Casein Kinase II Activity Is Required for Transferrin Receptor Endocytosis
1999; Elsevier BV; Volume: 274; Issue: 43 Linguagem: Inglês
10.1074/jbc.274.43.30550
ISSN1083-351X
AutoresLaura Cotlin, Masood Ahmed Siddiqui, Fiona Simpson, James F. Collawn,
Tópico(s)Retinal Development and Disorders
ResumoThe 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 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. transferrin receptor transferrin chicken embryo fibroblast biotinylated transferrin casein kinase I casein kinase II protein kinase C cyclic AMP-dependent protein kinase A major histocompatibility complex MHC class II invariant chain-transferrin receptor chimera The transferrin receptor (TR)1 binds the serum iron transport protein transferrin (Tf), internalizes through clathrin-coated pits, and facilitates Tf iron release in the sorting endosome. Efficient TR internalization requires a cytoplasmic tail tyrosine-containing motif, Tyr-Thr-Arg-Phe (1Collawn J.F. Stangel M. Kuhn L.A. Esekogwu V. Jing S. Trowbridge I.S. Tainer J.A. Cell. 1990; 63: 1061-1072Abstract Full Text PDF PubMed Scopus (388) Google Scholar, 2McGraw T.E. Maxfield F.R. Cell Regul. 1990; 1: 369-377Crossref PubMed Scopus (97) Google Scholar). Studies by Ohnoet al. (3Ohno H. Fournier M.-C. Poy G. Bonifacino J.S. J. Biol. Chem. 1996; 271: 29009-29015Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar) 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 μ2 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 (4Virshup D.M. Bennett V. J. Cell Biol. 1988; 106: 39-50Crossref PubMed Scopus (52) Google Scholar, 5Lin H.C. Moore M.S. Sanan D.A. Anderson R.G.W. J. Cell Biol. 1991; 114: 881-891Crossref PubMed Scopus (58) Google Scholar, 6Mahaffey D.T. Peeler J.S. Brodsky F.M. Anderson R.G.W. J. Biol. Chem. 1990; 265: 16514-16520Abstract Full Text PDF PubMed Google Scholar) and lattice assembly (7Zaremba S. Keen J.H. J. Cell Biol. 1983; 97: 1339-1347Crossref PubMed Scopus (152) Google Scholar) and, together with its direct interaction with receptor cytoplasmic tails, links the cell surface receptors to the clathrin-based endocytic machinery (reviewed in Refs. 8Keen J.H. Annu. Rev. Biochem. 1990; 59: 415-438Crossref PubMed Scopus (170) Google Scholar and 9Schmid S.L. Annu. Rev. Biochem. 1997; 66: 511-548Crossref PubMed Scopus (669) Google Scholar). 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 (10Fawcett D.W. J. Histochem. Cytochem. 1965; 13: 75-91Crossref PubMed Scopus (194) Google Scholar), starting at prophase and continuing through telophase (11Berlin R.D. Oliver J.M. Walter R.J. Cell. 1978; 15: 327-341Abstract Full Text PDF PubMed Scopus (119) Google Scholar, 12Berlin R.D. Oliver J.M. J. Cell Biol. 1980; 85: 660-671Crossref PubMed Scopus (92) Google Scholar). In A431 cells, the mitotic block appears to arrest clathrin assembly at various stages of invagination (13Pypaert M. Lucocq J.M. Warren G. Eur. J. Cell Biol. 1987; 45: 23-29PubMed Google Scholar), suggesting that the continuous activity of an enzyme (or enzymes) is required for vesicle formation (reviewed in Ref. 14Smythe E. Warren G. Eur. J. Biochem. 1991; 202: 689-699Crossref PubMed Scopus (137) Google Scholar). 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 (15Pypaert M. Mundy D. Souter E. Labbe J. Warren G. J. Cell Biol. 1991; 114: 1159-1166Crossref PubMed Scopus (48) Google Scholar). The role of kinases in receptor trafficking has been demonstrated in studies on the asialoglycoprotein receptor which show that a tyrosine kinase is required for endocytosis (16Fallon R.J. J. Biol. Chem. 1990; 265: 3401-3406Abstract Full Text PDF PubMed Google Scholar) and for phosphorylation of the receptor cytoplasmic tail (17Fallon R.J. Danaher M. Saylors R.L. Saxena A. J. Biol. Chem. 1994; 269: 11011-11017Abstract Full Text PDF PubMed Google Scholar). In vitro studies have also shown that components of the clathrin-coated vesicles can be phosphorylated by vesicle-associated kinases, but the functional significance of these results remains unclear (18Morris S.A. Mann A. Ungewickell E. J. Biol. Chem. 1990; 265: 3354-3357Abstract Full Text PDF PubMed Google Scholar). Furthermore, a number of cell surface proteins including CD4 (19Disanto J.P. Klein J.S. Flomenberg N. Immunogenetics. 