(Received for publication, August 24, 1994; and in revised form, December 9, 1994)
From the
Treatment with phorbol esters increases endocytosis of the
transferrin receptor in K562 cells (Klausner, R. D., Harford, J., and
van Renswoude, J.(1984) Proc. Natl. Acad. Sci. U. S. A. 81,
3005-3009). In this report, we demonstrate that this effect is
reversible within early times of protein kinase C activation (<2 h)
but that prolonged exposure to phorbol esters results in a net loss of
receptors. These effects are not due to the differentiation response of
K562 cells to phorbol esters since bryostatin-1 also down-regulates the
endocytosis of the transferrin receptor and shut downs receptor
synthesis, but does not induce differentiation (Hocevar, B. A., Morrow,
D. M., Tykocinski, M. L., and Fields, A. P.(1992) J. Cell Sci. 101, 671-679). We have characterized the early stages of
receptor down-regulation which occur due to stimulation of receptor
internalization from the cell surface. The fact that fluid-phase
pinocytosis is also enhanced upon protein kinase C activation indicates
that this effect is not specific for the transferrin receptor itself,
but is a rather general cellular response to tumor-promoting phorbol
esters. The fate of down-regulated transferrin receptors was followed
in morphological and subcellular fractionation studies that demonstrate
localization of this pool of receptors in early endocytic and recycling
compartments. Our results exclude the possibility that transferrin
receptor down-regulation results in trafficking of the receptor to
lysosomal compartments for degradation. This idea is consistent with
the observations that the time course of transferrin receptor
degradation is not enhanced in stimulated K562 cells, while transferrin
receptor synthesis is shut down. Our results rigorously demonstrate
that activation of protein kinase C down-regulates the K562 cell
transferrin receptor in two stages: acute regulation of early steps in
endocytosis that results in an immediate reduction of 40% in cell
surface number of receptors and a more chronic reduction in transferrin
receptor synthesis upon prolonged exposure to phorbol esters (>15
h).
It has been well-established that treatment of K562 cells with
the phorbol ester 4-phorbol 12-myristate 13-acetate (PMA) (
)perturbs endocytosis of the transferrin receptor, causing
an apparent loss of cell surface receptors(1, 2) .
This redistribution could be promoted by enhanced internalization,
reduced externalization, and/or targeting of transferrin receptors to a
compartment which does not participate in normal routes of receptor
traffic. Early studies characterizing the parameters of transferrin
receptor endocytosis in K562 cells suggested the existence of two
receptor pools, one surface-bound and the other intracellular. Klausner et al. (2) proposed that transferrin binding initiates
transferrin receptor internalization to intracellular compartments,
which in turn stimulates the release of an intracellular transferrin
receptor pool to the plasma membrane. PMA was thought to activate or
enhance an endogenous endocytic signal of this pathway. In this model,
contributions of two pools of receptors to recycling accounted for the
down-regulation of the transferrin receptor in the absence of available
ligand; PMA was considered to reproduce a ligand-induced endocytic
initiation signal, thus altering the release of intracellular
receptors, and presumably exerting this effect by the activation of
protein kinase C.
In contrast to the ligand-induced endocytic
transport mechanism, a mechanism of continuous transferrin receptor
cycling in K562 cells was later identified. These conflicting results
are attributed to the different methods utilized to determine cell
surface receptors. Klausner et al.(2) assessed
surface receptors with anti-transferrin receptor antibodies, a method
subject to variabilities such as ligand-induced perturbations of
antigenic determinants. In contrast, Watts (3) evaluated
surface-bound receptors by following internalization of I-labeled transferrin receptors as measured by resistance
to proteolytic digestion. Using this approach, it was observed that the
transferrin receptor was internalized with identical kinetics in the
presence or absence of available transferrin. These observations are
consistent with the principle that it is the receptors, independent of
available ligand, that are the true substrates for
endocytosis(4) . Finally, although PMA treatment caused
extensive phosphorylation of the receptor's cytoplasmic
tail(5, 6) , no conclusive evidence for this
modification as an endocytic signal has been
obtained(7, 8, 9, 10) .
