Journal of Molecular Biology
Volume 425, Issue 2, 23 January 2013, Pages 214-221
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N-Terminal Phosphorylation of p60 Katanin Directly Regulates Microtubule Severing

https://doi.org/10.1016/j.jmb.2012.11.022Get rights and content

Abstract

Proteins of the AAA (ATPases associated with various cellular activities) family often have complex modes of regulation due to their central position in important cellular processes. p60 katanin, an AAA protein that severs and depolymerizes microtubules, is subject to multiple modes of regulation including a phosphorylation in the N-terminal domain involved in mitotic control of severing. Phosphorylation decreases severing activity in Xenopus egg extracts and is involved in controlling spindle length. Here, we show that the evolutionarily divergent N-terminal domains of p60 have maintained hotspots of mitotic kinase regulation. By reconstituting in vitro severing reactions, we show that phosphomimetic modification at amino acid position 131 in Xenopus laevis p60 decreases severing and microtubule-stimulated ATPase activity without affecting the binding affinity of p60 for microtubules. At high concentrations of the phosphomimetic mutant p60, wild-type levels of activity could be observed, indicating a more switch-like threshold of activity that is controlled by regulating oligomerization on the microtubule. This provides a cellular mechanism for high local concentrations of p60, like those found on spindle poles, to maintain severing activity while most of the protein is inhibited. Overall, we have shown that the modular domain architecture of AAA proteins allows for precise control of cellular activities with simple modifications.

Graphical abstract

Highlights

► The N-terminal domain of p60 katanin is a hotspot for potential mitotic kinase regulation. ► Phosphomimetic p60 katanin displays lower activity with pure microtubules compared to wild type. ► The phosphomimetic mutation reduces microtubule-stimulated ATPase activity. ► p60 katanin's affinity for microtubules is unchanged by the phosphomimetic mutation. ► p60 katanin is regulated at the point of assembly on the microtubule

Introduction

Members of the AAA (ATPases associated with various cellular activities) family of proteins function in a diverse array of cellular processes including protein degradation, vesicle trafficking, protein quality control, and cell division.[1], [2], [3], [4] Typically, the highly conserved C-terminal catalytic domain is regulated by the N-terminal region, which is highly divergent and also mediates interactions with adaptor proteins that facilitate subcellular targeting or otherwise directly regulate AAA protein function.[3], [4] For example, two distinct cellular activities of the AAA protein p97/VCP are achieved by competitive binding of two different sets of adaptor proteins as well as a host of phosphorylations and acetylations at its N-terminus.[5], [6], [7] However, how the majority of AAA proteins are mechanistically regulated remains unknown.

Katanin is a heterodimeric microtubule-severing AAA ATPase composed of a catalytic p60 subunit containing the AAA domain and a targeting subunit p80.8 Like many AAA proteins, active katanin functions as a hexamer, though the exact mechanism of its microtubule-severing activity is unclear.9 In vivo, katanin plays critical roles in meiosis,10 cilia and flagella assembly,[11], [12], [13], [14], [15] and plant cell division[16], [17], [18] and has also been suggested to generate non-centrosomal microtubule arrays in neurons.19

Recently, we demonstrated another interesting role for katanin in governing meiotic spindle length differences between two closely related species of frogs, Xenopus laevis and Xenopus tropicalis.20 We found that suppression of microtubule severing by katanin is responsible in large part for the longer spindles assembled in X. laevis egg extracts relative to X. tropicalis. This difference in activity was not caused by differences in protein levels, but rather by the presence of an inhibitory Aurora B phosphorylation site at serine 131 in the N-terminus of X. laevis p60 katanin, which is absent from X. tropicalis p60.20 Addition of a recombinant mutant of X. laevis p60 katanin with serine 131 changed to alanine led to rapid microtubule severing in metaphase-arrested egg extracts, while a phosphomimetic mutant displayed reduced activity similar to the wild-type p60.20 Here, we investigate the biochemical basis of inhibitory phosphorylation at S131 by comparing the activities of wild-type and phosphomimetic mutants of p60 in vitro with pure microtubules. We show that phosphorylation of the N-terminal domain represents a novel mode of AAA ATPase regulation that may provide general insights into the mechanism and regulation of AAA proteins and microtubule-severing proteins in particular. Our results suggest a means for the cell to tune the activity of AAA proteins as a function of its local concentration.

Section snippets

A phosphomimetic mutant p60S131E displays concentration-dependent inhibition of severing in vitro

We previously identified serine 131 as a site of negative regulation in X. laevis p60 katanin. Missing from X. tropicalis, this site is conserved among many vertebrate species but absent from Drosophila melanogaster, Caenorhabditis elegans, and Strongylocentrotus purpuratus (sea urchin), indicating that regulatory control at this site may only be necessary under certain physiological contexts or that other selective pressures have caused the regulatory sites in this domain to be shifted within

Discussion

Given that basal ATP hydrolysis and microtubule binding are unaffected by the S131E mutation, we reasoned that the lack of microtubule-stimulated ATP hydrolysis must be due to either perturbed oligomerization or a disruption of an allosteric mechanism that normally stimulates ATPase activity upon microtubule binding. It is currently unknown whether ATPase stimulation occurs directly upon microtubule binding or indirectly as a consequence of microtubule-stimulated oligomerization. Our result

Acknowledgements

We thank Andy Martin and members of the Heald laboratory for helpful discussions and Dr. Liam Holt, Dr. Magdalena Strzelecka, and Dr. Marina Ellefson for critical reading of the manuscript. R.H. is supported by National Institutes of Health Grant R01GM098766.

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