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

Abstract

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...

Casein Kinase II-mediated Phosphorylation of NF-κB p65 Subunit Enhances Inducible Nitric-oxide Synthase Gene Transcription in Vivo

“…54) or in a dissociated form in different cellular compartments (53,55,56). Its regulation and function are not well understood.…”

“…Thus, the difference between the two cell lines is either located downstream of CKII or related to other parameters that modulate the enzyme activity, e.g. the subcellular distribution of its subunits (53).…”

Role of the Regulatory Domain of Protein Kinase D2 in Phorbol Ester Binding, Catalytic Activity, and Nucleocytoplasmic Shuttling

“…Regulatory domains not only modify the catalytic activity but also regulate the subcellular localization of protein kinases (Filhol et al, 2003). So far, very little is known about the subcellular localization of PKD2.…”

CK2 accumulation at the axon initial segment depends on sodium channel Nav1

“…Plasmids encoding GST-CK2a, GST-Nav1.2 1080-1203, GFPNav1.2 984-1203, myc-Kv2.1-Nav1.2 1080-1203 (Kv2.1-Nav1.2 1080-1203) and its point mutants (Kv2.1-Nav1.2 4SA) were previously described [5,7,10]. DNA sequence containing sequence for shRNA, a loop region of 9 nucleotides (TTCAAGAGA), and flanked by H1 and U6 promoter was chemically synthetized and inserted into pCR R 2.1-TOPO (Eurofins mwg/operon).…”