Thus,
although the effects of PMA on transferrin receptor endocytosis are
well documented, the precise mechanism of receptor down-regulation has
yet to be elucidated. Recently, our laboratory re-examined the
mechanism of PMA-induced transferrin receptor down-regulation in K562
cells using the In/Sur method of Wiley and Cunningham(4) . This
approach was undertaken in order to exclude the possibility of
ligand-induced endocytosis of the receptor or the internalization of a
separate pool of unoccupied receptors that could be modulated in
response to phorbol esters. These studies rigorously demonstrated that
the rate of transferrin receptor endocytosis is significantly and
specifically up-regulated in K562 cells exposed to phorbol esters: the
endocytic rate constant for transferrin internalization is nearly
doubled, 0.43 min compared to 0.28 min
for control cells(11) . In contrast, there is little
change in exocytic recycling of the receptor, arguing that the sole
basis for the redistribution of receptors is due the kinetic
alterations in endocytosis. Although this is consistent with the early
observations of Klausner et al.(2) , additional
changes in receptor traffic induced by phorbol esters cannot be
excluded. For example, down-regulated receptors might be diverted to
subcellular compartments that do not participate in the typical route
of receptor trafficking, thus creating two populations of intracellular
transferrin receptors in PMA-treated cells as initially
proposed(2) . Consideration of this idea is prompted by the
fact that treatment of K562 cells with phorbol esters also blocks
growth and induces differentiation. Several investigations have
demonstrated the multipotential nature of this cell
line(12, 13) , and it has been established that PMA
promotes stable megakaryocytic differentiation of the proerythroblastic
K562 cells (14) . It is possible that during this response,
transferrin receptors are simply cleared from the cell surface due to
morphological changes effected by the cellular differentiation program.
These changes might represent transport of the receptor to otherwise
inaccessible compartments along a new membrane traffic pathway, for
example, accelerated receptor degradation due to transport along the
lysosomal pathway. In light of these arguments, this investigation was
undertaken to specifically address the following questions: first, into
what compartments are transferrin receptors redistributed upon exposure
to PMA? Second, is down-regulation of the transferrin receptor a direct
action of phorbol esters on regulation of the endocytic pathway or is
this phenomenon simply a consequence of the differentiation program?
Finally, are degradative processes accelerated under these conditions
to account for the loss of receptors?
To assess the extent of receptor down-regulation,
binding of I-labeled transferrin to surface receptors was
measured (11) . K562 cells were incubated with appropriate
concentrations of
I-transferrin (0.5-500
nM) in the presence or absence of unlabeled ligand (25
µM) for 1 h at 4 °C. Cells were collected by
centrifugation, the cell pellet was rinsed with ice-cold PBS, and
cell-associated radioactivity was measured. The amount of nonspecific
I-transferrin associated with the cell pellet was
determined in the presence of unlabeled ligand and substracted from
each point. Scatchard analysis was employed to determine the number of
transferrin-binding sites.
Soluble receptor binding studies were
accomplished using the methods described by Klausner et
al.(15) . Briefly, K562 cells were solubilized in TBS (10
mM Tris, pH 7.4, 150 mM NaCl) containing 0.1% Triton
X-100. To 150-µl aliquots of the detergent-solublized cell extract,
50 µl of I-transferrin (0.5-500 nM)
and 50 µl of buffer with or without 50 µg/ml unlabeled
transferrin were added. After a 10-min incubation at room temperature,
250 µl of ice-cold 60% ammonium sulfate was added to the binding
reactions to precipitate the ligand-receptor complexes. After 5 min on
ice, the precipitate was collected by filtration through Whatman GF/C
filters and washed three times with 30% ammonium sulfate containing
0.8% BSA. Filters were dried, counted, and specific
I-transferrin binding was taken as the difference between
radioactivity associated in the absence and presence of unlabeled
transferrin.
Figure 1:
Down-regulation of K562 cell surface
transferrin receptors by PMA. K562 human erythroleukemia cells were
incubated in the absence () or presence (
) of 32.4 nM PMA for 2 h at 37 °C. The cells were then washed three times
in PBS at 4 °C, and cell surface
I-transferrin
binding was measured as described under ``Materials and
Methods.'' Briefly, K562 cells (0.5-1
10
cells) were resuspended in 25 mM HEPES, pH 7.4, 150
mM NaCl, 1 mg/ml BSA with indicated concentrations of
I-transferrin (0.5-50 nM), with or without
25 µM unlabeled transferrin (final volume 250 µl). The
binding reactions were incubated at 4 °C with constant mixing for
60 min and terminated upon centrifugation. The cell pellets were washed
briefly one time in ice-cold PBS and associated counts/minute was
measured by gamma counting. Shown is the specific amount of bound
I-transferrin determined from the difference in
cell-associated counts/minute in the absence and presence of unlabeled
transferrin. Data are from a single experiment representative of
similar experiments repeated on numerous
occasions.
