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

Xiaoying Shi, Barry Potvin, Tianmin Huang, Philip Hilgard, David C. Spray, Sylvia O. Suadicani, Allan W. Wolkoff, Pamela Stanley, Richard J. Stockert

Research output: Contribution to journalArticle

48 Citations (Scopus)

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), CK2α″. Western blot analysis of TRF1 proteins revealed a 60% reduction in total CK2α expression. Consistent with this finding, the hybrids HuH-7×HuH-7 and HuH-7×TRF1 expressed equivalent amounts of total CK2α. Immunoblots using antibodies against peptides unique to the previously described CK2 isoforms CK2α and CK2α′ and the novel CK2α″ isoform showed that, although TRF1 and parental HuH-7 cells expressed comparable amounts of CK2α and CK2α′, the mutant did not express CK2α″. Based on the genomic DNA sequence, RNA transcripts encoding CK2α″ apparently originate from alternative splicing of a primary transcript. Protein overexpression following transfection of TRF1 cells with cDNAs encoding either CK2α or the newly cloned CK2α″ 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 CK2α isoforms and that the pleiotropic TRF1 phenotype is a consequence of a reduction in total CK2 expression.

Original languageEnglish (US)
Pages (from-to)2075-2082
Number of pages8
JournalJournal of Biological Chemistry
Volume276
Issue number3
DOIs
StatePublished - Jan 19 2001

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Casein Kinase II
Hepatocellular Carcinoma
Membrane Proteins
Cells
Cell Line
Asialoglycoprotein Receptor
Protein Isoforms
Pseudomonas
Complementary DNA
Gap Junctions
Cell Surface Receptors
Phenotype
Telomeric Repeat Binding Protein 1
Phosphorylation
DNA sequences
Normal Distribution
Alternative Splicing
Normal distribution
Transferrin
Mannose

ASJC Scopus subject areas

  • Biochemistry

Cite this

A novel casein kinase 2 α-subunit regulates membrane protein traffic in the human hepatoma cell line HuH-7. / Shi, Xiaoying; Potvin, Barry; Huang, Tianmin; Hilgard, Philip; Spray, David C.; Suadicani, Sylvia O.; Wolkoff, Allan W.; Stanley, Pamela; Stockert, Richard J.

In: Journal of Biological Chemistry, Vol. 276, No. 3, 19.01.2001, p. 2075-2082.

Research output: Contribution to journalArticle

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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), CK2α″. Western blot analysis of TRF1 proteins revealed a 60{\%} reduction in total CK2α expression. Consistent with this finding, the hybrids HuH-7×HuH-7 and HuH-7×TRF1 expressed equivalent amounts of total CK2α. Immunoblots using antibodies against peptides unique to the previously described CK2 isoforms CK2α and CK2α′ and the novel CK2α″ isoform showed that, although TRF1 and parental HuH-7 cells expressed comparable amounts of CK2α and CK2α′, the mutant did not express CK2α″. Based on the genomic DNA sequence, RNA transcripts encoding CK2α″ apparently originate from alternative splicing of a primary transcript. Protein overexpression following transfection of TRF1 cells with cDNAs encoding either CK2α or the newly cloned CK2α″ 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 CK2α isoforms and that the pleiotropic TRF1 phenotype is a consequence of a reduction in total CK2 expression.",
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