Phosphorylation of Casein Kinase II by p34cdc2

Abstract

. In the present study, we have demonstrated that a glutathione S-transferase fusion protein encoding the C-terminal 126 amino acids of the ␣ subunit is phosphorylated by p34 cdc2 at the same sites as intact casein kinase II, indicating that the mitotic phosphorylation sites are localized within the C-terminal domain of ␣. Four residues within this domain, Thr-344, Thr-360, Ser-362, and Ser-370, conform to the minimal consensus sequence for p34 cdc2 phosphorylation. Synthetic peptides corresponding to regions of ␣ that contain each of these residues are phosphorylated by p34 cdc2 at these sites. Furthermore, alterations in the phosphorylation of the glutathione S-transferase proteins encoding the C-terminal domain of ␣ are observed when any of the four residues are mutated to alanine. When all four residues are mutated to alanine, the fusion protein is no longer phosphorylated by p34 cdc2 at any of the sites that are phosphorylated in mitotic cells. These results indicate that Thr-344, Thr-360, Ser-362, and Ser-370 are the sites on the ␣ subunit of casein kinase II that are phosphorylated in mitotic cells.Biochemical and genetic studies have demonstrated that the p34 cdc2 protein kinase is an indispensable regulator of events leading to the division of eukaryotic cells (for reviews see Refs. 1-4). To ensure that the division of cells is very precisely regulated, the activity of this protein serine/threonine kinase is exquisitely controlled through its interactions with regulatory cyclins and through phosphorylation of p34 cdc2 itself. p34 cdc2 is defined as a cyclin-dependent kinase since it is inactive unless it is associated with a regulatory cyclin subunit. Furthermore, p34 cdc2 is inhibited by phosphorylation of Tyr-15 and/or Thr-14 (5, 6) but requires phosphorylation of Thr-161 to be activated (7,8). CAK (the p34 cdc2 activating kinase) is responsible for phosphorylation of Thr-161 (9 -11), while the phosphorylation state of Thr-14 and/or Tyr-15 is at least in part controlled by the relative activities of the Wee1 protein kinase (12, 13) and cdc25 protein phosphatase (14,15).Concomitant with the activation of p34 cdc2 at the G 2 -M transition of eukaryotic cells is a massive burst of protein phosphorylation. Many of the events that are associated with entry into mitosis including nuclear envelope breakdown, transcriptional termination, nucleolar disassembly, cytoskeletal reorganization, and chromosome condensation appear to be associated with protein phosphorylation. While it is evident that p34 cdc2 directly phosphorylates a number of proteins at the G 2 -M transition, there are also indications that p34 cdc2 could indirectly regulate phosphorylation events through its phosphorylation of other protein kinases (16 -23).One protein serine/threonine kinase that could be regulated by p34 cdc2 is casein kinase II (CKII), 1 which has been shown to be dramatically phosphorylated in mitotic cells (19 -21). CKII is a tetrameric enzyme composed of two catalytic (␣ and/or ␣Ј-subunits) and two additional subunits (␤ s...

Mitotic Phosphorylation of DNA Topoisomerase II α by Protein Kinase CK2 Creates the MPM-2 Phosphoepitope on Ser-1469

“…A possible clue is that both subunits of CK2 are extensively phosphorylated by cdc2 kinase during mitosis (34). This is particularly dramatic for the catalytic ␣ subunit where 4 residues in the C-terminal domain are phosphorylated by cdc2 kinase resulting in substantial conformational modifications (35). This is consistent with our in vitro findings that the ability of CK2 to create the MPM-2 epitope is greatly stimulated in the presence of cdc2 kinase, although this FIG.…”

“…1A, lanes 2 and 3). Since CK2 is extensively phosphorylated by cdc2 kinase during mitosis (34,35), the influence of the simultaneous presence of both CK2 and cdc2 kinase was also determined. The results show that the presence of cdc2 kinase greatly stimulates the ability of CK2 to generate the MPM-2 phosphoepitope on topoisomerase II␣ (Fig.…”

“…Protein kinase CK2 is a serine/threonine kinase which has shown to be dramatically phosphorylated in mitotic cells (34,35). CK2 is the major kinase phosphorylating topoisomerase II in yeast (36) and a stable topoisomerase II-CK2 molecular complex has been demonstrated (12).…”

Protein kinase CK2 regulates CDC25B phosphatase activity

“…In 3T3 L1 fibroblast cells, the increased expression of CK2b was shown to cause accumulation in G2/M, suggesting a role of CK2 at this point of the cell cycle (Li et al, 1999). Furthermore, both types of CK2 subunits can be phosphorylated by CDK1 in vitro and in intact cells during mitosis (Litchfield et al, 1992;Bosc et al, 1995). In addition, CDK1 itself can be phosphorylated by CK2 (Russo et al, 1992) and CK2 has been implicated in the phosphorylation of cyclin B, which regulates its nuclear translocation (Li et al, 1997b).…”

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

“…It has also been demonstrated that CK2␣, but not CK2␣Ј, is phosphorylated in mitotic cells of mammalian and avian origin (16), suggesting that the functions of the two isoforms are independently regulated during cell division. The mitotic sites of phosphorylation of CK2␣ were identified as Thr 344 , Thr 360 , Ser 362 , and Ser 370 , all of which are located within the carboxyl-terminal domain of CK2␣ (17). There is also a "PXXP" motif adjacent to two of the phosphorylation sites.…”