1989; 30: 494-501Crossref PubMed Scopus (17) Google Scholar), Fc γ receptor (20Moraru I.I. Laky M. Stanescu T. Buzila L. Popescu L.M. FEBS Lett. 1990; 274: 93-95Crossref PubMed Scopus (19) Google Scholar), CD3 (21Dietrich J. Hou X. Wegener A.-M.K. Geisler C. EMBO J. 1994; 13: 2156-2166Crossref PubMed Scopus (193) Google Scholar), αvβ5 integrin receptor (22Panetti T.S. Wilcox S.A. Horzempa C. McKeown-Longo P.J. J. Biol. Chem. 1995; 270: 18593-18597Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), and TR (23Klausner R.D. Harford J. van Renswoude J. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 3005-3009Crossref PubMed Scopus (121) Google Scholar) are rapidly endocytosed following phorbol ester-mediated stimulation of protein kinase C (PKC). However, the PKC phosphorylation site in TR, Ser24, is not required for this effect (24Zerial M. Suomalainen M. Zanetti-Schneider M. Schneider C. Garoff H. EMBO J. 1987; 6: 2661-2667Crossref PubMed Scopus (29) Google Scholar, 25Davis R.J. Meisner H. J. Biol. Chem. 1987; 262: 16041-16047Abstract Full Text PDF PubMed Google Scholar), suggesting that PKC is acting at the level of the internalization machinery rather than the receptor itself (26Eichholtz T. Vossebeld P. van Overveld M. Ploegh H. J. Biol. Chem. 1992; 267: 22490-22495Abstract Full Text PDF PubMed Google Scholar, 27Schonhorn J.E. Akompong T. Wessling-Resnick M. J. Biol. Chem. 1995; 270: 3698-3705Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Additionally, cyclic AMP-dependent protein kinase A has also been reported to be required for the internalization of the urokinase-type plasminogen activator (28Goretzki L. Mueller B.M. J. Cell Sci. 1997; 110: 1395-1402Crossref PubMed Google Scholar), indicating that a number of kinases are somehow involved in receptor internalization. In yeast, casein kinase I (CKI) has been shown to be required for constitutive endocytosis of the pheromone receptor Ste3p (29Panek H.R. Stepp J.D. Engle H.M. Marks K.M. Tan P.K. Lemmon S.K. Robinson L.C. EMBO J. 1997; 16: 4194-4204Crossref PubMed Scopus (128) Google Scholar). Although a similar function for CKI has not been described in mammalian cells, one intriguing model proposed (29Panek H.R. Stepp J.D. Engle H.M. Marks K.M. Tan P.K. Lemmon S.K. Robinson L.C. EMBO J. 1997; 16: 4194-4204Crossref PubMed Scopus (128) Google Scholar) is that CKI activity regulates endocytosis by phosphorylating the adaptor complex, thereby activating the complex to promote coat assembly and endocytosis. However, since the adaptor complex in yeast is homologous to the recently identified AP-3 complex found at the endosome (30Simpson F. Peden A.A. Christopoulou L. Robinson M.S. J. Cell Biol. 1997; 137: 835-845Crossref PubMed Scopus (304) Google Scholar, 31Le Borgne R. Alconada A. Bauer U. Hoflack B. J. Biol. Chem. 1998; 273: 29451-29461Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar), it remains possible that CKI is required for vacuolar targeting rather than endocytosis. In the present study, we examined the role of protein kinases in TR internalization. Using pharmacological approaches and in vitro assays that reconstitute endocytosis, we found that compounds that inhibit tyrosine kinases and a number of serine/threonine kinases including PKA, PKC, and CKI have no effect on TR endocytosis. In contrast, we provide experimental evidence that suggests casein kinase II, or a