Purification and properties of a cAMP‐independent nuclear protein kinase from Dictyostelium discoideum

Abstract

A cyclic-AMP-independent nuclear protein kinase has been purified from Dictyostelium discoideum amoebae. The purification procedure involves chromatography of DEAE-Sephadex, phosphocellulose and heparinSepharose. The purified enzyme phosphorylates threonine and serine of acidic proteins as casein and phosvitin. Phosphorylation of casein is stimulated by spermine. The kinase requires Mg2 + and can utilize both ATP and GTP as phosphoryl donors. Heparin is a potent inhibitor of the enzyme, being the protein kinase activity fully inhibited at concentrations of 0.5 pg/ml. One polypeptide of molecular mass 38 kDa was the major protein band present in the purified kinase preparation as estimated by NaDodSO, denaturing polyacrylamide gel electrophoresis. This band belongs to the protein kinase because it is the only one that is observed associated with the protein kinase activity when the enzyme preparation is centrifuged in glycerol gradients. The 38-kDa polypeptide is also the major product of autophosphorylation of the enzyme preparation. The enzymatic properties allow to classify the enzyme as a type-I1 casein kinase. However, its structural properties are different from the mammalian type-I1 casein kinases and make the D. discoideum enzyme more similar to the plants type-I1 casein kinases.Phosphorylation/dephosphorylation of proteins represents an important mechanism in the control of cellular processes, including the regulation of gene expression in eukaryotic cells I1,21.Among the enzymes that catalyze the postranslational phosphorylation of proteins are cyclic-nucleotide-dependent an independent protein kinases. Extensive investigations of CAMP-dependent protein kinases have shown that these enzymes mediate all of the biological effects of cyclic AMP. In contrast, little is known about the physiological role of the CAMP-independent protein kinases, although several types of these kind of enzyme have been characterized in different biological systems, mainly higher eukaryotes [3].The nuclear phosphorylation of chromatin-associated proteins, both histones and non histones, have been implicated in the regulation of gene transcription [4, 51. A cyclic-nucleotideindependent, polyamine-sensitive protein kinase has been involved in the control of transcription of ribosomal RNA genes by means of phosphorylation of RNA polymerase I [6] and some transcriptional factors [7].In the slime mold Dictyostelium discoideum a lot of effort has been dedicated to the study of the involvement of CAMPdependent protein kinases in the expression of its differentiation program. CAMP is the chemical agent that mediates aggregation of the amoebae [8] and it is also implicated in the regulation of latter stages of differentiation [9]. CAMPdependent protein kinases have been identified in D. discoideum both in vegetative cells [lo] and during differentiation [I 1, 121. Besides, the characterization of the regulatory subunit of a CAMP-dependent protein kinase has also been carried out using a photo-affinity probe [13]. However, the character...

Molecular cloning of casein kinase II alpha subunit from Dictyostelium discoideum and its expression in the life cycle.

“…The rat testis enzyme was completely inhibited under the same conditions. Our results are at variance with those of a previous study (93), in which the heparin sensitivity of what appears to be the same Dictyostelium enzyme was found to be close to that of mammalian casein kinase II. We currently have no good explanation for this discrepancy, although it should be noted that certain basic peptides have the ability to reverse heparin inhibition of casein kinase II (13).…”

“…The deduced amino acid sequence of Dictyostelium casein kinase II a has strong homology with sequences of both (93), whereas autophosphorylation of the a subunit is reported to be much less than that of the 13 subunit in other species (1,27,30,43) unless polylysine is present (88).…”

“…For example, cyclic AMP (cAMP)-dependent protein kinase has been purified from D. discoideum (15, 20-22, 63, 73), and clones for the regulatory (R) (84) and catalytic (C) (9, 77) subunits of this protein kinase have been isolated. Myosin heavy-chain kinases (18,92), myosin lightchain kinase (105), protein kinase C (53,70), and casein kinases (86,93) have also been purified or partially purified from D. discoideum.…”

The relationship between the two forms of glycogen phosphorylase in Dictyostelium discoideum

“…Mgz * dependent nuclear enzyme, and is therefore presumably not equivalent to the cytosolic, Mnz * dependent kinase activity reported here (Renart et al 1984). The phosphorylation of the 'b' enzyme was also detected in vivo , suggesting that the observed in vitro phosphorylation of this enzyme was physiologically signiiicant (Fig 16).…”

Purification and properties of a casein kinase II-like enzyme from Neurospora crassa

“…No band of 32-31 kDa was observed in CK II preparation of N. crassa, either by staining or autoradiography. Such a band is absent in wheat germ enzyme [23] but present in Dictyostelium discoideum CK II [28]. As shown in Table 1, the enzyme activity was stimulated by polybasic proteins like protaminesulfate and inhibited by heparin and 2,3-diphosphoglycerate.…”