Similarities in structure and function of calf thymus and Drosophila casein kinase II.

112

A human cDNA encoding the alpha subunit of casein kinase II and a partial cDNA encoding the rat homologue were isolated by using a Drosophila casein kinase II cDNA probe. The 2.2-kb human cDNA contains a 1.2-kb open reading frame, 150 nucleotides of 5' leader, and 850 nucleotides of 3' noncoding region. Except for the first 7 deduced amino acids that are missing in the rat cDNA, the 328 amino acids beginning with the amino terminus are identical between human and rat. The Drosophila enzyme sequence is 90% identical with the human casein kinase II sequence, and there is only a single amino acid difference between the published partial bovine sequence and the human sequence. In addition, the C-terminus of the human cDNA has an extra 53 amino acids not present in Drosophila. Northern analysis of rat and human RNA showed predominant bands of 5.5, 3.1, and 1.8 kb. In rat tissues, brain and spleen had the highest levels of casein kinase II alpha subunit specific RNA, while skeletal muscle showed the lowest. Southern analysis of human cultured cell and tissue genomic DNA using the full-length cDNA probe revealed two bands with restriction enzymes that have no recognition sites within the cDNA and three to six bands with enzymes having single internal sites. These results are consistent with the possibility that two genes encode the alpha subunits.

Section: Discussionsupporting

confidence: 90%

“…The C-terminal 67 amino acids of human and rat are completely different from those of Drosophila, and it is in this region that the human and rat sequences differ at 6 positions. The additional C-terminal 53 amino acids account entirely for the larger size of the mammalian compared to the Drosophila a subunit (Dahmus et al, 1984).…”

Section: Resultsmentioning

confidence: 99%

Molecular cloning of the human casein kinase II .alpha. subunit

Meisner¹,

Heller-Harrison²,

Buxton³

et al. 1989

112

“…In addition to RNA polymerases, calf thymus contains protein kinases (Kranias et al, 1977;Kranias & Jungmann, 1978a,b;Dahmus, 1971 a-c;Gordon et al, 1983;Dahmus et al, 1984). It was of concern, therefore, that the calf thymus RNA polymerase II preparation used here was contaminated by a kinase which could interact with the photoprobes either by binding them at its active site or by transferring the radiolabel to polypeptide E of calf thymus RNA polymerase II.…”

Section: Discussionmentioning

confidence: 99%

Identification of a nucleoside triphosphate binding site on calf thymus RNA polymerase II

Freund¹,

McGuire²

1986

A nucleoside triphosphate binding site on calf thymus RNA polymerase II was identified by using photoaffinity analogues of adenosine 5'-triphosphate and guanosine 5'-triphosphate. Both radiolabeled 8-azidoadenosine 5'-triphosphate (8-N3ATP) and radiolabeled 8-azidoguanosine 5'-triphosphate (8-N3GTP) bound to a single polypeptide of this enzyme. This polypeptide has a molecular mass of 37 kilodaltons and an isoelectric point of 5.4. Ultraviolet (UV) irradiation was necessary for photolabeling to occur. In addition, no labeling occurred when the probe was prephotolyzed or when the enzyme was inactivated. Furthermore, photolabeling of the enzyme could be decreased by preincubation with natural substrates. To provide evidence that the radiolabeled polypeptide forms a part of the domain of the nucleoside triphosphate binding site, experiments were performed using unlabeled 8-N3ATP. Although this unlabeled analogue was not a substrate for RNA polymerase II, it photoinactivated the enzyme in the presence of UV irradiation, and it inhibited transcription elongation by the enzyme in a competitive manner in the absence of UV irradiation. As in the case with photolabeling, photoinactivation by 8-N3ATP could be decreased by natural substrates; in both cases, purine ribonucleoside triphosphates were more efficient than pyrimidine nucleoside triphosphates. Furthermore, photoinactivation was saturable at about the same concentration as the inhibition constant for 8-N3ATP. Collectively, these results provide evidence that the radiolabeled polypeptide in calf thymus RNA polymerase II is an essential component for activity and suggest that this polypeptide may be part of this enzyme's purine ribonucleoside triphosphate binding site.

“…The a subunits (M, 37 000-44 000) have been shown to be catalytic, while the (3 subunit (Mr 24000-28 000), which undergoes autophosphorylation in the presence of ATP or GTP, has a regulatory role (Hathaway et al, 1981;Feige et al, 1983; Cochet & Chambaz, 1983; Palen & Traugh, 1991). Until recently the structural relationship between the catalytic subunits of CK II was unclear (Dahmus et al, 1984). However, now with the availability of CK II sequences of these subunits, evidence has been presented showing that, in several species, the a and a' subunits are products of different genes (Chen-Wu et al, 1988;Meisner et al, 1989;Padmanabha et al, 1990; Lozeman et al, 1990;Litchfield et al, 1990;Maridor et al, 1991).…”

mentioning

confidence: 99%

Cloning and characterization of the casein kinase II .alpha. subunit gene from the lymphocyte-transforming intracellular protozoan parasite Theileria parva

Ok¹,

Sugimoto²,

Pa³

et al. 1992

Theileria parva is an obligate intracellular protozoan parasite which is the causative agent of East Coast fever, an acute, leukemia-like disease of cattle. The intralymphocytic stage of the parasite induces blastogenesis and clonal expansion of quiescent bovid lymphocytes. Experiments in our laboratory have shown a marked increase of casein kinase II- (CK II-) like activity in T. parva-transformed lymphocytes. We have also detected CK II activity in purified T. parva schizonts. To explore the significance of this increase, we used a Drosophila melanogaster CK II alpha cDNA probe [Saxena et al. (1987) Mol. Cell Biol. 7, 3409-3417] to isolate a T. parva genomic clone encoding a CK II catalytic subunit. The clone contains a 1.3-kb open reading frame coding for a predicted protein of 420 amino acids (M(r) 50,200). Northern blot analysis revealed a single transcript of 1.65 kb. The deduced T. parva CK II catalytic subunit sequence shows, over 321 residues comprising the C-terminus of the molecule, extensive identity with CK II alpha and alpha' sequences from both vertebrate and invertebrate organisms. The T. parva CK II subunit amino acid sequence displays 68% identity with the Drosophila alpha subunit and 67% with the Caenorhabditis elegans alpha subunit but only 58% and 56% sequence identity with the Saccharomyces cerevisiae alpha and alpha' subunits, respectively. Comparison of the T. parva sequence with higher eukaryotic alpha and alpha' sequences reveals that it is most identical with the alpha subunit. A unique component of the T. parva CK II alpha subunit is a 99 amino acid sequence at the N-terminus, which contains a sequence motif with features characteristic of signal peptides.