closely related kinase, is required. HeLa and 3T3-L1 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (HyClone, Logan, UT), 2 mml-glutamine, penicillin, and streptomycin. A431 cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (Atlanta Biologicals, Norcross, GA) and 2 mml-glutamine. Chicken embryo fibroblasts (CEFs) were grown in Dulbecco's modified Eagle's medium supplemented with 1% chicken serum and 1% fetal bovine serum (Atlanta Biologicals, Norcross, GA), 2% (v/v) tryptose phosphate broth (Difco, Detroit, MI), 2 mml-glutamine, penicillin, and streptomycin. Biotinylated transferrin (B-Tf) was prepared as described previously (32Lamaze C. Baba T. Redelmeier T.E. Schmid S.L. Mol. Biol. Cell. 1993; 4: 715-727Crossref PubMed Scopus (49) Google Scholar) using biotin-XX, SSE (6-((6-biotinoyl)amino)hexanoic acid, sulfosuccinimidyl ester, sodium salt) obtained from Molecular Probes (Eugene, OR). Pseudosubstrate peptides for protein kinase C (RFARKGALRQKNVHEVKN) (33House C. Kemp B.E. Science. 1987; 238: 1726-1728Crossref PubMed Scopus (783) Google Scholar), casein kinase I (DDDEESITRR) (34Agostinis P. Pinna L.A. Meggio F. Marin O. Goris J. Vandenheede J.R. Merlevede W. FEBS Letters. 1989; 259: 75-78Crossref PubMed Scopus (37) Google Scholar), and casein kinase II (RRREEETEEE) (35Kuenzel E.A. Krebs E.G. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 737-741Crossref PubMed Scopus (167) Google Scholar) were synthesized by Quality Controlled Biochemicals (Hopkinton, MA). The protein kinase A pseudosubstrate peptide TYADFIASGRTGRRNAI (36Glass D.B. Cheng H.C. Mende-Mueller L. Reed J. Walsh D.A. J. Biol. Chem. 1989; 264: 8802-8810Abstract Full Text PDF PubMed Google Scholar) was obtained from Calbiochem (La Jolla, CA). Control peptides containing the same amino acid composition but lacking the consensus phosphorylation sites were also prepared for CKI (STIRRDDDEE) and CKII (EEEEEERRRT) by Quality Controlled Biochemicals (Hopkinton, MA). Peptides were prepared as 20 mm stocks in KSHM buffer (100 mm potassium acetate, 85 mm sucrose, 20 mm Hepes, 1 mm magnesium acetate), the pH was adjusted to 7.4, and the peptides were stored at 4 °C. Genistein and H-89 were obtained from Biomol (Plymouth Meeting, PA). Genistein was prepared fresh for each experiment and H-89 was stored as a 48 mm stock solution in dimethyl sulfoxide at −20 °C. Herbimycin A was obtained from Life Technologies, Inc. (Gaithersburg, MD) and stored as a 1.75 mm stock solution in dimethyl sulfoxide at −20 °C. All other inhibitors were purchased from Calbiochem (La Jolla, CA). Calphostin incubation was performed in the presence of fluorescent light as described by the manufacturer (Calbiochem, La Jolla, CA). Wild-type, S24A, Δ3–18, 29–59 TR, Δ3–59 TR, and Ii-TR chimeras were expressed in CEF as described previously (1Collawn J.F. Stangel M. Kuhn L.A. Esekogwu V. Jing S. Trowbridge I.S. Tainer J.A. Cell. 1990; 63: 1061-1072Abstract Full Text PDF PubMed Scopus (388) Google Scholar, 37Kang S. Liang L. Parker C.D. Collawn J.F. J. Biol. Chem. 1998; 273: 20644-20652Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Cells were plated in triplicate at a density of 7.5 × 104cells/cm2 in 24-well tissue culture plates 24 h before the assay (Costar Corp., Cambridge, MA). Cells were then incubated in serum-free media for 1 h at 37 °C. Inhibitors were included during this incubation period for times ranging from 15 min to 1 h. Internalization rates were determined using the IN/SUR method (38Wiley H.S. Cunningham D.D. J. Biol. Chem. 1982; 257: 4222-4229Abstract Full Text PDF PubMed Google Scholar) as described previously (37Kang S. Liang L. Parker C.D. Collawn J.F. J. Biol. Chem. 1998; 273: 20644-20652Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). ATP levels were determined using an ATP detection kit (Calbiochem). Briefly, HeLa cells were incubated in the absence or presence of inhibitors for 60 min. An energy-depleting system consisting of 5 mm 2-deoxyglucose and 5 mm sodium azide was used as a control. Treated cells were placed on ice, scraped at 4 °C in HEPES buffer (25 mmHEPES, 10 mm MgCl2, and 0.02% NaN3), and aliquots were transferred to a cuvette and the volume adjusted to 200 μl with HEPES buffer. 