The CRAPome: a contaminant repository for affinity purification-mass spectrometry data.

Affinity purification coupled with mass spectrometry (AP-MS) is a widely used approach for the identification of protein-protein interactions. However, for any given protein of interest, determining which of the identified polypeptides represent bona fide interactors versus those that are background contaminants (for example, proteins that interact with the solid-phase support, affinity reagent or epitope tag) is a challenging task. The standard approach is to identify nonspecific interactions using one or more negative-control purifications, but many small-scale AP-MS studies do not capture a complete, accurate background protein set when available controls are limited. Fortunately, negative controls are largely bait independent. Hence, aggregating negative controls from multiple AP-MS studies can increase coverage and improve the characterization of background associated with a given experimental protocol. Here we present the contaminant repository for affinity purification (the CRAPome) and describe its use for scoring protein-protein interactions. The repository (currently available for Homo sapiens and Saccharomyces cerevisiae) and computational tools are freely accessible at http://www.crapome.org/.

Localization of phosphorylated CK2alpha to the mitotic spindle requires the peptidyl-prolyl isomerase Pin1.

CK2 is a serine/threonine kinase with many substrates, largely unknown modes of regulation and essential roles in mitotic progression. CK2α, a catalytic subunit of CK2, is phosphorylated in mitosis, and here we examine the effect of phosphorylation on CK2α localization. Using phosphospecific antibodies, we show that CK2α localizes to the mitotic spindle in a phosphorylation-dependent manner. Mitotic spindle localization requires the unique C-terminus of CK2α, and involves a novel regulatory mechanism in which phosphorylation of CK2α facilitates binding to the peptidyl-prolyl isomerase Pin1, which is required for CK2α mitotic spindle localization. This could explain how the constitutive activity of CK2α might be targeted towards mitotic substrates. Furthermore, because Pin1 has many important spindle substrates, this might represent a general mechanism for localization of mitotic signalling proteins.

Protein kinase CK2 is a constitutively active enzyme that promotes cell survival: strategies to identify CK2 substrates and manipulate its activity in mammalian cells.

Protein kinase CK2 is a constitutively active protein serine/threonine kinase that is ubiquitously expressed and essential for the survival of eukaryotic cells. On the basis of its elevated expression in a number of human cancers and its ability to promote tumorigenesis in transgenic mice, CK2 has emerged as a promising candidate for molecular-targeted therapy. Accordingly, there has been considerable interest in identifying the cellular events that are regulated by CK2 and the cellular substrates of CK2 that are responsible for mediating its actions in cells. Large-scale phosphoproteomics studies are revealing extensive lists of candidate CK2 substrates on the basis that these proteins are phosphorylated at sites conforming to the consensus for phosphorylation by CK2. However, efforts to validate the vast majority of these candidates as bona fide physiological CK2 substrates have been hindered by the lack of systematic strategies to identify its direct substrates and manipulate its activity in intact cells. To overcome these limitations, we describe experimental procedures for isolating CK2 from bacteria and from mammalian cells to enable in vitro phosphorylation of candidate substrates. We also outline strategies for manipulating the levels and activity of CK2 in intact cells. Collectively, the methods that are presented in this chapter should enable the identification and characterization of CK2 substrates and CK2-regulated processes both in vitro and in living cells.

Evidence for regulation of mitotic progression through temporal phosphorylation and dephosphorylation of CK2alpha.

Proper mitotic progression is crucial for maintenance of genomic integrity in proliferating cells and is regulated through an intricate series of events, including protein phosphorylation governed by a complex network of protein kinases. One kinase family implicated in the regulation of mitotic progression is protein kinase CK2, a small family of enzymes that is overexpressed in cancer and induces transformation in mice and cultured fibroblasts. CK2alpha, one isoform of the catalytic subunits of CK2, is maximally phosphorylated at four sites in nocodazole-treated cells. To investigate the effects of CK2alpha phosphorylation on mitotic progression, we generated phosphospecific antibodies against its mitotic phosphorylation sites. In U2OS cells released from S-phase arrest, these antibodies reveal that CK2alpha is most highly phosphorylated in prophase and metaphase. Phosphorylation gradually decreases during anaphase and becomes undetectable during telophase and cytokinesis. Stable expression of phosphomimetic CK2alpha (CK2alpha-4D, CK2alpha-4E) results in aberrant centrosome amplification and chromosomal segregation defects and loss of mitotic cells through mitotic catastrophe. Conversely, cells expressing nonphosphorylatable CK2alpha (CK2alpha-4A) show a decreased ability to arrest in mitosis following nocodazole treatment, suggesting involvement in the spindle assembly checkpoint. Collectively, these studies indicate that reversible phosphorylation of CK2alpha requires precise regulation to allow proper mitotic progression.