(Received for publication, May 9, 1995; and in revised form, October 12, 1995)
From the
Peptidylglycine -amidating monooxygenase (PAM) is a
bifunctional enzyme that catalyzes the COOH-terminal
-amidation of
neural and endocrine peptides through a two-step reaction carried out
sequentially by its monooxygenase and lyase domains. PAM occurs in
soluble and integral membrane forms. Metabolic labeling of stably
transfected hEK-293 and AtT-20 cells showed that
[
P]PO
was
efficiently incorporated into Ser and Thr residues of membrane PAM but
not into soluble PAM. Truncation of integral membrane PAM proteins
(which terminate with Ser
) at Tyr
eliminated their phosphorylation, suggesting that the
COOH-terminal region of the protein was the site of phosphorylation.
Recombinant PAM COOH-terminal domain was phosphorylated on Ser
and Ser
by protein kinase C (PKC). PAM-1 protein
recovered from different subcellular fractions of stably transfected
AtT-20 cells was differentially susceptible to calcium-dependent,
staurosporine-inhibitable phosphorylation catalyzed by endogenous
cytosolic protein kinase(s). Although phorbol ester treatment of
hEK-293 cells expressing PAM-1 stimulated the cleavage/release of a
bifunctional 105-kDa PAM protein, the effect was an indirect one since
it was also observed in hEK-293 cells expressing a truncated PAM-1
protein that was not phosphorylated. AtT-20 cells expressing PAM-1
lacking one of the PKC sites (PAM-1/Ser
Ala)
exhibited an altered pattern of PAM
PAM antibody internalization,
with the mutant protein targeted to lysosomes upon internalization.
Thus, phosphorylation of Ser
in the COOH-terminal
cytosolic domain of membrane PAM plays a role in a specific step in the
targeting of this protein.
Maturation of bioactive peptides in the nervous and endocrine
systems involves a series of post-translational processing steps that
occur as the precursor proteins and their products pass through the
secretory pathway. Peptide -amidation, which occurs late in the
pathway and is often essential for biological activity, is a two-step
reaction catalyzed in sequence by two enzymes contained within the
bifunctional enzyme, peptidylglycine
-amidating monooxygenase
(PAM; (
)EC 1.14.17.3)(1, 2) . The first
reaction is catalyzed by peptidylglycine
-hydroxylating
monooxygenase (PHM) and requires copper, ascorbate, and molecular
oxygen, and the second reaction is catalyzed by
peptidyl-
-hydroxyglycine
-amidating lyase (PAL) and generates
-amidated peptide and
glyoxylate(3, 4, 5, 6, 7) .
Alternative splicing of the single-copy PAM gene generates mRNAs encoding integral membrane and soluble PAM proteins in a tissue-specific and developmentally regulated manner(8, 9, 10) . PAM-1 includes Exon 16, a noncatalytic region separating PHM and PAL, a transmembrane domain and a COOH-terminal domain (Fig. 1); Exon 16 is absent from PAM-2. Exon 25 encodes a transmembrane domain and is absent from PAM-3; the splicing of Exon 25 determines the intracellular fate of the COOH-terminal domain. The COOH-terminal domain of integral membrane PAM is exposed to the cytoplasm, whereas the same domain in soluble bifunctional PAM resides in the lumen of the secretory compartment(12) . The COOH-terminal domain of PAM is essential for retrieval of integral membrane PAM from the cell surface in neuroendocrine and non-neuroendocrine cells (13, 14) and plays a modulatory role in the in vitro PHM activity of soluble bifunctional PAM-3(15) .
Figure 1:
PAM proteins studied. A, the
three alternatively spliced forms of PAM protein (PAM-1, -2, and -3)
examined, a mutant form of PAM-2 truncated at Gly (PAM-2/899) and a mutant form of PAM-1 truncated at Tyr
(PAM-1/936) are drawn to scale with their membrane topology in
the secretory compartment and apparent molecular mass indicated. N-CHO, N-linked oligosaccharide at Asn
; O-CHO, O-linked oligosaccharide; transmembrane
domain, filled box; CD, COOH-terminal domain; Exon 16, old Exon A; Exon 25/26, old Exons
B
/B
. The region of PAM-1 in which proteolytic
cleavage occurs in hEK-293 cells to release a 105-kDa soluble
bifunctional PAM protein is indicated by the large arrow. B,
the amino acid sequence of the COOH-terminal domain of PAM is
shown(8) ; likely sites for phoshorylation by PKC (11) , bold.