200 μl of releasing agent was added to each sample and the reaction was started by adding 100 μl of the luciferin-luciferase enzyme mixture. Luminescence was measured at 560 nm on a Turner Designs Luminometer (TD-20/20) and the ATP concentrations were determined from a standard calibration curve. The protein concentration of the lysate was determined using the Bradford Assay (Bio-Rad) and total ATP was measured as micromoles of ATP per mg of protein lysate. Rat brains snap-frozen in liquid nitrogen were obtained from Pel-Freeze (Rogers, AR), sliced into small pieces, and homogenized in a Dounce homogenizer containing an equal volume of KSHM buffer and protease inhibitors (Complete mini-inhibitors, Roche Diagnostic Corp. (Indianapolis, IN)). The homogenate was centrifuged for 20 min at 11,200 rpm in a Beckman J20 rotor, and the supernatant was centrifuged for an additional 45 min at 39,000 rpm using a Beckman Ti75 rotor. The resultant supernatant was snap-frozen in liquid nitrogen in 250-μl aliquots and stored at −80 °C. Stock cytosol protein concentrations ranged from 15 to 30 mg/ml. Permeabilized cells were prepared and endocytosis assays were preformed as described (32Lamaze C. Baba T. Redelmeier T.E. Schmid S.L. Mol. Biol. Cell. 1993; 4: 715-727Crossref PubMed Scopus (49) Google Scholar, 39Carter L.L. Redelmeier T.E. Woollenweber L.A. Schmid S.L. J. Cell Biol. 1993; 120: 37-45Crossref PubMed Scopus (139) Google Scholar). Permeabilized 3T3-L1 cells were used in place of A431 cells and were prepared by submerging plates in liquid nitrogen before scraping. After cytosol depletion, 10 μl of cells were incubated with pseudosubstrates or inhibitors for 10 min before addition to the reaction mixtures. Briefly, incubations were performed for 20 min at 37 °C in 40-μl reaction volumes containing an ATP-regenerating system (1 mm ATP, 1 mm GTP, 8 mm creatine phosphate, and 40 units/ml creatine phosphokinase), 6 mg/ml cytosol, and 2 μg/ml B-Tf and 0.2% bovine serum albumin in KSHM assay buffer. Reactions were stopped on ice, cells were pelleted in a microcentrifuge at 4 °C, and the supernatants were carefully removed. Control reactions contained an ATP-depleting system (5 mm glucose and 40 units/ml hexokinase) in place of the ATP-generating system and 6 mg/ml bovine serum albumin in place of the cytosol. The cell pellets were resuspended in avidin (50 μg/ml in 0.2% bovine serum albumin in KSHM buffer) and processed as described (32Lamaze C. Baba T. Redelmeier T.E. Schmid S.L. Mol. Biol. Cell. 1993; 4: 715-727Crossref PubMed Scopus (49) Google Scholar, 39Carter L.L. Redelmeier T.E. Woollenweber L.A. Schmid S.L. J. Cell Biol. 1993; 120: 37-45Crossref PubMed Scopus (139) Google Scholar). Results are presented as the total cell-associated B-Tf (determined from cells not treated with avidin) endocytosed in an ATP- and cytosol-dependent manner. 200 μg of dephosphorylated casein (Sigma) was incubated with either 62.5 ng of CKII (Calbiochem) or 28 ng of CKI (Promega) and 20 μm[γ-32P]ATP (NEN Life Science Products Inc.) in a total reaction volume of 40 μl of KSHM buffer. Inhibitors and pseudosubstrates were included at various concentrations, as specified in the figure legends. The phosphorylation reactions were performed at room temperature for 10 min and terminated with the addition of 10 μl of SDS sample buffer (1 m Tris-HCl, 50% glycerol, 10% SDS, 0.5% β-mercaptoethanol, 1% bromphenol blue, pH 6.8). Samples were