All of the data described above are consistent with previous work characterizing the down-regulation of transferrin receptors in K562 cells treated with phorbol esters(2, 11, 20) . The action of PMA on receptor down-regulation has been shown to enhance endocytosis of transferrin(2) , and work from our laboratory recently confirmed that the endocytic rate constant for receptor internalization is increased about 2-fold in stimulated cells(11) . The results presented here extend these previous findings by further documenting the reversible nature of this phenomenon and demonstrating that it is the biological action of phorbol esters to activate protein kinase C directly that alters endocytosis of the K562 cell transferrin receptors.
Does the activation of protein kinase C lead to other changes in K562 cell endocytosis or is this effect specific for transferrin receptors? The fact that the transferrin receptor is a substrate for phosphorylation by protein kinase C led to the original suggestion that perhaps this event triggered endocytosis of the receptor(2, 15) . However, there is little data to substantiate a role for transferrin receptor phosphorylation in internalization, particularly since mutagenesis studies have demonstrated that key serine residues phosphorylated by protein kinase C are not essential for receptor internalization(7, 8, 9, 10) . To specifically address this issue, the fluid-phase uptake of horseradish peroxidase by K562 cells was also examined under these conditions. Fig. 2compares endocytic uptake of horseradish peroxidase by control and PMA-treated cells. Exposure of K562 cells to the phorbol ester stimulates fluid-phase internalization of the probe about 2.5-fold, consistent with the idea that activation of protein kinase C results in a general cellular response to increase endocytosis. The finding that fluid-phase endocytosis is also enhanced by PMA treatment strongly argues that receptor down-regulation is not due to phosphorylation of transferrin receptor itself nor that this effect is specific for the transferrin receptor alone. Thus, the effects of phorbol esters are most likely exerted through protein kinase C regulation of elements involved in the endocytic pathway of K562 cells.
Figure 2:
PMA treatment stimulates fluid-phase
pinocytosis. The effects of phorbol esters on the fluid-phase
internalization of horseradish peroxidase by K562 cells were measured
as described under ``Materials and Methods.'' K562 cells were
first incubated in the presence or absence of 32.4 nM PMA for
2 h, then washed in PBS, and incubated for 30 min in 25 mM HEPES, pH 7.4, 150 mM NaCl, 1 mg/ml glucose, 1 mg/ml BSA
containing 1 mg/ml horseradish peroxidase. After fluid-phase uptake was
complete, cells were extensively washed in PBS, then lysed with 0.1%
Triton X-100, and cell-associated horseradish peroxidase activity was
assayed using o-dianisidine as substrate. Fluid-phase uptake
measurements (A
/min/mg cell protein) is
given in the mean ± S.D. from duplicate measurements from a
single experiment. Similar results were obtained on two other
occasions.
Figure 3:
FITC-transferrin is internalized to
endosomal domains in PMA-treated K562 cells. Confocal fluorescence
microscopy was performed to identify the cytolocalization of
endocytosed FITC-transferrin. K562 cells were treated with or without
32.4 nM PMA for 2 h at 37 °C, during which time 100 nM FITC-labeled transferrin was included in the incubation medium to
be internalized. The cells were then washed extensively with PBS, fixed
with 3.7% gluteraldehyde, and prepared for microscopy. Fluorescence
confocal microscopy was carried out as described under ``Materials
and Methods'' with optical sections (0.5 µm thick) taken at
the mid-plane of the cells at magnification 40. Images obtained
from two different experiments are shown. A 5-µm scale bar is
shown.
To verify the results of these morphological studies, we also
examined the profile of I-transferrin bearing
compartments in control and PMA-treated cells by subcellular
fractionation on self-forming Percoll density gradients(16) ;
this method is routinely employed to separate intracellular
compartments and in fact, the presence of radiolabeled transferrin is
commonly used as a marker to denote endocytic vesicles(23) .
