A novel casein kinase 2 alpha-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), CK2alpha". Western blot analysis of TRF1 proteins revealed a 60% reduction in total CK2alpha expression. Consistent with this finding, the hybrids HuH-7xHuH-7 and HuH-7xTRF1 expressed equivalent amounts of total CK2alpha. Immunoblots using antibodies against peptides unique to the previously described CK2 isoforms CK2alpha and CK2alpha' and the novel CK2alpha" isoform showed that, although TRF1 and parental HuH-7 cells expressed comparable amounts of CK2alpha and CK2alpha', the mutant did not express CK2alpha". Based on the genomic DNA sequence, RNA transcripts encoding CK2alpha" apparently originate from alternative splicing of a primary transcript. Protein overexpression following transfection of TRF1 cells with cDNAs encoding either CK2alpha or the newly cloned CK2alpha" 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 CK2alpha isoforms and that the pleiotropic TRF1 phenotype is a consequence of a reduction in total CK2 expression.
Shi
X
X
Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Potvin
B
B
Huang
T
T
Hilgard
P
P
Spray
D C
DC
Suadicani
S O
SO
Wolkoff
A W
AW
Stanley
P
P
Stockert
R J
RJ
D000595:Amino Acid Sequence; D001427:Bacterial Toxins / Q000494:pharmacology; D006528:Carcinoma, Hepatocellular / Q000378:metabolism* / Q000473:pathology; D047390:Casein Kinase II; D003001:Cloning, Molecular; D018076:DNA, Complementary; D017629:Gap Junctions / Q000378:metabolism; D005816:Genetic Complementation Test; D006801:Humans; D006822:Hybrid Cells; D008113:Liver Neoplasms / Q000378:metabolism* / Q000473:pathology; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000378:metabolism*; D021381:Protein Transport; D011549:Pseudomonas / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014407:Tumor Cells, Cultured
D001427:Bacterial Toxins; D018076:DNA, Complementary; D008565:Membrane Proteins; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases