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Regulation of tubulin heterodimer partitioning during interphase and mitosis
Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The microtubule cytoskeleton, which consists of dynamic polymers of alpha/beta tubulin heterodimers, organizes the cytoplasm and is essential for chromosome segregation during mitosis. My thesis addresses the significance and potential interplay between four distinct microtubule-regulatory proteins. The experimental approach included the development of a replicating vector system directing either constitutive expression of short hairpin RNAs or inducible ectopic expression, which allows stable depletion and/or conditional exchange of gene-products.

Based on the originally observed activities in frog egg extracts, MCAK and TOGp have been viewed as major antagonistic proteins that regulate microtubule-dynamics throughout the cell cycle. Surprisingly, while my thesis work confirmed an essential role of these proteins to ensure mitotic fidelity, tubulin subunits partitioning is not controlled by the endogenous levels of MCAK and TOGp in human somatic cells. Our major discovery in these studies is that the activities of both CaMKII and TOGp are essential for spindle bipolarity through a mechanism involving protection of spindle microtubules against MCAK activity at the centrosome.

In our search for the major antagonistic activities that regulates microtubule-dynamics in interphase cells, we found that the microtubule-destabilizing activity of Op18 is counteracted by MAP4. These studies also established Op18 and MAP4 as the predominant regulators of tubulin subunit partitioning in all three human cell model systems studied. Moreover, consistent with phosphorylation-inactivation of these two proteins during mitosis, we found that the microtubule-regulatory activities of both MAP4 and Op18 were only evident in interphase cells. Importantly, by employing a system for inducible gene product replacement, we found that site-specific phosphorylation-inactivation of Op18 is the direct cause of the demonstrated hyper-polymerization in response to T-cell antigen receptor triggering. This provides the first formally proven example of a signal transduction pathway for regulation of interphase microtubules.

Op18 is frequently upregulated in various types of human malignancies. In addition, a somatic mutation of Op18 has recently been identified in an adenocarcinoma. This thesis work revealed that the mutant Op18 protein exerts increased microtubule-destabilizing activity. The mutant Op18 protein was also shown to be partially resistant to phosphorylation-inactivation during mitosis, which was associated with increased chromosome segregation aberrancies. Interestingly, we also observed the same phenotype by overexpressing the wild type Op18 protein. Thus, either excessive levels of wild type Op18 or normal levels of mutated hyper-active Op18 seems likely to contribute to tumor progression by exacerbating chromosomal instability.

Place, publisher, year, edition, pages
Umeå: Molekylärbiologi (Medicinska fakulteten) , 2008. , 30 p.
Umeå University medical dissertations, ISSN 0346-6612 ; 1220
Keyword [en]
microtubule, mitotic spindle, signal transduction, phosphorylation, cancer
National Category
Biochemistry and Molecular Biology
URN: urn:nbn:se:umu:diva-1923ISBN: 978-91-7264-670-4OAI: diva2:142445
Public defence
2008-12-16, Major groove, 6L, Umeå, 10:00 (English)
Available from: 2008-11-18 Created: 2008-11-18 Last updated: 2010-01-18Bibliographically approved
List of papers
1. Differential functional interplay of TOGp/XMAP215 and the KinI kinesin MCAK during interphase and mitosis.
Open this publication in new window or tab >>Differential functional interplay of TOGp/XMAP215 and the KinI kinesin MCAK during interphase and mitosis.
2004 (English)In: EMBO J, ISSN 0261-4189, Vol. 23, no 3, 627-637 p.Article in journal (Refereed) Published
Abstract [en]