PAM proteins undergo various post-translational processing events
while moving through the secretory pathway. The NH-terminal
signal peptide is cleaved and Asn
is glycosylated in the
endoplasmic reticulum(12) . In cultured cardiomyocytes and
transfected hEK-293 cells, PAM-1, but not PAM-2, is
sialylated(13, 16) . Multiple sialylated and/or
sulfated O-linked oligosaccharides are present when Exon 16 is
included(17) . Endoproteolytic processing gives rise to smaller
soluble and membrane-associated PAM proteins in a tissue- and cell
type-specific manner(18, 19, 20) .
Tyr
in the COOH-terminal domain is sulfated only in
PAM-3(17) .
The COOH-terminal domain of PAM is highly conserved (63% amino acid identity for man and Xenopus laevis) and includes several conserved potential phosphorylation sites(8) . We set out to determine whether PAM was phosphorylated, and, if so, whether phosphorylation might play a role in regulating its routing. Metabolic labeling and immunoprecipitation of stably transfected hEK-293 and AtT-20 cells demonstrated that integral membrane PAM proteins were phosphorylated on Ser and Thr residues in the COOH-terminal domain. PAM proteins in different subcellular compartments were differentially susceptible to phosphorylation by endogenous protein kinases. A membrane PAM protein lacking one of the two PKC sites in recombinant PAM COOH-terminal domain was misrouted after internalization from the plasma membrane.
Pulse-chase experiments were carried out as described(20) . Phorbol 12-myristate 13-acetate (PMA, 1 µM final concentration) (Calbiochem) was diluted into CSFM from a 1 mg/ml (1.62 mM) stock in dimethyl sulfoxide, and staurosporine (1 µM final concentration) (Calbiochem) was diluted into CSFM from a 1 mM stock in dimethyl sulfoxide. Aliquots of cell extracts or media (50-100 µl) were diluted 5-fold in 50 mM sodium phosphate, pH 7.4, 1% Triton X-100 (Super E) containing protease inhibitors and protein phosphatase inhibitors and incubated with 10-20 µl of rabbit polyclonal antisera against PHM, PAL, or CD overnight at 4 °C(12, 14) . For immunoprecipitation with PAL antibody, extracts or media were boiled in 1% SDS for 5 min after addition of protease inhibitors and protein phosphatase inhibitors and then diluted 4-fold with Super E containing 0.5% Nonidet P-40 before addition of antisera. Immune complexes were isolated using protein A-Sepharose (Sigma), and immunoprecipitates were fractionated by SDS-PAGE and analyzed as described(14) . Films were densitized using an Abaton Scan 300/GS and NIH Image 1.35 software(14) .
In vitro phosphorylation of the resuspended
pellets was carried out at 30 °C for 1 h in a reaction volume of 50
µl containing 20 µl of resuspended differential centrifugation
pellet and 6 µCi of [-
P]ATP in 20
mM HEPES (pH 7.0), 10 mM MgCl
, and 0.25 M sucrose in the absence or presence of 0.5 mM
Ca
or cytosol (10 µl). Reactions were terminated
by adding 20 µl of 0.5 M EDTA. Samples were then
immunoprecipitated using rabbit polyclonal Ab571 (12, 15) and analyzed by SDS-PAGE and fluorography.
Antibody
internalization experiments were carried out using a rabbit polyclonal
antibody to PHM (Ab475) as described
previously(13, 14) ; primary antibody was visualized
using Cy3-AffiniPure F(ab`) donkey anti-rabbit
IgG(H+L) (Jackson Immunoresearch Laboratories, Inc., West Grove,
PA). Lysosomes were visualized simultaneously using rat monoclonal
antibody 1D4B (1:50) to lysosome-associated membrane protein 1 (LAMP-1) (29) (Developmental Studies Hybridoma Bank, Dept. of
Pharmacology and Molecular Sciences, Johns Hopkins University School of
Medicine) and FITC-goat F(ab`)
anti-rat IgG(H+L)
(CalTag Laboratories, Inc., San Francisco, CA). Confocal microscopy was
carried out using a Bio-Rad MRC 600 microscope. Cells were visualized
by the simultaneous collection of the two fluorescent signals; optical
sections were 1 µm thick, and each image was a composite of eight
frames.
