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Casein kinase II activity is required for transferrin receptor endocytosis
L F Cotlin1, M A Siddiqui, F Simpson
1Department of Cell Biology, University of Alabama at Birmingham, MCLM 392, UAB Station, Birmingham, Alabama 35294-0005, USA.
Abstract:
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.
Insights
Protein kinase inhibitors affect transferrin receptor (TR) internalization. Casein kinase II (CKII) activity is required for TR endocytosis, as shown by experiments with H-89 and CKII inhibitors.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Transferrin receptor (TR) internalization is a crucial cellular process for iron uptake.
- The specific kinases regulating TR endocytosis remain incompletely understood.
Purpose of the Study:
- To investigate the role of protein kinases in regulating transferrin receptor (TR) internalization.
- To identify the specific kinase involved in constitutive TR endocytosis.
Main Methods:
- Treatment of various cell lines (HeLa, A431, 3T3-L1, chicken embryo fibroblasts) with protein kinase inhibitors.
- Utilized a permeabilized cell endocytosis assay with specific kinase inhibitors and purified kinase.
- Examined the internalization of wild-type TR and a TR mutant lacking an internalization signal, as well as a TR chimera.
Main Results:
- Serine/threonine kinase inhibitor H-89, but not tyrosine kinase or protein kinase C inhibitors, blocked TR endocytosis.
- H-89 inhibition was rapid, reversible, and specific for endocytosis, affecting both TR and a TR chimera.
- Casein kinase II (CKII) pseudosubstrate inhibitors significantly blocked TR internalization in a cell-free assay, while other kinase inhibitors had no effect.
- Addition of purified CKII reversed the inhibition, confirming its role in TR endocytosis.
Conclusions:
- Casein kinase II (CKII) activity is essential for the constitutive endocytosis of the transferrin receptor.
- These findings identify CKII as a key regulator of TR internalization, providing insights into cellular iron homeostasis.