CK2 is a highly conserved, ubiquitously expressed protein serine/threonine kinase present in all eukaryotes. Circumscribed as having a vast array of substrates located in a number of cellular compartments, CK2 has been implicated in critical cellular processes such as proliferation, apoptosis, differentiation, and transformation. Despite advances in elucidating its substrates and involvement in cellular regulation, its precise mode of regulation remains poorly defined. In this respect, there are currently conflicting views as to whether CK2 is constitutively active or modulated in response to specific stimuli. Perhaps an important consideration in resolving these apparent discrepancies is recognition of the existence of many discrete CK2 subpopulations that are distinguished from one another by localization or association with distinct cellular components. The existence of these subpopulations brings to light the possibility of each population being regulated independently rather than the entire cellular CK2 content being regulated globally. Logically, each local population may then be regulated in a distinct manner to carry out its precise function(s). This review will examine those mechanisms including regulated expression and assembly of CK2 subunits, phosphorylation of CK2, and interactions with small molecules or cellular proteins that could contribute to the local regulation of distinct CK2 populations.

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...

CKIP-1 is a recently identified interaction partner of protein kinase CK2 with a number of protein-protein interaction motifs, including an N-terminal pleckstrin homology domain. To test the hypothesis that CKIP-1 has a role in targeting CK2 to specific locations, we examined the effects of CKIP-1 on the localization of CK2. These studies demonstrated that CKIP-1 can recruit CK2 to the plasma membrane. Furthermore, the pleckstrin homology domain of CKIP-1 was found to be required for interactions with CK2 and for the recruitment of CK2 to the plasma membrane. In this regard, point mutations in this domain abolish membrane localization and compromise interactions with CK2. In addition, replacement of the pleckstrin homology domain with a myristoylation signal was insufficient to elicit any interaction with CK2. An investigation of the lipid binding of CKIP-1 reveals that it has broad specificity. A comparison with other pleckstrin homology domains revealed that the pleckstrin homology domain of CKIP-1 is distinct from other defined classes of pleckstrin homology domains. Finally, examination of CK2␣ for a region that mediates interactions with CKIP-1 revealed a putative HIKE domain, a complex motif found exclusively in proteins that bind pleckstrin homology domains. However, mutations within this motif were not able to abolish CKIP-1-CK2 interactions suggesting that this motif by itself may not be sufficient to mediate interactions. Overall, these results provide novel insights into how CK2, a predominantly nuclear enzyme, is targeted to the plasma membrane, and perhaps more importantly how it may be regulated. CK21 (formerly casein kinase II) is a ubiquitously expressed extraordinary conserved Ser/Thr kinase found in all eukaryotic cells (1-3). CK2 has been shown to be essential for viability in a variety of models ranging from yeast to mammalian cells (4 -6). An elevated CK2 activity has been detected in leukemic cells, healthy tissues with high mitotic index, and in a variety of human cancers (7-9). In addition, CK2 exhibits oncogenic activity when overexpressed in transgenic mice (10). The majority of CK2 is found in the nucleus of logarithmically growing cells (11-13); however, there have been indications that the nuclear/cytoplasmic distribution of CK2 is regulated in a cell cycle-dependent manner (14). Antibodies that interfere with the nuclear uptake of CK2 inhibit mitogenic stimulation upon micro-injection into cells (15), indicating the importance of the nature of CK2 localization for its biological function. To date, CK2 has been shown to phosphorylate and/or interact with a broad range of proteins located in a variety of cellular compartments, including nuclear proteins (16 -20), cytoplasmic proteins (21-23), and proteins localized at the plasma membrane (24 -26).This tetrameric enzyme is composed of two catalytic (␣ and ␣Ј) and two regulatory subunits (␤). The two catalytic subunits are products of separate genes and show greater than 90% sequence identity over their N-terminal 330 amino acids (27). ...