Figure 2:
Phosphorylation of PAM proteins in hEK-293
cells. A, duplicate wells of hEK-293 cells expressing the PAM
proteins indicated were incubated with
[S]methionine/cysteine or
[
P]PO
for 6 h;
cell extracts were immunoprecipitated with PHM antibody and
fractionated by SDS-PAGE. The lower molecular weight band observed in
the immunoprecipitate of
[
P]PO
-labeled
PAM-2 cells was not observed consistently and is thought to be
unrelated to PAM-2. Exposure times for fluorography (
S)
and autoradiography (
P) were 16-20 h for both
isotopes. B, 120-kDa PAM-1 protein was subjected to
phosphoamino acid analysis as described under ``Materials and
Methods.''
AtT-20
cells stably expressing PAM-1, PAM-2, or PAM-3 were metabolically
labeled with [P]PO
or [
S]methionine/cysteine for 6 h (Fig. 3A). As in hEK-293 cells, PAM-1 and PAM-2 were
phosphorylated while PAM-3 was not. AtT-20 cells, unlike hEK-293 cells,
cleave PAM-1 into monofunctional PHM (45-kDa) and membrane-bound PAL
(70-kDa) proteins; it is apparent from immunoprecipitates prepared
using antisera to the COOH-terminal cytosolic domain that only the
70-kDa PAL fragment was phosphorylated. AtT-20 cells expressing a PAM-1
protein truncated at Tyr
(PAM-1/936) did not yield a
phosphorylated PAM protein (Fig. 3A). Phosphoamino acid
analysis of acid-hydrolyzed AtT-20 PAM-1 labeled with
[
P]PO
revealed
phosphoserine with lesser amounts of phosphothreonine and no
phosphotyrosine (Fig. 3B).
Figure 3: Phosphorylation of PAM proteins in AtT-20 cells. A, duplicate wells of AtT-20 cells expressing the PAM proteins indicated were incubated as described in Fig. 2. Cell extracts were immunoprecipitated with PHM antibody and/or COOH-terminal domain antibody as indicated. Samples were fractionated by SDS-PAGE. B, phosphoamino acid analysis of 120-kDa PAM-1 was carried out as described in Fig. 2.
Figure 4:
In vitro phosphorylation of
recombinant PAM COOH-terminal domain by PKC. A, recombinant
COOH-terminal domain peptide (1 µg) (see Fig. 5A for sequence) was incubated with purified PKC and
[-
P]ATP (see ``Materials and
Methods'') for 1 h at 30 °C. The desalted reaction mixture was
analyzed by SDS-PAGE and autoradiography. B, recombinant
COOH-terminal domain (20 µg; 2 nmol) was phosphorylated by PKC in vitro with 1 µCi of [
-
P]ATP
(0.33 pmol) and digested with trypsin (1 µg) for 16 h. Peptides
were fractionated by reverse phase HPLC using a trifluoroacetic
acid/acetonitrile system, and aliquots were counted using a liquid
scintillation counter; the early and late eluting tryptic
phosphopeptides are referred to as Peaks I and II,
respectively.
Figure 5:
Amino acid sequence analysis of PAM
COOH-terminal domain phosphorylated by PKC. A, the sequence of
recombinant PAM COOH-terminal domain peptide is shown. B and C, This peptide (40 µg) was phosphorylated by PKC in
vitro with 1 µCi of [-
P]ATP and 1
mM ATP and digested with trypsin (1 µg) for 16 h. Peaks I and II (as in Fig. 4B) were
separated using the pH 7 phosphate perchlorate buffer system described
under ``Materials and Methods'' and subjected to Edman
degradation. The yield of product at each cycle is plotted; peak I yielded two overlapping sequences.
Recombinant PAM
COOH-terminal domain protein phosphorylated in vitro by PKC
with [-
P]ATP was subjected to tryptic
digestion and fractionation by reverse phase HPLC (Fig. 4B). Major and minor
P-labeled
tryptic peptides (Peaks I and II) were detected. In order to identify
these tryptic phosphopeptides, phosphorylation of recombinant PAM
COOH-terminal domain by PKC was driven to completion by using a higher
concentration of ATP. Tryptic peptides were fractionated by reverse
phase HPLC using a phosphate-perchlorate system (pH 7.0) to resolve
phosphorylated and nonphosphorylated peptides(26) . Under these
conditions, approximately equal amounts of phosphopeptides I and II
were produced (data not shown); both phosphopeptides were subjected to
Edman degradation.
