trans-SNARE complexes. The multisubunit tethering homotypic fusion and vacuole protein sorting (HOPS) complex, which is essential for the fusion of vacuolar membranes, was previously shown to have direct affinity for Vam7p. The functional significance of this interaction, however, has been unclear. Using a fully reconstituted in vitro fusion reaction, we now show that HOPS facilitates membrane fusion by recruiting Vam7p for fusion. In the presence of HOPS, unlike with other tethering agents, very low levels of added Vam7p suffice to induce vigorous fusion. This is a specific recruitment of Vam7p rather than an indirect stimulation of SNARE complex formation through tethering, as HOPS does not facilitate fusion with a low amount of a soluble form of another vacuolar SNARE, Vti1p. Our findings establish yet another function among the multiple tasks that HOPS performs to catalyze the fusion of yeast vacuoles.
| S-EPMC3843000 | biostudies-literature