analyzed by SDS-polyacrylamide gel electrophoresis, and casein phosphorylation was quantitated on a PhosphorImager (Molecular Dynamics). To determine the role of kinases in TR internalization, endocytosis was monitored in HeLa and A431 cells after pretreatment with tyrosine kinase and PKC inhibitors. Using the IN/SUR method for measuring the internalization rate (38Wiley H.S. Cunningham D.D. J. Biol. Chem. 1982; 257: 4222-4229Abstract Full Text PDF PubMed Google Scholar), TR endocytosis was found to be rapid in both HeLa and A431 cells under control conditions (k e = 0.123 min−1and 0.99 min−1, respectively, TableI) and comparable to TR internalization rates measured in other cells types (37Kang S. Liang L. Parker C.D. Collawn J.F. J. Biol. Chem. 1998; 273: 20644-20652Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). After pretreatment of the HeLa and A431 cells at 37 °C for 1 h or more with a number of tyrosine kinase and PKC inhibitors, the results indicate that TR endocytosis was unaffected (Table I). Interestingly, however, when the genistein concentration was increased to 500 μm (the concentration required to block asialoglycoprotein receptor endocytosis (16Fallon R.J. J. Biol. Chem. 1990; 265: 3401-3406Abstract Full Text PDF PubMed Google Scholar)), 5-fold higher than is normally used to inhibit tyrosine kinases (40Wu-Wong J.R. Berg C.E. Wang J. Chiou W.J. Fissel B. J. Biol. Chem. 1999; 274: 8103-8110Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar), TR endocytosis was inhibited by more than 50% (data not shown). This suggests that a kinase, not necessarily a tyrosine kinase, was required for TR endocytosis.Table IEffect of various tyrosine and serine/threonine kinase inhibitors on transferrin receptor internalizationDrug pretreatmentHeLa cellsA431 cellsInternalization rateInternalization efficiencyInternalization rateInternalization efficiencymin−1%min−1%Control (none)0.123 ± 0.017 (7)aMean ± S.E., number in parentheses is the number of independent experiments.100bPercentage of internalization efficiency of untreated controls was arbitrarily set to 100%.0.099 ± 0.028 (6)100Tyrosine kinase inhibitors Genistein (100 μm)0.120 ± 0.041 (4)980.079 ± 0.004 (4)80 Herbimycin A (8 μm)0.124 ± 0.006 (3)1010.116 ± 0.024 (3)117 Tyrphostin A25 (50 μm)0.113 ± 0.020 (3)920.086 ± 0.030 (3)87 Tyrphostin B46 (50 μm)0.118 ± 0.007 (3)960.081 ± 0.019 (3)82PKC inhibitors Calphostin C (100 nm)0.128 ± 0.032 (6)1040.118 ± 0.035 (4)119 Iso-H-7 (50 μm)0.109 ± 0.005 (5)89not tested— Bisindomaleimide (200 nm)0.112 ± 0.006 (3)910.105 ± 0.010 (3)106a Mean ± S.E., number in parentheses is the number of independent experiments.b Percentage of internalization efficiency of untreated controls was arbitrarily set to 100%. Open table in a new tab When a serine/threonine kinase inhibitor, H-89, was tested, TR endocytosis was dramatically inhibited in both HeLa and A431 cells (Fig. 1). After as little as a 15-min pretreatment with 150 μm H-89, TR internalization decreased by ∼50% (average of 47 and 55% in HeLa and A431, respectively). Inhibition of endocytosis was confirmed in steady-state distribution assays at 37 °C in HeLa cells as well, which demonstrated that the TR surface expression increased from 20.4 ± 0.2 to 38.8 ± 6.1% (mean ± S.D. from three experiments). Calculation of the estimated internalization rate from the steady-state distribution (1Collawn J.F. Stangel M. Kuhn L.A. Esekogwu V. Jing S. Trowbridge I.S. Tainer J.A. Cell. 1990; 63: 1061-1072Abstract Full Text PDF PubMed Scopus (388) Google Scholar) suggests that after H-89 treatment, the TR internalization rate was 44% of the untreated control, indicating that recycling was not affected. To confirm that H-89 was not indirectly inhibiting TR internalization by altering cell viability, the capacity of the cells to recover after H-89 