Casein Kinase II Activity Is Required for Transferrin Receptor Endocytosis

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. The transferrin receptor (TR)1 binds the serum iron transport protein transferrin (Tf), internalizes through clathrincoated pits, and facilitates Tf iron release in the sorting endosome. Efficient TR internalization requires a cytoplasmic tail tyrosine-containing motif, Tyr-Thr-Arg-Phe (1, 2). Studies by Ohno et al. (3) using yeast two-hybrid analysis demonstrate that the TR cytoplasmic tail signal interacts with one of the four subunits of the AP-2 adaptor complex, the 2 chain. This interaction provides a mechanism for promoting TR clustering in clathrin-coated pits and subsequent internalization. The AP-2 adaptor complex is also required for clathrin recruitment (4 -6) and lattice assembly (7) and, together with its direct interaction with receptor cytoplasmic tails, links the cell surface receptors to the clathrin-based endocytic machinery (reviewed in Refs. 8 and 9).Despite the extensive characterization of many of the proteins involved in endocytosis, little is known about how the clathrin-based endocytic machinery is regulated. What is evident, however, is endocytosis via clathrin-coated pits is blocked during mitosis (10), starting at prophase and continuing through telophase (11,12). In A431 cells, the mitotic block appears to arrest clathrin assembly at various stages of invagination (13), suggesting that the continuous activity of an enzyme (or enzymes) is required for vesicle formation (reviewed in Ref. 14). Using in vitro reconstitution assays, mitotic cytosol has been shown to inhibit invagination of clathrin-coated pits and one of the factors responsible is cdc2 kinase (15).The role of kinases in receptor trafficking has been demonstrated in studies on the asia...

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“…In spite of these changes, the internalization rate of Tf was not affected by 20 M H89, neither in N2a (control, k e ϭ 0.013 min Ϫ1 ; H89, k e ϭ 0.017 min Ϫ1 , n ϭ 6) nor in HeLa cells (control, k e ϭ 0.107 min Ϫ1 ; H89, k e ϭ 0.109 min Ϫ1 , n ϭ 6) ( Figure 11B). This is consistent with previous reports in HeLa cells and A431 cells in which the internalization rate of Tf was not affected by low concentrations of H89, being only decreased at higher concentrations (300 M) due to casein kinase II inhibition (Cotlin et al, 1999). Treatment with either H89 (20 M) or Myr-PKI peptide (100 M) for 60 min did not modify the cell surface 125 I-diTf binding activity (our unpublished data), indicating that recycling of continuously internalized TfR was also not altered.…”

“…This correlated with the IC 50 of 13 M estimated for the effect of H89 on 125 I-EGF binding ( Figure 8C). Ligand-binding activity decreased within the range of 5-20 M H89, whereas at higher H89 concentrations this effect was gradually lost until it became completely abolished at 300 M. Thus, other serine-threonine kinases inhibited at these high H89 concentrations (Chijiwa et al, 1990;Cotlin et al, 1999) are probably required for the endocytosis of EGFR acting downstream of PKA. The time course of PKA inhibition was also congruent with an involvement of PKA in the endocytic effect of H89.…”

Genetic Ablation of Phosphatidylinositol Transfer Protein Function in Murine Embryonic Stem Cells

“…In all cases, 60% of the TfR is present in intracellular compartments, whereas 40% of the TfR resides at the cell surface ( Figure 7A). These steady-state distributions of TfR are in close agreement with those recorded for other cell types (Jing et al, 1990;Cotlin et al, 1999).…”

AP2 Adaptor Complex-Dependent Internalization of CD5: Differential Regulation in T and B Cells

“…In addition to SHIP described above, another key regulator of AP2-mediated endocytosis may be CK2, an important regulator of receptor endocytosis (59). We and others have shown that CK2 constitutively associates with and is activated by CD5 (32,60).…”