Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 103 (14), 5391-6

Phosphoproteome Analysis of the Human Mitotic Spindle

Affiliations

Phosphoproteome Analysis of the Human Mitotic Spindle

Marjaana Nousiainen et al. Proc Natl Acad Sci U S A.

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.

Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.
Fig. 1.
Analytical strategy for the mapping of phosphorylation sites in spindle proteins. Mitotic spindles were isolated and proteins subsequently separated by SDS/PAGE. The gel was cut into 18 slices and trypsinized. The samples were batch-incubated with Fe3+-IMAC resin, and eluted peptides were analyzed by nano-LC-ESI-MS/MS and nano-ESI-MS/MS. IMAC flow-through and samples not enriched by IMAC were analyzed by nano-LC-ESI-MS/MS.
Fig. 2.
Fig. 2.
Example of MALDI-TOF MS spectra of a phosphopeptide-containing sample before and after IMAC enrichment. (A) Before IMAC enrichments. (B) IMAC flow-through. (C) IMAC eluate. Peaks marked by filled triangles in C were identified as phosphopeptides in subsequent analyses.

Similar articles

  • Proteome Analysis of the Human Mitotic Spindle
    G Sauer et al. Mol Cell Proteomics 4 (1), 35-43. PMID 15561729.
    The accurate distribution of sister chromatids during cell division is crucial for the generation of two cells with the same complement of genetic information. A highly d …
  • The Clathrin-dependent Spindle Proteome
    SR Rao et al. Mol Cell Proteomics 15 (8), 2537-53. PMID 27174698.
    The mitotic spindle is required for chromosome congression and subsequent equal segregation of sister chromatids. These processes involve a complex network of signaling m …
  • The Plk1-dependent Phosphoproteome of the Early Mitotic Spindle
    A Santamaria et al. Mol Cell Proteomics 10 (1), M110.004457. PMID 20860994.
    Polo-like kinases regulate many aspects of mitotic and meiotic progression from yeast to man. In early mitosis, mammalian Polo-like kinase 1 (Plk1) controls centrosome ma …
  • Interplay Between Spindle Architecture and Function
    KJ Helmke et al. Int Rev Cell Mol Biol 306, 83-125. PMID 24016524. - Review
    The mitotic spindle performs the universal and crucial function of segregating chromosomes to daughter cells, and all spindles share common characteristics that facilitat …
  • Regulated Assembly of the Mitotic Spindle: A Perspective From Two Ends
    L Cassimeris et al. Curr Issues Mol Biol 5 (3), 99-112. PMID 12866832. - Review
    Chromosome segregration and cell division requires the regulated assembly of the mitotic spindle apparatus. This mitotic spindle is composed of condensed chromosomes atta …
See all similar articles

Cited by 135 PubMed Central articles

See all "Cited by" articles

Publication types

LinkOut - more resources

Feedback