The results presented in Fig. 4demonstrate that the
fractionation profiles of control and PMA-treated cells are identical:
I-transferrin appears in fractions containing membrane
vesicles of light buoyant density as described previously(16) .
Most importantly, the radiolabeled ligand does not appear in dense
compartments separated on this gradient, in particular, those fractions
containing lysosomes as marked by
-hexosaminidase activity (panel B). The observations made in these subcellular
fractionation experiments support the conclusion that the endocytic
pathway of the transferrin receptor is not altered by activation of
protein kinase C and that down-regulated receptors reside in endosomal
compartments of the K562 cells.
Figure 4:
Sub-cellular fractionation profiles of
PMA-treated and control K562 cells. PMA-treated and control K562 cells
were incubated at 37 °C for 1 h in the presence of 5 nMI-transferrin. The cells were then collected, washed
with PBS, and disrupted in TEA-sucrose using a ball-bearing homogenizer
in order to obtain a post-nuclear supernatant. PNS fractions (400
µl) were mixed with 7.6 ml of 22% Percoll in TEA-sucrose and the
entire mixture was then underlayed with a 0.5-ml 60% sucrose cushion.
The samples were centrifuged in a Ti50 rotor for 90 min at 17,000
revolutions/min at 4 °C and fractionated from the interface of the
sucrose cushion (fractions from bottom of the tube are on the left). 100 µl of each fraction was taken to measure
I-transferrin content (panel A). Aliquots (10
µl) were also assayed for
-hexosaminidase activity with
methylumbelliferyl-
-N-acetylglucosaminide (1 mM)
in MacIlvaine buffer (pH 4.5) containing 0.1% Nonidet P-40 and 0.1%
gelatin. After 30 min at 37 °C, trichloroacetic acid was added to
the reaction mixture (5% final concentration), and clarified
supernatants were assayed for released methylumbelliferone (excitation
365 nm, emission 450 nm). Panel B shows activity profiles of
lysosomal
-hexosaminidase (in fluorescence units). There was no
significant difference between the subcellular fractionation profiles
for control (
) and PMA-treated K562 cells
(
).
Recently,
Hocevar et al.(24) demonstrated that exposure of K562
cells to the macrocyclic lactone, bryostatin-1, activates protein
kinase C but does not induce differentiation. Although structurally
unrelated to phorbol esters(25) , bryostatin-1 appears to
activate protein kinase C in an isotype-specific manner and, in K562
cells, bryostatin-1 promotes differential translocation of the
isotype of protein kinase C to the
nucleus(24) . We therefore took advantage of the specificity of
bryostatin-1's action to investigate whether transferrin receptor
down-regulation would occur in the absence of differentiation.
Fig. 5demonstrates that similar to the effects of phorbol
esters, bryostatin-1 induces 50% loss of cell surface receptors in
K562 cells. Panel A presents the Scatchard plots of
I-transferrin binding to cells exposed to 50 nM bryostatin-1 compared to control (untreated cells). Although in
this particular experiment K562 cells were treated with bryostatin-1
overnight, preliminary experiments determined that down-regulation of
transferrin receptors was maximal within 4 h of exposure to the protein
kinase C activator (data not shown). For comparison, panel B shows Scatchard analysis of transferrin binding to K562 cells
after overnight exposure to PDBu. For both treatments, activation of
protein kinase C provoked greater than 50% loss of binding sites.
Within this time period of exposure to phorbol esters, other studies
have established that K562 cells initiate expression of the
megakaryocytic phenotype(14) , consistent with the cessation of
cell growth. In contrast, K562 cells treated with bryostatin-1 continue
to grow and fail to differentiate as noted by the lack of gpIIIa
expression or induction of granulocyte/macrophage-colony-stimulating
factor and interleukin-6 secretion(24) . In these experiments,
the growth of the K562 cells was monitored to confirm that PDBu-treated
cells ceased to divide and that bryostatin-1-treated cells were similar
to control (untreated) K562 cells in their rate of growth (data not
shown). Therefore, the observation that both bryostatin-1 and phorbol
esters down-regulate the transferrin receptor indicates that activation
of protein kinase C regulates endocytosis of K562 cells through
pathways independent of those involved in cellular differentiation.