Peak I yielded two overlapping sequences
Lys-Gly-Tyr-Ser-Arg and Gly
-Tyr-Ser-Arg (Fig. 5, A and B); Ser
was
identified as the phosphorylation site since it was the only Ser or Thr
residue in the peptide, and recovery of Ser at cycle 4 of the Edman
degradation was low, consistent with the low recovery of
phenylthiohydantoin-phosphoserine(30) . Edman degradation of
peak II (Fig. 5C) provided the sequence
Gly
-Ser-Gly-Gly-Leu-Asn-Leu-Gly-Asn-Phe-Phe-Ala-Ser-Arg;
Ser was recovered in good yield at the 2nd cycle, but little Ser was
recovered at the 13th cycle (Ser
; underlined) (Fig. 5C). Thus, PKC first catalyzed the
phosphorylation of Ser
; more extensive phosphorylation
included Ser
.
Figure 6:
Phosphorylation of PAM-1 localized in
different subcellular compartments. Differential centrifugation
fractions were prepared from AtT-20 cells as described under
``Materials and Methods.'' A, an equal percentage of
each fraction was subjected to Western blot analysis using an antibody
to Exon 16 (Ab629) that recognizes PHM and PAL. B,
differential centrifugation fractions resuspended in isotonic buffer
were incubated with [-
P]ATP in the absence
or presence of 0.5 mM Ca
or cytosol (10
µl), and phosphorylated PAM proteins were analyzed after
immunoprecipitation with an antibody to PAL and SDS-PAGE. C,
staurosporine (1 µM; Stau) was added to the
phosphorylation reaction with aliquots of the 30,000 and 40,000 rpm
pellets, and phosphorylated PAM proteins were analyzed as in B.
Each pellet was resuspended
and incubated with [-
P]ATP under 4
conditions: no additions, Ca
only, AtT-20 cytosol
without or with added Ca
(Fig. 6B).
PAM-1 and the 70-kDa PAL protein (PALm) generated when 45-kDa PHM is
produced were phosphorylated in all subcellular fractions under all
conditions examined. In the absence of added Ca
or
cytosol, the most extensive phosphorylation of PAM-1 and PALm was
observed in the fraction containing the highest concentration of PAM,
the 20,000 rpm pellet (Fig. 6B). Addition of
Ca
in the absence of cytosol had little effect on the
phosphorylation of PAM in any of the fractions. Addition of cytosol to
the 20,000 rpm fraction resulted in a substantial inhibition of the
phosphorylation of PAM-1 and PALm; no effect of cytosol alone was
observed with the other fractions. When cytosol was present along with
Ca
, phosphorylation of PAM-1 and PALm in the 30,000
rpm and 40,000 rpm pellets was stimulated substantially. In the
presence of cytosol and Ca
, the relatively small
amount of PAM protein in the 40,000 rpm pellet was phosphorylated much
more efficiently than PAM proteins in other subcellular fractions.
The Ca requirement for cytosol-dependent
phosphorylation of PAM proteins in the 30,000 rpm and 40,000 rpm
pellets suggested a role for Ca
-stimulated protein
kinase(s). Since PKC phosphorylated purified PAM COOH-terminal domain in vitro (Fig. 4A), a role for endogenous PKC
was examined using staurosporine, a PKC inhibitor (Fig. 6C). Inclusion of 1 µM staurosporine
during the incubation with [
-
P]ATP
completely blocked the effect of Ca
and cytosol on
PAM phosphorylation in both the 30,000 and 40,000 rpm pellets and even
reduced phosphorylation below the levels observed in the absence of
cytosol. Thus, a staurosporine-inhibitable PKC-like enzyme could be the
Ca
-dependent cytosolic factor that phosphorylates PAM
in the 30,000 and 40,000 rpm pellets.
