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CaMKIIgamma-mediated inactivation of the Kin I kinesin MCAK is essential for bipolar spindle formation.
Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine). (Gullberg)
Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine). (Gullberg)
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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.

Place, publisher, year, edition, pages
2005. Vol. 24, no 6, 1256-1266 p.
Keyword [en]
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: urn:nbn:se:umu:diva-16502DOI: 10.1038/sj.emboj.7600601PubMedID: 15775983OAI: diva2:156175
Available from: 2007-10-04 Created: 2007-10-04Bibliographically approved
In thesis
1. Regulation of tubulin heterodimer partitioning during interphase and mitosis
Open this publication in new window or tab >>Regulation of tubulin heterodimer partitioning during interphase and mitosis
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
microtubule, mitotic spindle, signal transduction, phosphorylation, cancer
National Category
Biochemistry and Molecular Biology
urn:nbn:se:umu:diva-1923 (URN)978-91-7264-670-4 (ISBN)
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

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Holmfeldt, PerStenmark, SonjaGullberg, Martin
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