Abstract. We have developed specific antibodies to synthetic peptide antigens that react with the individual subunits of casein kinase 11 (CKII) . Using these antibodies, we studied the localization of CKII in asynchronous HeLa cells by immunofluorescence and immunoelectron microscopy. Further studies were done on HeLa cells arrested at the Gl/S transition by hydroxyurea treatment . Our results indicate that the CKII a and ß subunits are localized in the cytoplasm C ASEIN kinase II (CKII)' is a ubiquitous protein serlne/threonine kinase found in eukaryotic cells (Edelman et al . 1987 and highly conserved among eukaryotic organisms, including Drosophila, yeast, C. elegans, bovine, and human (Saxena et al ., 1987;Chen-Wu et al ., 1988;Hu and Rubin, 1990a ;Lozeman, 1990). Casein kinase II from several species share a common polypeptide subunit structure, a2ß2, with a of M 37,000-44,000 and ß of M 24,000-28,000 by electrophoresis (Edelman et al ., 1987) . Two forms of a are known designated a (M 41,000-44,000) and a' (M, 37,000-42,000) . The a and a' subunits are thought to be the catalytic subunits based on their kinase activity in the absence of the ß subunit and sequences common to other protein kinases (Hathaway et al ., 1981; Cochet and Chambaz, 1983 ;Chen-wu et al ., 1988 ;Meisner et al., 1989;. The function of the ß subunit is unknown, and it shares no extensive homology to other known protein sequences (Jakobi et al ., 1989) . The ß subunit has a high degree of polarity with clusters ofnegative charges in the amino-terminal region and positive charge clusters in the carboxy-terminal region (Takio et al., 1987) . The basic compounds, spermine, spermidine, and polylysine, stimulate activity by interacting at least in part with the ß subunit (Traugh et al ., 1990) . This subunit may have a regulatory role in the holoenzyme (Takio et al., 1987), and evidence supporting this has been found in A-431 cells (Ackerman et al ., 1990). The comparative studies using the native CKII holoenzyme and the bacterially expressed a subunit show that the expressed a is inhibited by heparin, but it is not stimulated by polyamines and has 9% of K t of the holoenzyme (Hu and Rubin, 1990b during interphase and are distributed throughout the cell during mitosis . Further electron microscopic investigation revealed that CKII a subunit is associated with spindle fibers during metaphase and anaphase. In contrast, the CKII a' subunit is localized in the nucleus during GI and in the cytoplasm during S. Taken together, our results suggest that CKII may play significant roles in cell division control by shifting its localization between the cytoplasm and nucleus .further suggests that CKII holoenzyme is stabilized by interaction with the ß subunit .

Synthetic peptide substrates for the cell division cycle regulated protein kinase, p34cdc2, have been developed and characterized. These peptides are based on the sequences of two known substrates of the enzyme, Simian Virus 40 Large T antigen and the human cellular recessive oncogene product, p53. The peptide sequences are H-A-D-A-Q-H-A-T-P-P-K-K-K-R-K-V-E-D-P-K-D-F-OH (T antigen) and H-K-R-A-L-P-N-N-T-S-S-S-P-Q-P-K-K-K-P-L-D-G-E-Y-NH2 (p53), and they have been employed in a rapid assay of phosphorylation in vitro. Both peptides show linear kinetics and an apparent Km of 74 and 120 microM, respectively, for the purified human enzyme. The T antigen peptide is specifically phosphorylated by p34cdc2 and not by seven other protein serine/threonine kinases, chosen because they represent major classes of such enzymes. The peptides have been used in whole cell lysates to detect protein kinase activity, and the cell cycle variation of this activity is comparable to that measured with specific immune and affinity complexes of p34cdc2. In addition, the peptide phosphorylation detected in mitotic cells is depleted by affinity adsorption of p34cdc2 using either antibodies to p34cdc2 or by immobilized p13, a p34cdc2-binding protein. Purification of peptide kinase activity from mitotic HeLa cells yields an enzyme indistinguishable from p34cdc2. These peptides should be useful in the investigation of p34cdc2 protein kinase and their regulation throughout the cell division cycle.