A Comprehensive Analysis of Polo-like Kinase 4's Regulation and Role in Centriole Biogenesis

Persistent Link:
http://hdl.handle.net/10150/318808
Title:
A Comprehensive Analysis of Polo-like Kinase 4's Regulation and Role in Centriole Biogenesis
Author:
Klebba, Joseph Earl
Issue Date:
2014
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
Plk4 has been termed a `suicide kinase' because it promotes its own destruction to regulate protein levels. We identified numerous autophosphorylated residues within a region of Plk4 called the Downstream regulatory element (DRE). We find that phosphorylation of a single residue is sufficient for Slimb recruitment and phosphorylation of the surrounding residues builds a high affinity Slimb-binding site. These autophosphorylation events are dependent on Plk4 homodimerization, although the domains that mediate this dimerization are unknown. We show that Plk4 homodimerization is mediated by interactions between the PB1-PB2 cassette. We find that like all Polo kinases, Plk4 encodes a mechanism of kinase autoinhibition. Unlike other Polo kinases, which rely on external inputs for relief of inhibition, Plk4 is self-sufficient in relieving kinase inhibition. This relief of autoinhibition is regulated by PB3 of Plk4 and is dependent on homodimerization, thereby making homodimerization a necessary step in formation of the Slimb phosphodegron on Plk4. Polo Boxes are known as multifunctional domains, and the Polo Boxes of Plk4 are no different. We identified numerous Slimb-mediated ubiquitination sites on PB1 as well as PB2. Furthermore, the PB1-PB2 cassette mediates the interaction between Plk4 and the N-terminus of Asterless. In Drosophila cells, Plk4 requires Asterless for centriolar localization and Asterless overexpression drives centriole amplification in a Plk4 dependent manner. This is a fascinating result as endogenous Plk4 protein levels are undetectable in S2 cells, making it hard to envision a scenario where overexpression of Asterless could shuttle a non-existent Plk4 population to the centriole to initiate duplication. We found that in addition to shuttling Plk4 to the centriole, Asterless stabilizes Plk4, likely protecting Plk4 at the centriole to allow it to `license' the centriole for duplication. Moreover, we show that Asterless encodes two distinct Plk4 binding sites: the previously described N-terminal binding site as well as a novel C-terminal binding site. We found that the interaction between the C-terminal of Asterless and Plk4 is necessary for centriole duplication while the interaction between the N-terminal of Asterless and Plk4 is expendable. Together these findings provide significant insight into Plk4 biology and the mechanisms which limit its activity.
Type:
text; Electronic Dissertation
Keywords:
Cell Biology & Anatomy
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Graduate College; Cell Biology & Anatomy
Degree Grantor:
University of Arizona
Advisor:
Rogers, Gregory C.

Full metadata record

DC FieldValue Language
dc.language.isoen_USen
dc.titleA Comprehensive Analysis of Polo-like Kinase 4's Regulation and Role in Centriole Biogenesisen_US
dc.creatorKlebba, Joseph Earlen_US
dc.contributor.authorKlebba, Joseph Earlen_US
dc.date.issued2014-
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractPlk4 has been termed a `suicide kinase' because it promotes its own destruction to regulate protein levels. We identified numerous autophosphorylated residues within a region of Plk4 called the Downstream regulatory element (DRE). We find that phosphorylation of a single residue is sufficient for Slimb recruitment and phosphorylation of the surrounding residues builds a high affinity Slimb-binding site. These autophosphorylation events are dependent on Plk4 homodimerization, although the domains that mediate this dimerization are unknown. We show that Plk4 homodimerization is mediated by interactions between the PB1-PB2 cassette. We find that like all Polo kinases, Plk4 encodes a mechanism of kinase autoinhibition. Unlike other Polo kinases, which rely on external inputs for relief of inhibition, Plk4 is self-sufficient in relieving kinase inhibition. This relief of autoinhibition is regulated by PB3 of Plk4 and is dependent on homodimerization, thereby making homodimerization a necessary step in formation of the Slimb phosphodegron on Plk4. Polo Boxes are known as multifunctional domains, and the Polo Boxes of Plk4 are no different. We identified numerous Slimb-mediated ubiquitination sites on PB1 as well as PB2. Furthermore, the PB1-PB2 cassette mediates the interaction between Plk4 and the N-terminus of Asterless. In Drosophila cells, Plk4 requires Asterless for centriolar localization and Asterless overexpression drives centriole amplification in a Plk4 dependent manner. This is a fascinating result as endogenous Plk4 protein levels are undetectable in S2 cells, making it hard to envision a scenario where overexpression of Asterless could shuttle a non-existent Plk4 population to the centriole to initiate duplication. We found that in addition to shuttling Plk4 to the centriole, Asterless stabilizes Plk4, likely protecting Plk4 at the centriole to allow it to `license' the centriole for duplication. Moreover, we show that Asterless encodes two distinct Plk4 binding sites: the previously described N-terminal binding site as well as a novel C-terminal binding site. We found that the interaction between the C-terminal of Asterless and Plk4 is necessary for centriole duplication while the interaction between the N-terminal of Asterless and Plk4 is expendable. Together these findings provide significant insight into Plk4 biology and the mechanisms which limit its activity.en_US
dc.typetexten
dc.typeElectronic Dissertationen
dc.subjectCell Biology & Anatomyen_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.disciplineCell Biology & Anatomyen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorRogers, Gregory C.en_US
dc.contributor.committeememberRogers, Gregory C.en_US
dc.contributor.committeememberGregorio, Carolen_US
dc.contributor.committeememberMartinez, Jesseen_US
dc.contributor.committeememberMontfort, Williamen_US
dc.contributor.committeememberMcDermott, Kimberlyen_US
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