treatment was tested. In this experiment, HeLa cells were treated for 1 h in 150 μm H-89, after which the media was exchanged with fresh inhibitor-free media every hour. After a 4-h recovery period, TR internalization was then compared with untreated cells. As shown in Fig. 2 A, TR internalization returned to 116% of control values, demonstrating that the H-89 inhibition was reversible. To assess the possible cytotoxic effects of H-89 using another approach, we measured cellular ATP concentrations immediately after a 1-h pretreatment in 150 μm H-89. As shown in Fig. 2 B, the treated cells had 95% of the control levels of ATP. Taken together, the results suggested that H-89 treatment was not causing a general cytotoxic effect that indirectly inhibited TR endocytosis. As a further control, we treated cells with 5 mm 2-deoxyglucose and 5 mm sodium azide and found that intracellular ATP levels dropped 55%, confirming that changes in ATP levels were easily monitored (Fig. 2 B). To determine if H-89 treatment also affected TR internalization in nontransformed cells, endocytosis was examined in primary CEFs. This experimental system also allowed us to test human TR mutants and chimeras (Fig. 3 A) in order to determine the structural features of the TR cytoplasmic tail required for H-89 inhibition. Pretreatment of cells expressing the wild-type TR with 150 μm H-89 inhibited TR endocytosis by more than 80% (Fig. 3 B). The only phosphorylation site in the TR cytoplasmic tail is serine 24, a PKC phosphorylation site (41Davis R.J. Johnson G.L. Kelleher D.J. Anderson J.K. Mole J.E. Czech M.P. J. Biol. Chem. 1986; 261: 9034-9041Abstract Full Text PDF PubMed Google Scholar). To confirm that this site was not important for the H-89 inhibition, internalization assays were performed on CEF expressing a mutant TR lacking this site (Fig. 3 A). As shown in Fig. 3 C, S24A was inhibited to the same extent as wild-type TR, suggesting that the presence of the phosphorylation site on the receptor was not required for the inhibitory effect. To determine if the H-89 inhibition was specific for the TR, we tested two additional mutants, a TR cytoplasmic tail deletion mutant, Δ3–18, 29–59 TR, that lacks most of the cytoplasmic tail but contains the internalization signal (1Collawn J.F. Stangel M. Kuhn L.A. Esekogwu V. Jing S. Trowbridge I.S. Tainer J.A. Cell. 1990; 63: 1061-1072Abstract Full Text PDF PubMed Scopus (388) Google Scholar), and a TR chimera that contains the MHC class II invariant chain cytoplasmic tail in place of the TR cytoplasmic tail (Invariant chain-TR, Fig. 3 A) (37Kang S. Liang L. Parker C.D. Collawn J.F. J. Biol. Chem. 1998; 273: 20644-20652Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). As shown in Fig. 3 C, endocytosis of both the deletion mutant and the MHC class II invariant chain chimera were dramatically inhibited by H-89 treatment, suggesting that the wild-type TR is not the only cell surface molecule affected. Furthermore, since the MHC class II invariant chain contains a di-leucine internalization signal rather than a tyrosine-based signal (37Kang S. Liang L. Parker C.D. Collawn J.F. J. Biol. Chem. 1998; 273: 20644-20652Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 42Odorizzi C.G. Trowbridge I.S. Xue L. Hopkins C.R. Davis C.D. Collawn J.F. J. Cell Biol. 1994; 126: 317-330Crossref PubMed Scopus (160) Google Scholar), this indicates that receptors containing either class of internalization signal are equally inhibited by H-89 treatment. One possible concern with the H-89 inhibition was that the H-89 was having an indirect effect on membrane dynamics, in which case pinocytosis would be affected as well. To test for this, we monitored the internalization of a TR mutant that lacked the endocytosis signal, Δ3–59 TR (Fig. 3 A), after H-89 treatment. The results in Fig. 