XMAP215/TOGp family members and KinI kinesins are conserved microtubule (MT)-regulatory proteins, and have been viewed as possessing prominent antagonistic stabilizing/destabilizing activities that must be balanced. Here, interdependencies between TOGp and the KinI kinesin MCAK were analyzed in human leukemia cells. A system was established that permits inducible overexpression in homogeneous cell populations that simultaneously synthesize interfering short hairpin RNAs. We present evidence that the functional interplay of TOGp and MCAK proteins is manifested as three distinct phenotypes during the cell cycle. The first involves a role for TOGp in protecting spindle MTs from MCAK activity at the centrosome, which appears essential to prevent the formation of disorganized multipolar spindles. The second phenotype involves TOGp-dependent counteraction of excessive MCAK activity during mitosis, which recapitulates the previously established plus-end specific counteractive activities in vitro. The third involves an unexpected destabilization of the interphase MTs by overexpressed TOGp, a phenotype that requires endogenous MCAK. We hypothesize that TOGp-dependent prevention of MCAK-mediated spindle disorganization, as evidenced by depletion experiments, reflects a primary physiological role for TOGp in human somatic cells.

Humans, Interphase/*physiology, K562 Cells, Kinesin/*metabolism, Microtubule-Associated Proteins/*metabolism, Mitosis/*physiology, Mitotic Spindle Apparatus/*metabolism
urn:nbn:se:umu:diva-16504 (URN)10.1038/sj.emboj.7600076 (DOI)14749730 (PubMedID)
Available from: 2007-10-04 Created: 2007-10-04Bibliographically approved
2. CaMKIIgamma-mediated inactivation of the Kin I kinesin MCAK is essential for bipolar spindle formation.
Open this publication in new window or tab >>CaMKIIgamma-mediated inactivation of the Kin I kinesin MCAK is essential for bipolar spindle formation.
Show others...
2005 (English)In: EMBO J, ISSN 0261-4189, Vol. 24, no 6, 1256-1266 p.Article in journal (Refereed) Published
Abstract [en]

MCAK, a member of the kinesin-13 family, is a microtubule (MT) depolymerase that is necessary to ensure proper kinetochore MT attachment during spindle formation. Regulation of MCAK activity and localization is controlled in part by Aurora B kinase at the centromere. Here we analyzed human cells depleted of the ubiquitous Ca(2+)/calmodulin-dependent protein kinase IIgamma isoform (CaMKIIgamma) by RNA interference and found that CaMKIIgamma was necessary to suppress MCAK depolymerase activity in vivo. A functional overlap with TOGp, a MT regulator known to counteract MCAK, was suggested by similar CaMKIIgamma- and TOGp-depletion phenotypes, namely disorganized multipolar spindles. A replicating vector system, which permits inducible overexpression in cells that simultaneously synthesize interfering short hairpin RNAs, was used to dissect the functional interplay between CaMKIIgamma, TOGp, and MCAK. Our results revealed two distinct but functionally overlapping mechanisms for negative regulation of the cytosolic/centrosomal pool of MCAK. These two mechanisms, involving CaMKIIgamma and TOGp, respectively, are both essential for spindle bipolarity in a normal physiological context, but not in MCAK-depleted cells.

Ca(2+)-Calmodulin Dependent Protein Kinase/genetics/*physiology, Cell Division/genetics/physiology, Humans, Kinesin/*metabolism, Microtubule-Associated Proteins/genetics/physiology, Mitotic Spindle Apparatus/*metabolism, Nucleic Acid Conformation, RNA Interference, RNA; Small Interfering/genetics
urn:nbn:se:umu:diva-16502 (URN)10.1038/sj.emboj.7600601 (DOI)15775983 (PubMedID)
Available from: 2007-10-04 Created: 2007-10-04Bibliographically approved
3. Aneugenic activity of Op18/stathmin is potentiated by the somatic Q18-->e mutation in leukemic cells.
Open this publication in new window or tab >>Aneugenic activity of Op18/stathmin is potentiated by the somatic Q18-->e mutation in leukemic cells.
2006 (English)In: Mol Biol Cell, ISSN 1059-1524, Vol. 17, no 7, 2921-2930 p.Article in journal (Refereed) Published
Abstract [en]

