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Molecular identification of a calcium-inhibited catalytic subunit of casein kinase type 2 from Paramecium tetraurelia.

ABSTRACT: We have previously described the occurrence in Paramecium of a casein kinase (CK) activity (EC 2.7.1.37) with some unusual properties, including inhibition by Ca(2+) (R. Kissmehl, T. Treptau, K. Hauser, and H. Plattner, FEBS Lett. 402:227-235, 1995). We now have cloned four genes, PtCK2alpha1 to PtCK2alpha4, all of which encode the catalytic alpha subunit of type 2 CK (CK2) with calculated molecular masses ranging from 38.9 to 39.4 kDa and pI values ranging from 8.8 to 9.0. They can be classified into two groups, which differ from each other by 28% on the nucleotide level and by 18% on the derived amino acid level. One of them, PtCK2alpha3, has been expressed in Escherichia coli and characterized in vitro. As we also have observed with the isolated CK, the recombinant protein preferentially phosphorylates casein but also phosphorylates some Paramecium-specific substrates, including the exocytosis-sensitive phosphoprotein pp63/parafusin. Characteristically, Ca(2+) inhibits the phosphorylation at elevated concentrations occurring during stimulation of a cell. Reconstitution with a recombinant form of the regulatory subunit from Xenopus laevis, XlCK2beta, confirms Ca(2+) sensitivity also under conditions of autophosphorylation. This is unusual for CK2 but correlates with the presence of two EF-hand calcium-binding motifs, one of which is located in the N-terminal segment essential for constitutive activity, as well as with an aberrant composition of normally basic domains recognizing acidic substrate domains. Immunogold localization reveals a considerable enrichment in the outermost cell cortex layers, excluding cilia. We discuss a potential role of this Ca(2+)-inhibited PtCK2alpha species in a late step of signal transduction.

SUBMITTER: Vetter D

PROVIDER: S-EPMC326640 | biostudies-literature | 2003 Dec

REPOSITORIES: biostudies-literature

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Molecular identification of a calcium-inhibited catalytic subunit of casein kinase type 2 from Paramecium tetraurelia.

Vetter Daniel D Kissmehl Roland R Treptau Tilman T Hauser Karin K Kellermann Josef J Plattner Helmut H

Eukaryotic cell 20031201 6

We have previously described the occurrence in Paramecium of a casein kinase (CK) activity (EC 2.7.1.37) with some unusual properties, including inhibition by Ca(2+) (R. Kissmehl, T. Treptau, K. Hauser, and H. Plattner, FEBS Lett. 402:227-235, 1995). We now have cloned four genes, PtCK2alpha1 to PtCK2alpha4, all of which encode the catalytic alpha subunit of type 2 CK (CK2) with calculated molecular masses ranging from 38.9 to 39.4 kDa and pI values ranging from 8.8 to 9.0. They can be classifie ...[more]

PMID: 14665457

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Project description:BACKGROUND: A Paramecium tetraurelia pilot genome project, the subsequent sequencing of a Megabase chromosome as well as the Paramecium genome project aimed at gaining insight into the genome of Paramecium. These cells display a most elaborate membrane trafficking system, with distinct, predictable pathways in which actin could participate. Previously we had localized actin in Paramecium; however, none of the efforts so far could proof the occurrence of actin in the cleavage furrow of a dividing cell, despite the fact that actin is unequivocally involved in cell division. This gave a first hint that Paramecium may possess actin isoforms with unusual characteristics. The genome project gave us the chance to search the whole Paramecium genome, and, thus, to identify and characterize probably all actin isoforms in Paramecium. RESULTS: The ciliated protozoan, P. tetraurelia, contains an actin multigene family with at least 30 members encoding actin, actin-related and actin-like proteins. They group into twelve subfamilies; a large subfamily with 10 genes, seven pairs and one trio with > 82% amino acid identity, as well as three single genes. The different subfamilies are very distinct from each other. In comparison to actins in other organisms, P. tetraurelia actins are highly divergent, with identities topping 80% and falling to 30%. We analyzed their structure on nucleotide level regarding the number and position of introns. On amino acid level, we scanned the sequences for the presence of actin consensus regions, for amino acids of the intermonomer interface in filaments, for residues contributing to ATP binding, and for known binding sites for myosin and actin-specific drugs. Several of those characteristics are lacking in several subfamilies. The divergence of P. tetraurelia actins and actin-related proteins between different P. tetraurelia subfamilies as well as with sequences of other organisms is well represented in a phylogenetic tree, where P. tetraurelia sequences only partially cluster. CONCLUSION: Analysis of different features on nucleotide and amino acid level revealed striking differences in isoforms of actin and actin-related proteins in P. tetraurelia, both within the organism and in comparison to other organisms. This diversification suggests unprecedented specification in localization and function within a unicellular eukaryote.

Dataset Information

Molecular identification of a calcium-inhibited catalytic subunit of casein kinase type 2 from Paramecium tetraurelia.

Publications

Molecular identification of a calcium-inhibited catalytic subunit of casein kinase type 2 from Paramecium tetraurelia.

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