Figure 5:
Bryostatin-1 down-regulates K562 cell
surface transferrin receptors. K562 cells were incubated with 50 nM bryostatin-1 overnight, then prepared for I-transferrin binding studies as described for Fig. 1and Table 1. Panel A presents the results
of Scatchard analysis for treated (
) and control (
) cells.
I-Transferrin binding measurements were also performed
with K562 cells treated with bryostatin-1 overnight then incubated for
an additional 2 h with 100 nM staurosporine (
)
comparison, Panel B shows Scatchard analysis of
I-transferrin binding to K562 cells treated overnight
with 50 nM PDBu (
) and for an additional 2 h with 100
nM staurosporine (
). Shown are data obtained from an
individual experiment with both PDBu and bryostatin-1 treatments
performed in parallel. The experiment was repeated three times, with
data summarized in Table 2.
To
investigate this phenomenon, the total number of transferrin receptors
in K562 cells treated overnight with phorbol esters was measured in a
soluble receptor binding assay. As shown by the results of Fig. 6, K562 cells exposed to PDBu overnight display about a 50%
loss in detectable I-transferrin binding activity
relative to control (panel A). In contrast, no decrease in
binding activity is observed after 2 h of treatment. In a similar
manner, overnight treatment with bryostatin-1 was also observed to
induce a loss in
I-transferrin-binding sites (panel
B). Thus, while short times of protein kinase C alters only the
number of cell surface transferrin receptors, prolonged activation
leads to a reduction in both cell surface and intracellular receptors.
Figure 6:
Transferrin binding to total solubilized
receptors. -Transferrin binding to detergent-solublized
receptors was assayed using ammonium sulfate to precipitate
receptor-ligand complexes(3) . K562 cells were treated for 16 h
in the absence (
) or presence of either 50 nM PDBu
(
) or 50 nM bryostatin-1 (
). As a control, K562
cells were incubated for only 2 h with 100 nM PDBu (
).
Briefly, the cells were then washed in PBS, and detergent extracts were
prepared in TBS containing 0.1% Triton X-100. Binding of
I-transferrin to detergent-solublized receptors (cell
surface and intracellular) was performed as described under
``Materials and Methods'' with nonspecific binding measured
in the presence of excess unlabeled transferrin. Shown is measured
amount of transferrin bound (pmol/10
cells) to total
soluble receptors as a function of
[
I-transferrin]. Results from one of three
separate experiments is shown, with
50% loss of total transferrin
receptors after overnight treatment with either PDBu or bryostatin-1
demonstrated on each occasion.
One explanation for the apparent loss of receptors is that it may
reflect an increase in transferrin receptor degradation. However, as
demonstrated by the results of Fig. 3and Fig. 4, PMA
does not appear to provoke altered routes of endocytic traffic such
that the transferrin receptor becomes retargeted to lysosomal
compartments for degradation. To directly ascertain the fate of
down-regulated receptors, we performed cell surface iodination to label
K562 cell surface transferrin receptors, then induced down-regulation
of these receptors upon exposure to PMA. After the extended times of
incubation indicated in Fig. 7, K562 cells were solubilized, and
the I-labeled receptors were immunoprecipitated to be
analyzed by SDS-PAGE and subsequent autoradiography. As shown by the
results of Fig. 7, the time course of cell surface transferrin
receptor degradation is unaffected in the PMA-treated K562 cells. For
both control and PMA-treated cells, the receptor half-life appears to
be on the order of about 8 h. The fact that the time course of receptor
degradation is unaffected in PMA-treated cells rigorously excludes the
possibility that in addition to its immediate effects on endocytosis,
activation of protein kinase C also redirects membrane traffic to
degradative lysosomal compartments, consistent with the observations
discussed above ( Fig. 3and Fig. 4).
Figure 7:
Time course of cell surface transferrin
receptor degradation. K562 cells were surface iodinated by the
lactoperoxidase-glucose oxidase coupled method described under
``Materials and Methods.'' After surface labeling, the cells
were resuspended in -minimal essential medium and incubated in the
presence or absence of 32.4 nM PMA for 0-16 h, at which
time the cells were collected to be processed for immunoprecipitation
of
I-labeled transferrin receptors. Immunoprecipitates
were analyzed by SDS-PAGE and the results of autoradiography of the gel
(4 day exposure) are presented. The experiment was repeated four times
with similar results.