Figure 7:
Effect of phorbol ester treatment on
phosphorylation of PAM-1. A, hEK-293 cells expressing PAM-1
were metabolically labeled with
[P]PO
for 6 h
under the following conditions: PMA (1 µM final) was added
during the final hour of incubation with
[
P]PO
;
staurosporine (1 µM final) was added during the entire
period of incubation with
[
P]PO
; for
desensitization of PKC, cells were incubated with CSFM containing 1
µM PMA for 18 h prior to incubation with
[
P]PO
and 1
µM PMA was included in the medium during incubation with
[
P]PO
. Duplicate
wells were incubated with [
S]methionine/cysteine
for 6 h. Cell extracts were immunoprecipitated with CD antibody (12) and analyzed by SDS-PAGE and autoradiography (
P) or fluorography (
S). B,
autoradiograms and fluorograms from two independent experiments were
densitized, and average values of
P-labeled 120-kDa or
22-kDa PAM proteins normalized to
S-labeled PAM-1 (120
kDa) were plotted.
Under basal conditions, a small fraction of the PAM-1 produced in hEK-293 cells is cleaved at or near the cell surface to release a soluble 105-kDa bifunctional PAM protein and generate an integral membrane COOH-terminal fragment (Fig. 1)(13) . The 105-kDa PAM protein is not detected in the cells. Based on its apparent molecular mass and recognition by antisera to the COOH-terminal domain of PAM, the 22-kDa phosphoprotein observed here consists of a small portion of the luminal domain, the transmembrane domain, and the COOH-terminal domain of PAM. The increased level of phosphorylated 22-kDa PAM COOH-terminal protein upon PMA treatment may reflect increased cleavage producing the 22-kDa PAM COOH-terminal protein and/or preferential phosphorylation of the 22-kDa PAM COOH-terminal protein by activated PKC.
Figure 8:
Effect of phorbol ester treatment on
cleavage/release of PAM proteins from hEK-293 cells. Duplicate wells of
hEK-293 cells expressing PAM-1, PAM-3, or PAM-2/899 were labeled with
[S]methionine/cysteine for 15 min and chased for
the indicated times with (+) or without(-) PMA (1
µM). Chase times were selected based on the experimentally
determined endoplasmic reticulum to Golgi transit time (t
1 h) and secretion rate of PAM proteins
from hEK-293 cells (
5% of cellular content of PAM-1/h;
50% of
cellular content of PAM-3/h;
25% of cellular content of
PAM-2/899/h)(13) . Chase media and final extracts were
immunoprecipitated with PHM antibody and analyzed by SDS-PAGE and
fluorography; films were densitized to quantify the effects of PMA. The
same type of experiment was repeated twice with similar results. The
doublet observed in the medium of PAM-2/899 cells presumably reflects
endoproteolytic cleavage at different sites near the transmembrane
domain.
In a number of
other cell types, PMA treatment increases the flux of membrane proteins
through the constitutive secretory pathway(32, 33) .
To determine whether PMA treatment affected the secretion of soluble
PAM proteins, hEK-293 cells expressing PAM-3 were pulse-labeled with
[S]methionine/cysteine for 15 min and chased for
90 min with or without PMA. A shorter chase time was chosen to evaluate
the effect of PMA on PAM-3 secretion because little PAM-3 remains in
the hEK-293 cells after a 2-h chase(13) . PMA had no effect on
the secretion of PAM-3 from hEK-293 cells (Fig. 8B).
The effect of PMA on release of PAM proteins is limited to membrane
forms of PAM. PMA could act directly by stimulating phosphorylation of
integral membrane PAM or indirectly by affecting membrane flux or
proteolytic cleavage. To determine whether the effect was direct or
indirect, hEK-293 cells expressing integral membrane PAM lacking all of
the identified phosphorylation sites (PAM-2/899) and shown not to
undergo phosphorylation (Fig. 2A) were treated with PMA (Fig. 8C). PMA treatment significantly (5-fold; n = 2) increased the cleavage/release of soluble PAM
derived from PAM-2/899. Thus, the PMA effect on cleavage/release of
soluble PAM derived from integral membrane PAM was not mediated
directly by phosphorylation of the PAM protein.
Figure 9:
Analysis of PAM-1/Ser
Ala mutant. A, AtT-20 cells expressing PAM-1 or PAM-1/S937A
were fixed and visualized with a mouse monoclonal antibody to the
COOH-terminal domain of PAM; scale bar, 20 µm; nuclei (N) are seen as clear area. B, AtT-20 cells
expressing PAM-1 or PAM-1/S937A were incubated with antibody to PHM for
10 min; excess antibody was washed away and the cells were either fixed
immediately or returned to 37 °C for 10 or 30 min before fixation.