CKIP-1 is a pleckstrin homology domain-containing protein that induces alterations of the actin cytoskeleton and cell morphology when expressed in human osteosarcoma cells. CKIP-1 interacts with the heterodimeric actin-capping protein in cells, so we postulated that this interaction was responsible for the observed cytoskeletal and morphological effects of CKIP-1. To test this postulate, we used peptide "walking arrays" and alignments of CKIP-1 with CARMIL, another CP-binding protein, to identify Arg-155 and Arg-157 of CKIP-1 as residues potentially required for its interactions with CP. CKIP-1 mutants harboring Arg-155 and Arg-157 substitutions exhibited greatly decreased CP binding, while retaining wild-type localization, the ability to interact with protein kinase CK2, and self-association. To examine the phenotype associated with expression of these mutants, we generated tetracycline-inducible human osteosarcoma cells lines expressing R155E,R157E mutants of CKIP-1. Examination of these cell lines reveals that CKIP-1 R155E,R157E did not induce the distinct changes in cell morphology and the actin cytoskeleton that are characteristic of wild-type CKIP-1 demonstrating that the interaction between CKIP-1 and CP is required for these cellular effects. CKIP-14 was identified in a yeast two-hybrid screen for novel interaction partners of protein kinase CK2 (1). The cDNA for CKIP-1 codes for a protein of ϳ46 kDa with an amino-terminal pleckstrin homology (PH) domain and a carboxyl-terminal leucine-rich region as well as five putative PXXP motifs. The PH domain of CKIP-1 is required for phospholipid binding in vitro and for plasma membrane localization in cells (1, 2). Furthermore, this domain is necessary for interactions with protein kinase CK2, because mutants lacking the PH domain fail to interact with the kinase. Additionally, we have demonstrated that a subpopulation of protein kinase CK2 is targeted to the plasma membrane by CKIP-1 in cells (2). This targeting of CK2 is lost when the PH domain of CKIP-1 is replaced by a myristoylation recognition sequence, even though the CKIP-1 mutant still localizes to the plasma membrane. These results suggest that CKIP-1 may function in an analogous manner to protein kinase A anchoring proteins, which target cAMP-dependent protein kinase A (3-6). In addition to this potential role as a CK2-targeting protein, CKIP-1 appears to have roles independent of CK2. Recent reports have shown that CKIP-1 functions in muscle cell differentiation (7) and AP-1 regulation and apoptosis (8).To investigate the cellular functions of CKIP-1, we generated cell lines with tetracycline-regulated expression of FLAG-CKIP-1. Induction of FLAG-CKIP-1 in these cells caused changes in cellular morphology, as well as increases in F-actin and total cellular levels of actin (9). To determine the mechanistic basis for these observations, we performed a proteomic screen using Tandem affinity purification (10) and large-scale immunoprecipitations to identify CKIP-1 interaction partners. We identified the h...

Protein kinase CK2 represents a small family of highly conserved protein kinases involved in a complex series of cellular events. Furthermore, CK2 has been localised to many discrete cellular sites and has an extensive and diverse array of substrates and interaction partners in cells. Despite considerable investigation, the precise mechanism(s) of regulation of CK2 in cells remains poorly understood. In consideration of the prospect that cells contain many distinct sub-populations of CK2 that are distinguished on the basis of localisation and/or interactions with other cellular components, one possibility is that there may be differential regulation of specific sub-populations of CK2. With this in mind, some of the individual sub-populations of CK2 may be regulated through particular protein-protein interactions that may play a role in recruiting CK2 into the vicinity of its substrates and/or modulating its ability to phosphorylate specific cellular targets. In this respect, here we examine two CK2-interacting proteins, namely Pin1 and CKIP-1 that have been shown to participate in the modulation of CK2 specificity or the subcellular localisation of CK2, respectively. One aspect of this work has been focused on the prospect that Pin1 interacts with CK2 in response to UV stimulation in a manner analogous to the phosphorylation-dependent interactions of CK2 that occur following the mitotic phosphorylation of CK2. A second aspect of this work involves an examination of the structural basis for interactions between CK2 and CKIP-1 with emphasis on a putative HIKE domain in CK2.