Since the half-life of cell surface transferrin receptors is unchanged in K562 cells exposed to PMA, the simplest explanation for the results of Fig. 6is that in response to protein kinase C, transferrin receptor synthesis is completely shut down resulting in a net loss of receptors. This interpretation is consistent with the results of Ho et al.(28) which demonstrate that activation of protein kinase C in HL60 cells dramatically eliminates transferrin receptor mRNA after 12 h of exposure to PMA. To examine whether phorbol esters promote a similar effect in K562 cells, RNA was prepared from cells treated with 50 nM PMA for various times of incubation up to 16 h. The results of the Northern blot analysis shown in Fig. 8indicate that activation of protein kinase C leads to a rapid loss of transferrin receptor message, with a significant decrease in mRNA levels observed within 4 h of exposure to the phorbol ester. We therefore propose that for K562 cells, phorbol esters induce down-regulation of transferrin receptors in two stages: an acute phase during which the rate of endocytosis is stimulated, although the pattern of transferrin receptor traffic is unchanged, followed by a more chronic down-regulation of transferrin receptor synthesis resulting in a net loss of receptors.
Figure 8: Northern blot analysis of K562 cell transferrin receptor mRNA after treatment with PMA. Total RNA was isolated from K562 cells incubated for the indicated times with 50 nM PMA, and 20-µg samples were electrophoresed on 1% formaldehyde agarose gels and blotted to nylon membranes to be UV-cross-linked. The Northern blot was hybridized with radiolabeled probes for transferrin receptor mRNA and 36B4 mRNA (control) as indicated. Identical results were obtained in a similar experiment performed on a separate occasion.
The effects of phorbol esters on the receptor-mediated
endocytosis of transferrin have been studied in many different cell
lines, including A431 cells(6) , mouse 3T3 and Ltk cells(7, 8) , and Chinese hamster ovary
cells(9, 10) . In each of these studies, activation of
protein kinase C was documented to increase transferrin receptor
phosphorylation, but either little change in surface number of
receptors was observed(7) , or increased numbers of transferrin
receptors appearing on the cell surface were
reported(8, 9, 10) . It was determined that
the latter effect is promoted by increased rates of exocytosis such
that receptor recycling is stimulated and transferrin receptors become
redistributed to the cell surface(9) . Despite the fact that
receptor phosphorylation was initially implicated to play a role in the
membrane trafficking of the transferrin
receptor(1, 5, 15) , site-directed
mutagenesis studies that altered or deleted the key serine residue
phosphorylated by protein kinase C (serine 24) revealed little change
in the uptake of ligand or kinetics of transferrin receptor
internalization-recycling(7, 8, 9) . These
observations have led to the current view that receptor phosphorylation
does not play a role in the receptor-mediated endocytosis and recycling
of transferrin.
In contrast to observations made for these other cell lines, endocytosis of the transferrin receptor by erythroid or leukemic cell lines is stimulated by PMA(2, 5, 20, 29) . As shown by the work presented here, as well as in many past investigations(2, 11, 29) , PMA-stimulated K562 cells display a pattern of increased internalization resulting in a decreased number of cell surface transferrin receptors. Since none of these cell lines have been utilized in site-directed mutagenesis studies examining receptor phosphorylation, the role for serine 24 in PMA-stimulated internalization has not been directly assessed. The fact that phorbol esters will provoke differentiation of these cell lines further complicates the situation since differentiation-dependent changes in transferrin receptor endocytosis and the state of receptor phosphorylation have also been reported(30) . Finally, although down-regulation of the transferrin receptor was determined to result from enhanced rates of endocytosis(2, 11) , the fate of these redistributed receptors remained poorly defined. All of these uncertainties prompted this investigation of the pattern of transferrin receptor traffic in K562 cells in order to gain a better understanding of the factors involved in PMA-stimulated internalization.
