The Pleckstrin Homology Domain of CK2 Interacting Protein-1 Is Required for Interactions and Recruitment of Protein Kinase CK2 to the Plasma Membrane

Abstract

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). ...

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.…”

“…CKIP-1 exhibits a very broad spectrum of binding to phosphoinositols through its PH domain (19). In myoblast C2C12 cells, CKIP-1 binds to PI3P (phosphoinositol-3-phosphate) at the plasma membrane in response to insulin and regulates muscle differentiation; however, PI3K inhibition or coexpression of PI3K (⌬p85), a dominant negative mutant of PI3K, leads to nuclear accumulation of CKIP-1 (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).…”

The Role of CKIP-1 in Cell Morphology Depends on Its Interaction with Actin-capping Protein

“…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.…”

“…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.…”