Identification and Characterization of CKIP-1, a Novel Pleckstrin Homology Domain-containing Protein That Interacts with Protein Kinase CK2

Abstract

The catalytic subunits of protein kinase CK2, CK2␣ and CK2␣, are closely related to each other but exhibit functional specialization. To test the hypothesis that specific functions of CK2␣ and CK2␣ are mediated by specific interaction partners, we used the yeast twohybrid system to identify CK2␣-or CK2␣-binding proteins. We report the identification and characterization of a novel CK2-interacting protein, designated CKIP-1, that interacts with CK2␣, but not CK2␣, in the yeast two-hybrid system. CKIP-1 also interacts with CK2␣ in vitro and is co-immunoprecipitated from cell extracts with epitope-tagged CK2␣ and an enhanced green fluorescent protein fusion protein encoding CKIP-1 (i.e. EGFP-CKIP-1) when they are co-expressed. CK2 activity is detected in anti-CKIP-1 immunoprecipitates performed with extracts from non-transfected cells indicating that CKIP-1 and CK2 interact under physiological conditions. The CKIP-1 cDNA is broadly expressed and encodes a protein with a predicted molecular weight of 46,000. EGFP-CKIP-1 is localized within the nucleus and at the plasma membrane. The plasma membrane localization is dependent on the presence of an amino-terminal pleckstrin homology domain. We postulate that CKIP-1 is a non-enzymatic regulator of one isoform of CK2 (i.e. CK2␣) with a potential role in targeting CK2␣ to a particular cellular location. Protein kinase CK2 (CK2)1 is an essential, highly conserved, protein serine/threonine kinase present in all eukaryotic cells (reviewed in Refs. 1-6). CK2 has been reported to phosphorylate a broad range of cellular proteins located in a variety of cellular compartments (mainly the nucleus and cytoplasm) and is involved in important cellular processes such as transcription, translation, morphogenesis, and cell cycle progression (reviewed in Refs. 1-7). These observations support an important role for CK2 in a variety of cellular functions; however, its specific roles and mode of regulation in cells remain poorly understood. Moreover, these results suggest that CK2 is involved in a complex array of interactions with a wide selection of cellular proteins that are present in a broad variety of cellular locations.CK2 is a tetrameric protein comprised of two regulatory subunits (CK2␤) and two catalytic subunits (CK2␣ and/or CK2␣Ј). CK2␣ and CK2␣Ј are the products of separate genes, and their amino acid sequences are highly conserved between higher eukaryotes (reviewed in Ref. 7). In fact, in mammals and birds, CK2␣ and CK2␣Ј exhibit greater than 90% identity over their 330 amino-terminal amino acids (7). This aminoterminal sequence identity is in stark contrast to the unrelated carboxyl-terminal sequences of CK2␣ and CK2␣Ј that exhibit no obvious similarity (8 -10). This sequence divergence between the carboxyl-terminal domains of CK2␣ and CK2␣Ј suggests that important functional differences that exist between the two different catalytic isozymes result from these unique sequences (11).Previous studies have failed to demonstrate significant catalytic differences between CK2␣ and...

Protein kinase CK2 interacts with the splicing factor hPrp3p

“…Moreover, in addition to being a binding partner, hPrp3p is also a substrate for the CK2 holoenzyme consisting of two regulatory and two catalytic a-or a 0 subunits, but not for the catalytic subunits alone. Binding partners for the individual subunits of CK2 were already detected earlier including hsp90, PP2a, Pim-1, CKIP-1, cyclin H, NAP1 and Egr-1 (Jain et al, 1996;Li et al, 1999;Bosc et al, 2000;Faust et al, 2002;Messenger et al, 2002). Some of these binding partners are also substrates for the CK2 kinase.…”

Phosphorylation regulates the stability of the regulatory CK2β subunit

“…Myc-tagged proteins were also detected with the same antibodies on immunoblots. HA-tagged proteins were detected with biotinylated anti-HA 3F10 rat monoclonal antibodies (Roche Biochemicals) using enhanced chemiluminescence (Supersignal from Pierce Chemical Company) as described Bosc et al, 2000). Anti-b-tubulin (a generous gift from Dr L Dagnino, London, Canada) was utilized as a loading control for immunoblot analysis of extracts prepared from cells treated with the proteasome inhibitor MG132.…”

The Role of the Pleckstrin Homology Domain-containing Protein CKIP-1 in Activation of p21-activated Kinase 1 (PAK1)

“…CKIP-1 was originally identified as a CK2␣-interacting protein. However, it has not been demonstrated whether CKIP-1 is involved in phosphorylation of CK2 substrates (18,19,23). Our data show for the first time that CKIP-1 delivers CK2␣ to PAK1 in response to EGF, and that this process is regulated by PI3K, demonstrating a mechanistic link between CK2 and PI3K signaling.…”

“…Subcellular localization of CKIP-1 is dependent on the cell type. CKIP-1 localizes in the nucleus in osteosarcoma Saos-2, mouse preosteoblastic MC-3T3, and rodent fibroblast Rat-1 cells, but is also found at the plasma membrane in monkey kidney COS-7 cells (18). Therefore, it has been suggested that CKIP-1, as a scaffolding protein, may shuttle between the nucleus and the plasma membrane and redistributes signaling complexes between these two compartments in a PI3K-dependent manner (24).…”

“…The pleckstrin homology (PH) domain-containing protein CKIP-1, identified as a CK2␣-interacting protein (18), recruits CK2␣ to the plasma membrane (19). Accumulating evidence indicates that CKIP-1 is involved in various cellular processes including cell proliferation, differentiation, morphology, and migration in various human cell lines including human osteosarcoma U2-OS and macrophage (20 -22).…”