Internalized PAM
PAM antibody complex was visualized with
FITC-tagged goat anti-rabbit immunoglobulin; magnification as in A.
At steady state, a small fraction of the
membrane PAM in AtT-20 cells is on the plasma membrane; the PAM
proteins on the cell surface are rapidly internalized and enter the
endocytic pathway(13, 14, 21, 34) .
Antibody internalization experiments were carried out to compare the
manner in which these two PAM proteins traverse the endocytic pathway (Fig. 9B). AtT-20 cells expressing PAM-1 or PAM-1/S937A
were incubated with antibody to PHM; the antibody bound to the small
fraction of the PAM protein accessible on the plasma membrane. The
cells were then washed and allowed to internalize the PAMPAM
antibody complex for different amounts of time. After 10 min of chase,
antibody internalization by cells expressing PAM-1 and PAM-1/S937A
appeared quite similar; the PAM
PAM antibody complex accumulated
in the perinuclear region. After a longer chase, the PAM
PAM
antibody complex remained localized in the perinuclear region in AtT-20
cells expressing PAM-1. In contrast, the PAM
PAM antibody complex
in AtT-20 cells expressing PAM-1/S937A was targeted to vesicular
structures distributed more widely throughout the cytoplasm.
The
PAMPAM antibody complex internalized by AtT-20 cells expressing
PAM-1/S937A assumed a distribution resembling that of lysosomes. To
evaluate this possibility, AtT-20 cells expressing both PAM proteins
were allowed to internalize PAM antibody for 30 min, and the
internalized antibody and lysosomes were then visualized simultaneously
by confocal microscopy (Fig. 10). In AtT-20 cells expressing
PAM-1, the internalized PAM antibody was localized primarily in
structures distinct from lysosomes. In AtT-20 cells expressing
PAM-1/S937A, much of the internalized PHM antibody was localized in
structures recognized by the LAMP antibody. These results suggest that
phosphorylation at Ser
plays a crucial role in
trafficking of membrane PAM after internalization from the plasma
membrane.
Figure 10: Internalized PAM antibody and LAMP. AtT-20 cells expressing PAM-1 or PAM-1/S937A were allowed to internalize PHM antibody for 30 min, fixed, and visualized simultaneously with a rat monoclonal antibody to LAMP and a FITC-tagged secondary antibody and a Cy3-tagged donkey anti-rabbit immunoglobulin. Dual-channel images (PHM antibody in red and LAMP antibody in green) were merged into co-localization images; areas of overlap appear yellow in color. The scale bar is 10 µ.
In this study we demonstrated that PAM proteins were
phosphorylated on Ser and Thr residues within their COOH-terminal
domain only when this domain was exposed to the cytosol because of the
presence of a transmembrane domain. Phosphorylation of membrane PAM was
eliminated when the protein was truncated at Tyr. Since
recombinant PAM COOH-terminal domain was phosphorylated in vitro on Ser
and Ser
by PKC, we decided to
investigate a role for PKC and phosphorylation in the trafficking of
membrane PAM.
Our in vitro phosphorylation studies using
subcellular fractions of AtT-20 cells indicated that the PAM proteins
localized to different subcellular compartments at steady state were
differentially susceptible to phosphorylation. Phosphorylation of
120-kDa PAM-1 and PALm in the particulate fractions occurred in the
absence of cytosol. Phosphorylation of PAM proteins in the secretory
granule-enriched 20,000 rpm fraction was inhibited by the addition of
cytosol. In contrast, PAM proteins in the 30,000 and 40,000 rpm
fractions were phosphorylated in a Ca-dependent
manner by protein kinases(s) in the cytosol. Although the identity of
the kinase(s) responsible for the phosphorylation of PAM was not
investigated, kinase activity was dependent on the addition of
Ca
and was inhibited in the presence of
staurosporine. These results indicate that PAM proteins in this
subcellular compartment differ in their ability to undergo additional
phosphorylation. This could reflect prior phosphorylation of the
relevant sites or the presence of inhibitors of phosphorylation. In any
case, these results suggest that phosphorylation of PAM could
differentially affect its function depending upon its subcellular
localization.
