Phosphoproteome analysis of the human mitotic spindle

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. mass spectrometry ͉ mitosis ͉ molecular cell biology ͉ phosphorylation ͉ proteomics A t the transition from interphase to mitosis, the microtubule network undergoes a profound change that culminates in the formation of the spindle apparatus. The mitotic spindle then serves to segregate the chromosomes to opposite poles of the cell and to define the plane of cell division. A large number of proteins associate with this microtubule-based structure and regulate its dynamic formation and function (1, 2). Several spindle proteins, including XKCM1 and XMAP215 family members (3, 4), have been identified that either stabilize or destabilize microtubules by mediating the rapid changes between polymerization and depolymerization (5, 6). These proteins play an important role in spindle formation, as illustrated by the striking change in microtubule half-life from 5-10 min in interphase to less than 1 min in mitosis (7), which results in the short and unstable microtubules characteristic of mitosis (5, 6). Another important group of spindle proteins comprises motors of the kinesin and dynein families that are essential for mitotic progression (5,6,8,9). They push the spindle poles away from each other during early mitosis and play crucial roles in capturing chromosomes and positioning them at the metaphase plate. Subsequently, motors also contribute to central spindle formation and cytokinesis. In addition, numerous structural prote...

Temporal changes in Hec1 phosphorylation control kinetochore–microtubule attachment stability during mitosis

“…Previous proteomics studies using isolated mitotic HeLa cell spindles or human embryonic stem cells have demonstrated that Hec1 is phosphorylated on Ser55 and Ser69 in vivo by undetermined kinases (Nousiainen et al, 2006;Malik et al, 2009;Brill et al, 2009;Olsen et al, 2010). We now include Ser44, Ser15 and Ser8 to the list of known in vivo N-terminal Hec1 phosphorylation sites (supplementary material Fig.…”

Covalent capture of kinase-specific phosphopeptides reveals Cdk1-cyclin B substrates

“…We compared our results with several large phosphoproteomic datasets (6)(7)(8). Interestingly, at least 36 of our sites, or 50%, are known to be phosphorylated in vivo in HeLa cells.…”

“…Prediction of high-likelihood kinase substrates can sometimes be achieved by using knowledge of the substrate sequence motif preferences of individual kinases, but only when these preferences are strong (4,5). Finally, thousands of in vivo phosphorylation sites from metazoan organisms have been identified in proteomic screens (6)(7)(8)(9), but for most of these sites the responsible upstream kinases remain unknown.…”

Phosphorylation by Aurora-B Negatively Regulates Survivin Function During Mitosis

“…1A, upper panel, labeled 1 in schematic), the abundance of which was reduced in the presence of lambda phosphatase (lower panel). As survivin is also phosphorylated by CDK1 during mitosis, 36,37 it is possible that CDK1 is a major contributor to the abundance of protein in this spot. Nevertheless, the data clearly demonstrate that survivin is phosphorylated in prometaphase cells.…”