A novel casein kinase 2 α-subunit regulates membrane protein traffic in the human hepatoma cell line HuH-7

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-7xTRF1 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 alpha ", Western blot analysis of TRF1 proteins revealed a 60% reduction in total CK2 alpha expression. Consistent with this finding, the hybrids HuH-7xHuH-7 and HuH-7xTRF1 expressed equivalent amounts of total CK2 alpha. Immunoblots using antibodies against peptides unique to the previously described CK2 isoforms CK2 alpha and CK2 alpha' and the novel CK2 alpha " isoform showed that, although TRF1 and parental HuH-7 cells expressed comparable amounts of CK2 alpha and CK2 alpha', the mutant did not express CK2 alpha " Eased on the genomic DNA sequence, RNA transcripts encoding CK2 alpha " apparently originate from alternative splicing of a primary transcript. Protein overexpression following transfection of TRF1 cells with cDNAs encoding either CK2 alpha or the newly cloned CK2 alpha " 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 alpha isoforms and that the pleiotropic TRF1 phenotype is a consequence of a reduction in total CK2 expression.