Searching interaction partners of protein kinase CK2β subunit by two-hybrid screening

Self Cite

126

Protein kinase CK2 is a multifunctional enzyme which has long been described as a stable heterotetrameric complex resulting from the association of two catalytic (␣ or ␣) and two regulatory (␤) subunits. To track the spatiotemporal dynamics of CK2 in living cells, we fused its catalytic ␣ and regulatory ␤ subunits with green fluorescent protein (GFP). Both CK2 subunits contain nuclear localization domains that target them independently to the nucleus. Imaging of stable cell lines expressing low levels of GFP-CK2␣ or GFP-CK2␤ revealed the existence of CK2 subunit subpopulations exhibiting differential dynamics. Once in the nucleus, they diffuse randomly at different rates. Unlike CK2␤, CK2␣ can shuttle, showing the dynamic nature of the nucleocytoplasmic trafficking of the kinase. When microinjected in the cytoplasm, the isolated CK2 subunits are rapidly translocated into the nucleus, whereas the holoenzyme complex remains in this cell compartment, suggesting an intramolecular masking of the nuclear localization sequences that suppresses nuclear accumulation. However, binding of FGF-2 to the holoenzyme triggers its nuclear translocation. Since the substrate specificity of CK2␣ is dramatically changed by its association with CK2␤, the control of the nucleocytoplasmic distribution of each subunit may represent a unique potential regulatory mechanism for CK2 activity.Protein kinase CK2 is a ubiquitous serine/threonine protein kinase, generally described as a stable ␣ 2 ␤ 2 tetramer, where ␣ and ␤ are the catalytic and regulatory subunits, respectively (3). Although its signaling function has long remained obscure, the importance of CK2 is suggested by the evolutionary conservation of the enzyme and by the fact that the disruption of both Saccharomyces cerevisiae genes encoding CK2 catalytic subunits is a lethal event (29). In addition to its role in embryonic development and terminal differentiation, the enzyme is required for normal cell cycle progression (20,30). At last, a function of CK2 in cell survival has recently emerged (1).Many of the identified CK2 substrates that are critical for cell proliferation and viability are localized in different cellular compartments. However, there is controversy as to the localization of CK2 and where its substrates are phosphorylated. Although the current prevailing view of CK2 is a tetrameric enzyme, accumulating evidence also indicates that free populations of both CK2 subunits can exist and exert specific functions in the cell (18, 37). At least in vitro, CK2␤ exerts a central role in modulating the catalytic activity of CK2 (26). Consequently, it is suspected that in vivo, the substrate specificity of the enzyme is likely to be determined both by subcellular localization and by affinity for its regulatory subunit that brings the kinase in proximity to the substrate.In a previous study, the behavior of CK2 subunits fused to GFP was characterized in living cells (25). The expressed fusion proteins were functional and interacted with endogenous CK2. Both subunits were mostl...

Section: Discussionmentioning

confidence: 90%

Live-Cell Fluorescence Imaging Reveals the Dynamics of Protein Kinase CK2 Individual Subunits

Filhol

Nueda

Martel

et al. 2003

Self Cite

126

“…Our results demonstrate that the heterodimeric actin capping protein is a novel CKIP-1-interacting protein. In a similar respect, it is noteworthy that yeast twohybrid studies to identify CK2-interacting proteins previously led to the identification of CP as a potential interacting partner of protein kinase CK2 (19).…”

Section: Resultsmentioning

confidence: 97%

The Pleckstrin Homology Domain-Containing Protein CKIP-1 Is Involved in Regulation of Cell Morphology and the Actin Cytoskeleton and Interaction with Actin Capping Protein

Canton

Olsten

Kim

et al. 2005

CKIP-1 is a pleckstrin homology domain-containing protein that interacts with protein kinase CK2. To elucidate the functions of CKIP-1, we generated human osteosarcoma cell lines with tetracycline-regulated expression of Flag-CKIP-1. Flag-CKIP-1 expression resulted in distinct changes in cellular morphology. Therefore, we examined the actin profile by immunofluorescence, quantitative measurement of phalloidin binding, and immunoblot analysis. These studies demonstrate that Flag-CKIP-1 expression resulted in increases in F-actin staining and protein levels of ␤-actin. To elucidate the mechanisms behind the observed phenotype, we utilized tandem affinity purification to isolate CKIP-1 interacting proteins. Mass spectrometry analysis led to the identification of the actin capping protein subunits, CP␣ and CP␤, as novel CKIP-1 interaction partners. Interactions were confirmed by coimmunoprecipitation and by colocalization. Furthermore, we demonstrate that Ser9 of CP␣ is phosphorylated by protein kinase CK2 in vitro, that CP␣ is phosphorylated in vivo, and that treatment with a CK2-specific inhibitor results in a decrease in CP␣ phosphorylation. Finally, we demonstrate that CKIP-1 and CK2 inhibit the activity of actin capping protein at the barbed ends of actin filaments. Overall, our results are consistent with CKIP-1 playing a role in the regulation of the actin cytoskeleton through its interactions with actin capping protein.The phosphorylation and dephosphorylation of proteins that is mediated by a complex network of protein kinases is a key mechanism intimately associated with the control of various aspects of cellular regulation (reviewed in references 23 and 34). One component of these regulatory kinase networks is protein kinase CK2. Protein kinase CK2 (formerly casein kinase II) is a ubiquitous and highly conserved protein serine/ threonine kinase that is essential for viability in eukaryotic organisms (7,26,52). Although its precise functions remain poorly defined, there is mounting evidence to suggest that CK2 plays an important role in control of cell proliferation and transformation (reviewed in references 31 and 37). A number of studies have shown alteration in the expression of CK2 in a variety of tumor or leukemic cells (11,17,53,59). Furthermore, the targeted overexpression of CK2␣ in the T cells of transgenic mice results in lymphocyte transformation. In these mice, there is evidence for collaboration between the dysregulated expression of CK2␣ and the c-Myc and Tal-1 oncogenes in lymphoma development (25, 51). Taken together, these studies demonstrate a role for CK2 as a component of the kinase networks that regulate the growth and division of cells. Despite the importance of CK2 in various aspects of cell regulation and its role in transformation, many questions regarding its regulation in cells remain.An emerging paradigm in signal transduction is the importance of the clustering and targeting of signaling molecules. A notable example of this targeting is the interaction between the pleiotropic c...