Op18/stathmin (Op18) is a phosphorylation-regulated microtubule destabilizer that is frequently overexpressed in tumors. The importance of Op18 in malignancy was recently suggested by identification of a somatic Q18-->E mutation of Op18 in an adenocarcinoma. We addressed the functional consequences of aberrant Op18 expression in leukemias by analyzing the cell cycle of K562 cells either depleted of Op18 by expression of interfering hairpin RNA or induced to express wild-type or Q18E substituted Op18. We show here that although Op18 depletion increases microtubule density during interphase, the density of mitotic spindles is essentially unaltered and cells divide normally. This is consistent with phosphorylation-inactivation of Op18 during mitosis. Overexpression of wild-type Op18 results in aneugenic activities, manifest as aberrant mitosis, polyploidization, and chromosome loss. One particularly significant finding was that the aneugenic activity of Op18 was dramatically increased by the Q18-->E mutation. The hyperactivity of mutant Op18 is apparent in its unphosphorylated state, and this mutation also suppresses phosphorylation-inactivation of the microtubule-destabilizing activity of Op18 without any apparent effect on its phosphorylation status. Thus, although Op18 is dispensable for mitosis, the hyperactive Q18-->E mutant, or overexpressed wild-type Op18, exerts aneugenic effects that are likely to contribute to chromosomal instability in tumors.

Aneugens/metabolism, Cell Cycle/genetics, Chromosomal Instability, Humans, Leukemia/*genetics/metabolism/pathology, Microtubules/metabolism, Mitosis/genetics, Mitotic Spindle Apparatus/*metabolism, Mutation, Phosphorylation, RNA Interference, Stathmin/antagonists & inhibitors/*genetics/*metabolism, Tubulin/metabolism
urn:nbn:se:umu:diva-16501 (URN)10.1091/mbc.E06-02-0165 (DOI)16624860 (PubMedID)
Available from: 2007-10-04 Created: 2007-10-04Bibliographically approved
4. Interphase-specific phosphorylation-mediated regulation of tubulin dimer partitioning in human cells.
Open this publication in new window or tab >>Interphase-specific phosphorylation-mediated regulation of tubulin dimer partitioning in human cells.
2007 (English)In: Molecular biology of the cell, ISSN 1059-1524, Vol. 18, no 5, 1909-1917 p.Article in journal (Refereed) Published
Abstract [en]

The microtubule cytoskeleton is differentially regulated by a diverse array of proteins during interphase and mitosis. Op18/stathmin (Op18) and microtubule-associated protein (MAP)4 have been ascribed opposite general microtubule-directed activities, namely, microtubule destabilization and stabilization, respectively, both of which can be inhibited by phosphorylation. Here, using three human cell models, we depleted cells of Op18 and/or MAP4 by expression of interfering hairpin RNAs and we analyzed the resulting phenotypes. We found that the endogenous levels of Op18 and MAP4 have opposite and counteractive activities that largely govern the partitioning of tubulin dimers in the microtubule array at interphase. Op18 and MAP4 were also found to be the downstream targets of Ca(2+)- and calmodulin-dependent protein kinase IV and PAR-1/MARK2 kinase, respectively, that control the demonstrated counteractive phosphorylation-mediated regulation of tubulin dimer partitioning. Furthermore, to address mechanisms regulating microtubule polymerization in response to cell signals, we developed a system for inducible gene product replacement. This approach revealed that site-specific phosphorylation of Op18 is both necessary and sufficient for polymerization of microtubules in response to the multifaceted signaling event of stimulation of the T cell antigen receptor complex, which activates several signal transduction pathways.

Base Sequence, Ca(2+)-Calmodulin Dependent Protein Kinase/metabolism, Cell Line, DNA/genetics, Dimerization, Humans, Interphase/*physiology, Jurkat Cells, K562 Cells, Microtubule-Associated Proteins/antagonists & inhibitors/genetics/metabolism, Microtubules/metabolism, Phosphorylation, Protein Structure; Quaternary, Protein-Serine-Threonine Kinases/metabolism, Signal Transduction, Stathmin/antagonists & inhibitors/genetics/metabolism, Transfection, Tubulin/*chemistry/*metabolism
urn:nbn:se:umu:diva-16490 (URN)10.1091/mbc.E07-01-0019 (DOI)17344472 (PubMedID)
Available from: 2007-10-03 Created: 2007-10-03Bibliographically approved

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