PMA treatment of hEK-293 cells expressing PAM-1 failed to increase the level of phosphorylation of PAM-1. However, it increased the cleavage/release of a soluble bifunctional PAM protein derived from PAM-1. Since truncation of the COOH-terminal domain of integral membrane PAM eliminated its phosphorylation without abolishing the PMA effect on cleavage/release of soluble bifunctional PAM, this effect of PKC must be an indirect one not involving phosphorylation of PAM. Lack of a PMA effect on the release of soluble PAM-3 from hEK-293 cells indicated that the effect was confined to integral membrane proteins.
The proteolytic release of soluble fragments derived from
the -amyloid protein precursor(35) , tumor necrosis factor
receptor (36) , pro-TGF-
(37, 38) , and
CSF-1 receptor (39) is also promoted by activators of PKC. As
observed for PAM-1, this effect does not involve the conversion of
-amyloid protein precursor(40) ,
pro-TGF-
(38) , and CSF-1 receptor (39) into better
substrates for proteolysis by direct phosphorylation. The proteolytic
enzyme(s) mediating these cleavages have not been identified; mutant
CHO cells selected for an inability to cleave pro-TGF-
also fail
to cleave many other surface proteins, suggesting the involvement of a
common factor(37, 41) . PMA treatment may activate a
proteolytic enzyme(s) or increase the accessibility of membrane
proteins to the compartment containing the proteolytic
enzyme(s)(41) . Participation of PKC in regulating the
cleavage/release of PAM raises the intriguing possibility that there is
a physiological significance to the regulated release of PAM from
integral membrane PAM. In primary atrial myocyte cultures, phorbol
ester treatment also stimulates the cleavage/release of PAM proteins
derived from PAM-1 and PAM-2(42) .
The COOH-terminal domain
of integral membrane PAM contains information critical for the
intracellular trafficking of PAM in endocrine cells (14, 21, 34) and in fibroblast-like
cells(13) . Truncation of most of the COOH-terminal domain
resulted in mistargeting of integral membrane PAM to the cell surface
and loss of the ability of cells to internalize PAM that has reached
the cell surface. More detailed mutagenesis studies indicated that
signals mediating the internalization of PAM from the plasma membrane
are somewhat distinct from signals mediating the localization of PAM in
the trans-Golgi network region of the cell(34) .
Mutation of the PKC site at Ser to Ala in the
COOH-terminal domain of PAM-1 resulted in a protein whose steady state
localization in AtT-20 cells was very similar to that of wild type
PAM-1. Nevertheless, when an antibody internalization paradigm was used
to look specifically at the small fraction of PAM-1 undergoing
internalization, mutation of this site was found to have a profound
effect. While the PAM
PAM antibody complex internalized by AtT-20
cells expressing PAM-1 was rapidly accumulated in the perinuclear
region of the cells, the PAM
PAM antibody complex internalized by
AtT-20 cells expressing PAM-1/S937A was first accumulated in the
perinuclear region and then dispersed to structures that appear to be
lysosomes.
In endocrine cells, integral membrane PAM proteins are
thought to be retrieved from immature secretory granules via
constitutive-like vesicles with very little membrane PAM actually
reaching the cell surface under basal conditions. Membrane PAM protein
that reaches the plasma membrane is thought to enter the endocytic
pathway(14, 34) . We propose that phosphorylation of
Ser is crucial for a step in the endocytic pathway that
directs PAM proteins in the recycling pathway; in the absence of
phosphorylation at this site, the protein appears to enter lysosomes.
If the Ca
-stimulated, staurosporine-inhibitable
process that phosphorylates PAM proteins in subcellular fractions
functions in cells, recycling of PAM proteins may be regulated in
concert with Ca
-stimulated exocytosis.
Phosphorylation has been implicated in the trafficking of integral
membrane proteins such as the mannose
6-phosphate(43, 44) , epidermal growth
factor(45) , and polymeric immunoglobulin (26) receptors. Phosphorylation of the mannose 6-phosphate
receptor is crucial for its interaction with AP-1 Golgi adaptor
proteins(46) . Interaction of the COOH-terminal domain of
membrane PAM with cytosolic factors involved in the trafficking of
membrane proteins may involve phosphorylation of the COOH-terminal
region of PAM. Additional studies on cytosolic protein kinases that
phosphorylate PAM and proteins that interact with the COOH-terminal
domain of PAM will provide insight into the role of phosphorylation in
the trafficking of membrane PAM.