“…This might lead to an unbalanced phosphorylation of CK2␣-and holoenzyme-specific substrates in CK2␤ Ϫ/Ϫ cells and to changes in the phosphorylation pattern of substrates relevant for cell survival. Furthermore, CK2␤ might have acquired other tasks in higher organisms that it does not perform in yeast, which might be reflected by the fact that CK2␤ can interact with many cellular proteins (9,21). It is worth noting that in the two yeast species that have been analyzed, different phenotypes were observed when CK2␤ was knocked out.…”

Section: Discussionmentioning

confidence: 99%

Disruption of the Regulatory β Subunit of Protein Kinase CK2 in Mice Leads to a Cell-Autonomous Defect and Early Embryonic Lethality

Buchou

Vernet

Blond

et al. 2003

Self Cite

241

202

Protein kinase CK2 is a ubiquitous protein kinase implicated in proliferation and cell survival. Its regulatory ␤ subunit, CK2␤, which is encoded by a single gene in mammals, has been suspected of regulating other protein kinases. In this work, we show that knockout of the CK2␤ gene in mice leads to postimplantation lethality. Mutant embryos were reduced in size at embryonic day 6.5 (E6.5). They did not exhibit signs of apoptosis but did show reduced cell proliferation. Mutant embryos were resorbed at E7.5. In vitro, CK2␤ ؊/؊ morula development stopped after the blastocyst stage. Attempts to generate homozygous embryonic stem (ES) cells failed. By using a conditional knockout approach, we show that lack of CK2␤ is deleterious for mouse ES cells and primary embryonic fibroblasts. This is in contrast to what occurs with yeast cells, which can survive without functional CK2␤. Thus, our study demonstrates that in mammals, CK2␤ is essential for viability at the cellular level, possibly because it acquired new functions during evolution.Protein kinase CK2 is a pleiotropic and highly conserved protein kinase with more than 300 substrates described to date. It seems to be involved in controlling a large panel of normal cellular functions such as gene expression, protein synthesis, cell cycle, and proliferation, as well as pathological processes such as carcinogenesis and viral tumorigenesis (12, 33). Recently, its function in protecting cells against apoptosis has been reported (1).CK2 is a tetrameric holoenzyme generally composed of two catalytic subunits, ␣ and ␣Ј, and two regulatory ␤ subunits which combine to form an ␣␣Ј␤ 2 , ␣ 2 ␤ 2 , or ␣Ј 2 ␤ 2 heterotetramer. The catalytic CK2 subunits ␣ and ␣Ј belong to the eukaryotic protein kinase superfamily. In contrast, the regulatory ␤ subunit is a unique protein encoded by a single gene in mammals (3) and does not belong to a known protein family.CK2␤ has several functions in the holoenzyme complex. Reconstitution experiments with recombinant purified subunits have demonstrated that CK2␤ modulates the activity of CK2. Depending on the substrate, CK2␤ activates or downregulates the activity of the catalytic subunit (24). CK2␤ also confers stability to the holoenzyme complex (18) and seems to mediate interaction with a number of substrates (19).The crystal structure elucidations of the isolated CK2␤ subunit (5) and of the holoenzyme complex (28) indicate that the ␤ subunit exists as a dimer and is the building block of the CK2 holoenzyme bridging the two catalytic subunits. The crystal structure is also consistent with the suggested flexible role of the ␤ subunit as a docking partner for other protein kinases and other interacting partners in the cell (28).Functional and biochemical studies have indicated that fractions of both the catalytic and regulatory subunits may exist separately. A population of CK2␣ that binds to protein phosphatase 2A is free of CK2␤ (16). Moreover, CK2␤ fractions devoid of the catalytic subunit, but probably involved in complexes with other prote...