Casein kinase II activity is required for transferrin receptor endocytosis.
The effect of protein kinase inhibitors on transferrin receptor (TR) internalization was examined in HeLa, A431, 3T3-L1 cells, and primary chicken embryo fibroblasts. We show that TR endocytosis is not affected by tyrosine kinase or protein kinase C inhibitors, but is inhibited by one serine/threonine kinase inhibitor, H-89. Inhibition occurred within 15 min, was completely reversible after H-89 withdrawal, and was specific for endocytosis rather than pinocytosis since a TR mutant lacking an internalization signal was not affected. Interestingly, H-89 also inhibited the internalization of a TR chimera containing the major histocompatibility complex class II invariant chain cytoplasmic tail, indicating that the effect was not specific for the TR. Since H-89 inhibits a number of kinases, we employed a permeabilized cell endocytosis assay to further characterize the kinase. In permeabilized 3T3-L1 cells, addition of pseudosubstrate inhibitor peptides of casein kinase II (CKII) blocked TR internalization by more than 50%, whereas pseudosubstrates of cyclic AMP-dependent kinase A, protein kinase C, and casein kinase I had no effect. Furthermore, addition of purified CKII to the cell-free reactions containing CKII pseudosubstrates reversed the endocytosis block, suggesting that CKII or a CKII-like activity is required for constitutive endocytosis.
Cotlin
L F
LF
Department of Cell Biology, University of Alabama at Birmingham, MCLM 392, UAB Station, Birmingham, Alabama 35294-0005, USA.
Siddiqui
M A
MA
Simpson
F
F
Collawn
J F
JF
D016475:3T3 Cells; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D047390:Casein Kinase II; D002642:Chick Embryo; D016384:Consensus Sequence; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005347:Fibroblasts / Q000378:metabolism; D006367:HeLa Cells; D000949:Histocompatibility Antigens Class II / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011990:Receptors, Transferrin / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D000949:Histocompatibility Antigens Class II; D010455:Peptides; D011990:Receptors, Transferrin; D011994:Recombinant Proteins; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C