The results presented here define that activation of protein kinase C down-regulates the K562 cell transferrin receptor in two distinct phases. The first acute phase of phorbol ester treatment results in enhanced rates of endocytosis(2, 11) ; this effect is reversible within the first few hours of exposure and is the direct result of protein kinase C activation since the kinase-specific inhibitor staurosporine restores cell surface transferrin receptor numbers to basal levels. The fact that fluid-phase uptake of horseradish peroxidase is also stimulated under these conditions indicates that the effect of protein kinase C activation is not specific for the transferrin receptor itself. Although this does not exclude a role for serine 24 phosphorylation in the process of down-regulation, this observation does suggest that the target of protein kinase C action most likely resides in elements of the endocytic apparatus. This idea is consistent with proposals first made by Backer and King (31) concerning the PMA-triggered internalization of receptors independent of their phosphorylation states.
A second point to be made concerning this initial stage of receptor down-regulation is that it does not occur as a consequence of the induction of cellular differentiation. Our results demonstrate that bryostatin-1, a protein kinase C activator that does not provoke differentiation of K562 cells(24) , displays all of the properties of phorbol esters; that is, exposure to bryostatin-1 accelerates endocytosis and down-regulates transferrin receptors from the surface of K562 cells. This response clearly distinguishes receptor down-regulation from pathways of cellular differentiation and further substantiates a role for protein kinase C in the regulation of endocytosis. Finally, the pattern of transferrin receptor membrane traffic is unaltered in PMA-stimulated K562 cells, excluding the possibilities that down-regulated receptors become rerouted to new intracellular compartments or internalized into lysosomal, degradative pathways. Our combined results support the hypothesis that phosphorylation of factor(s) involved in early stages of receptor-mediated endocytosis is responsible for the PMA-stimulated down-regulation of transferrin receptors in K562 cells.
In addition to these early and immediate effects of protein kinase C activation, prolonged treatment of K562 cells with phorbol esters or bryostatin-1 results in decreased synthesis of transferrin receptors. The second chronic phase of receptor down-regulation promotes not only a decrease in the number of transferrin receptors appearing on the cell surface, but a general loss of both cell surface and intracellular receptors as well. While one explanation for this overall loss of receptors during this chronic phase of down-regulation is that PMA-stimulated internalization results in transferrin receptor traffic to the lysosome, the results of our morphological and subcellular fractionation experiments do not support this idea. Instead, based on the results of our Northern blot analysis we conclude that prolonged activation of protein kinase C leads to a marked reduction in the level of transferrin receptor mRNA. This effect is identical to the regulation of transferrin receptor synthesis observed in HL-60 leukemia cells, which also display transferrin receptor down-regulation in response to PMA, exhibiting decreased receptor mRNA, and a pattern of monocytic differentiation(28) . Since the level of receptor message observed in their studies temporally correlated with the cessation of HL-60 cell growth, Ho et al.(28) concluded that the reduction in transferrin receptor levels was not a consequence of cellular differentiation. Rather, these investigators proposed that transferrin receptor down-regulation may signal programmed inhibition of cell growth, which is necessary for terminal differentiation. Our results do not support this idea since bryostatin-1 also reduces transferrin receptor levels; thus, receptor synthesis may be regulated by protein kinase C independent of the acquisition of the differentiated phenotype. This suggests that the phenomenon of receptor down-regulation is not responsible for the inhibition of cell growth associated with terminal differentiation, and in fact the bryostatin-1-treated K562 cells continue to divide.
Our results clearly support the model that activation of protein kinase C down-regulates the K562 cell receptor in the two stages outlined above: 1) immediate stimulation of early steps in endocytosis, and 2) a secondary decline of transferrin receptor synthesis. Of particular interest is the immediate and reversible action of protein kinase C to clear transferrin receptors from the cell surface. Limited evidence for a role of phosphorylation in the endocytic pathway exists(31) ; however, in vitro studies have documented that inhibitors of protein phosphatases, okadaic acid and microcystin-LR, block endocytic vesicle fusion(32) . Although the latter studies by Woodman et al.(32) determined that this effect was not exerted via cdc2 kinase, the actual kinase involved in in vitro endosome fusion remains unknown. It should be noted that in vivo studies have also indicated that okadaic acid inhibits endocytosis in HeLa cells(33) , consistent with the notion that elements of the endocytic pathway can be modulated by phosphorylation. Our study demonstrates that the down-regulation of K562 cell transferrin receptors provides an excellent paradigm to study the role of phosphorylation and protein kinase C in endocytosis, and future work in this area encompassing in vitro studies may provide further insights into the regulation of vesicle traffic.