3 C show that internalization of the Δ3–59 TR mutant was not affected by this treatment, indicating that inhibition was not due to nonspecific membrane effects. H-89 inhibits a number of kinases including PKA, PKC, CKI, and CKII by competing for the ATP-binding site (43Chijiwa T. Mishima A. Hagiwara M. Sano M. Hayashi K. Inoue T. Naito K. Toshioka T. Hidaka H. J. Biol. Chem. 1990; 265: 5267-5272Abstract Full Text PDF PubMed Google Scholar). To determine which kinase was being inhibited, we tested pseudosubstrate peptide inhibitors of several serine/threonine kinases in an in vitro assay that reconstitutes endocytosis (32Lamaze C. Baba T. Redelmeier T.E. Schmid S.L. Mol. Biol. Cell. 1993; 4: 715-727Crossref PubMed Scopus (49) Google Scholar, 39Carter L.L. Redelmeier T.E. Woollenweber L.A. Schmid S.L. J. Cell Biol. 1993; 120: 37-45Crossref PubMed Scopus (139) Google Scholar, 44Jost M. Simpson F. Kavran J.M. Lemmon M.A. Schmid S.L. Curr. Biol. 1998; 8: 1399-1402Abstract Full Text Full Text PDF PubMed Google Scholar). Upon addition of exogenous cytosol and an ATP-regenerating system, B-Tf becomes sequestered in deeply invaginated coated pits and vesicles. Under these conditions, the B-Tf is inaccessible to avidin and is scored as internalized Tf (32Lamaze C. Baba T. Redelmeier T.E. Schmid S.L. Mol. Biol. Cell. 1993; 4: 715-727Crossref PubMed Scopus (49) Google Scholar). For the initial characterization, we confirmed that H-89 inhibited B-Tf internalization in permeabilized 3T3-L1 cells. At 150 μmH-89, B-Tf uptake was inhibited by 38.1 ± 4.9% (mean ± S.D). At 300 and 500 μm, B-Tf internalization was inhibited by 66.2 ± 5.8 and 95.7 ± 3.5%, respectively (Fig. 4 A). Next, we tested TR endocytosis after H-89 treatment in intact 3T3-L1 cells using the standard IN/SUR assay and 125I-labeled Tf. As shown in Fig.4 B, TR endocytosis was blocked by ∼54% after a 15-min pretreatment with 150 μm H-89, demonstrating that H-89 inhibited TR endocytosis in 3T3-L1 cells in both assays. Since the IC50 of H-89 for CKII is 137 μm (43Chijiwa T. Mishima A. Hagiwara M. Sano M. Hayashi K. Inoue T. Naito K. Toshioka T. Hidaka H. J. Biol. Chem. 1990; 265: 5267-5272Abstract Full Text PDF PubMed Google Scholar) (compared with 0.048 μm for PKA, 32 μm for PKC, and 38 μm for CKI (43Chijiwa T. Mishima A. Hagiwara M. Sano M. Hayashi K. Inoue T. Naito K. Toshioka T. Hidaka H. J. Biol. Chem. 1990; 265: 5267-5272Abstract Full Text PDF PubMed Google Scholar)), similar to the concentration that inhibited TR internalization in HeLa and A431 cells,in vitro phosphorylation reactions were performed in the presence and absence of H-89 using purified CKII and casein as the substrate (Fig. 4 C). The results show that H-89 at concentrations of 150, 300, and 500 μm inhibited CKII activity 43, 56, and 62%, respectively. This indicated that the loss of CKII activity in the in vitro phosphorylation assay paralleled the loss of B-Tf internalization after H-89 treatment, suggesting a possible link. Next, we tested the ability of pseudosubstrate peptide inhibitors of PKA, PKC, CKI, and CKII to block B-Tf internalization in permeabilized 3T3-L1 cells. During a 10-min preincubation period when cytosol, ATP, GTP, and an ATP-regenerating system are added, we introduced pseudosubstrate peptide inhibitors of PKA, PKC, CKI, or CKII at 2-fold higher concentrations than the IC50 of the peptides for the kinases. As shown in Fig.5 A, the PKA, PKC, and CKI pseudosubstrate peptides had little effect on B-Tf endocytosis, whereas the CKII peptide inhibited endocytosis by more than 70%. Comparison of the pseudosubstrate peptide to a control peptide containing the same amino acid composition but lacking the consensus site for CKII, indicated that the pseudosubstrate peptide effect was specific an
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