A Novel Casein Kinase 2 α-Subunit Regulates Membrane Protein Traffic in the Human Hepatoma Cell Line HuH-7

Abstract

A previously isolated endocytic trafficking mutant (TRF1) isolated from HuH-7 cells is defective in the distribution of subpopulations of cell-surface receptors for asialoorosomucoid (asialoglycoprotein receptor (ASGR)), transferrin, and mannose-terminating glycoproteins. The pleiotropic phenotype of TRF1 also includes an increased sensitivity to Pseudomonas toxin and deficient assembly and function of gap junctions. HuH-7؋TRF1 hybrids exhibited a normal subcellular distribution of ASGR, consistent with the TRF1 mutation being recessive. A cDNA expression library derived from HuH-7 mRNA was transfected into TRF1 cells, which were subsequently selected for resistance to Pseudomonas toxin. Sequence analysis of a recovered cDNA revealed a unique isoform of casein kinase 2 (CK2), CK2␣؆. Western blot analysis of TRF1 proteins revealed a 60% reduction in total CK2␣ expression. Consistent with this finding, the hybrids HuH-7؋HuH-7 and HuH-7؋TRF1 expressed equivalent amounts of total CK2␣. Immunoblots using antibodies against peptides unique to the previously described CK2 isoforms CK2␣ and CK2␣ and the novel CK2␣؆ isoform showed that, although TRF1 and parental HuH-7 cells expressed comparable amounts of CK2␣ and CK2␣, the mutant did not express CK2␣؆. Based on the genomic DNA sequence, RNA transcripts encoding CK2␣؆ apparently originate from alternative splicing of a primary transcript. Protein overexpression following transfection of TRF1 cells with cDNAs encoding either CK2␣ or the newly cloned CK2␣؆ restored the parental HuH-7 phenotype, including Pseudomonas toxin resistance, cell-surface ASGR binding activity, phosphorylation, and the assembly of gap junctions. This study suggests that HuH-7 cells express at least three CK2␣ isoforms and that the pleiotropic TRF1 phenotype is a consequence of a reduction in total CK2 expression.Receptor-mediated endocytosis, a universal mechanism for the uptake of macromolecules by cells, is initiated by the binding of ligand to specific cell-surface receptors, followed by a complex series of intracellular vesicular transfers (1). The asialoglycoprotein receptor (ASGR) 1 is a prototype of the class of receptors that constitutively enter cells via clathrin-coated pits and traffic the endocytic pathway, recycling between early endosomal compartments and the cell surface (2). Two subpopulations of ASGR, States 1 and 2, were originally identified based on the kinetics of ligand transport and the identification of parallel endocytic pathways (3). Changes in both the phosphorylation (4) and acylation (5) status of the receptor have been suggested to affect the transition between these states. The existence of receptor subpopulations has also been suggested for other class II recycling receptors, including the low density lipoprotein, mannose 6-phosphate, and ␣ 2 -macroglobin receptors (6). A mutant affecting endocytic traffic (TRF1) was previously isolated from HuH-7 cells using a dual selection protocol (7). To avoid the selection of receptor-minus mutants, selection pressure was ap...

The Minor Subunit Splice Variants, H2b and H2c, of the Human Asialoglycoprotein Receptor Are Present with the Major Subunit H1 in Different Hetero-oligomeric Receptor Complexes

“…The lack of this latter isoform resulted in a 60% reduction of total CK2 expression and hypophosphorylation of ASGP-Rs in the Trf1 mutant. Transfection of a cDNA encoding the CK2␣Љ isoform into the Trf1 mutant restored these cells to the parental phenotype, in which ASGP-Rs were phosphorylated normally and both the State 1 and State 2 endocytic pathways were active (52). Hence the expression of the CK2␣Љ isoform is required to maintain a functional State 2 endocytic pathway in HuH-7 cells.…”

“…An exciting discovery was recently made by Shi et al (52), who found that both Trf1 and HuH-7 cells expressed similar amounts of two CK2 catalytic subunit isoforms, CK2␣ and CK2␣Ј, but that the Trf1 mutant did not express a third and novel isoform, CK2␣Љ. The lack of this latter isoform resulted in a 60% reduction of total CK2 expression and hypophosphorylation of ASGP-Rs in the Trf1 mutant.…”

Phosphorylation of the HuR ligand APRIL by casein kinase 2 regulates CD83 expression

“…Although more than 300 substrates of CK2 have been identified to date, little is known about the spatiotemporal substrate specificity of the different holoenzyme complexes, which contain various combinations of two individual catalytic subunits derived from the respective a, a 0 and a 00 isoforms, and a dimer of two non-catalytic, presumably regulatory b subunits [42,59,60]. It therefore appears that the myriad of regulatory effects caused by CK2 is reflected in a large and, with respect to its subunit composition, highly diverse population of holoenzymes.…”

Divalent cation hinder the solubilization of a tubulin kinase activity from Trypanosoma cruzi epimastigotes

“…In humans, Shi et al (2001) have reported a third catalytic subunit isoform, α''. Although both the isolated catalytic subunits and the holoenzyme are constitutively active, the ß subunits profoundly affect many properties of CK2, whose precise function is still poorly understood (Pinna, 2002).…”