Phosphoproteome analysis of the human mitotic spindle

doi:10.1073/pnas.0507066103

Phosphoproteome analysis of the human mitotic spindle

Department of Cell Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany

Edited by Stephen J. Elledge, Harvard Medical School, Boston, MA, and approved January 8, 2006 (received for review August 15, 2005)

Abstract

During cell division, the mitotic spindle segregates the sister chromatids into two nascent cells, such that each daughter cell inherits one complete set of chromosomes. Errors in spindle formation can result in both chromosome missegregation and cytokinesis defects and hence lead to genomic instability. To ensure the correct function of the spindle, the activity and localization of spindle associated proteins has to be tightly regulated in time and space. Reversible phosphorylation has been shown to be one of the key regulatory mechanisms for the organization of the mitotic spindle. The relatively low number of identified in vivo phosphorylation sites of spindle components, however, has hampered functional analysis of regulatory spindle networks. A more complete inventory of the phosphorylation sites of spindle-associated proteins would therefore constitute an important advance. Here, we describe the mass spectrometry-based identification of in vivo phosphorylation sites from purified human mitotic spindles. In total, 736 phosphorylation sites were identified, of which 312 could be attributed to known spindle proteins. Among these are phosphorylation sites that were previously shown to be important for the regulation of spindle-associated proteins. Importantly, this data set also comprises 279 novel phosphorylation sites of known spindle proteins for future functional studies. This inventory of spindle phosphorylation sites should thus make an important contribution to a better understanding of the molecular mechanisms that regulate the formation, function, and integrity of the mitotic spindle.

Footnotes

^‡To whom correspondence should be addressed. E-mail: rkoerner{at}biochem.mpg.de
↵*Present address: Laboratory of Applied Environmental Chemistry, University of Kuopio, P.O. Box 181, 50100 Mikkeli, Finland.
↵ ^†Present address: Institute for Biochemistry, University of Göttingen, Humboldtallee 23, D-37073 Göttingen, Germany.
Author contributions: H.H.W.S., E.A.N., and R.K. designed research; M.N., G.S., and R.K. performed research; M.N. contributed new reagents/analytic tools; M.N., H.H.W.S., G.S., and R.K. analyzed data; and H.H.W.S., E.A.N., and R.K. wrote the paper.
Conflict of interest statement: No conflicts declared.
This paper was submitted directly (Track II) to the PNAS office.
Data deposition: The data set reported in this paper has been deposited in the Proteomics Identification Database (PRIDE), www.ebi.ac.uk/pride (accession nos. 1628–1631).
Abbreviations:

Abbreviations:

ESI,

electrospray ionization;

IMAC,

immobilized metal ion chromatography;

MS/MS,

tandem mass spectrometry;

LC,

liquid chromatography.