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(Received for publication, August 16,
1995; and in revised form, November 20, 1995) From the
Phosphorylation of prolactin by endogenous protein kinases
within isolated secretory granules was shown to result in the
production of both phosphoserine and phosphothreonine residues. The
majority of the radiolabel was determined to be present in the C
terminus of the molecule after specific cleavage with glandular
kallikrein. Glandular kallikrein cleaves in three places at the C
terminus, liberating three small peptides, only one of which contains a
phosphorylatable residue. Sequencing of this phosphopeptide showed it
to be Arg
For many years prolactin (PRL) ( Functional studies from this laboratory have
determined that monophosphorylated PRL is an antagonist to
non-phosphorylated PRL in two cell systems where non-phosphorylated PRL
promotes cell proliferation(8, 9) . It is therefore
important to establish the sites of PRL phosphorylation so that these
may be reproduced in vitro for further analysis of this
antagonism which operates through a single
receptor(9, 10) . In our earlier studies, we used a
variety of purified protein kinases in an attempt to identify potential
phosphorylation sites(1) . This approach, however, while
illustrating that PRL is a very readily phosphorylated molecule, did
not narrow the search because such a variety of protein kinases with
very different consensus recognition sequences were found capable of
phosphorylating PRL. For PRL from other species, only protein kinase A
(PKA) has been tried and shown to successfully phosphorylate ovine,
chicken, and turkey PRL(7) . In this article we present
evidence that PRL is phosphorylated on both a serine and threonine
residue and that only the serine phosphorylation, which is the major
site, can be re-produced by PKA.
For two-dimensional phosphopeptide
mapping, horizontal electrophoresis was for 2 h at 450 V with cooling
water at 6 °C on a 20 Samples for phosphoamino acid analysis, produced by trypsinization
(above), were transferred into vacuum hydrolysis tubes for hydrolysis
at 110 °C in a heating block for 1 h. Hydrolysates were dried and
mixed with standards (2 µg each of phosphotyrosine, phosphoserine,
and phosphothreonine) (Sigma) in water and applied to a 20
For HPLC and sequencing
analysis of the radiolabeled kallikrein peptide, granule
phosphorylation reactions were increased proportionately to contain
Figure 1:
Sequence of rat PRL.
Sequence taken from Cooke et al. (31). Predicted tryptic
cleavage sites are shown by small arrows. Kallikrein cleavage
sites are shown by large arrows as per Powers and
Hatala(19) .
Since most PRL secretion occurs via a regulated pathway, the
hormone is packaged in dense granules for storage prior to exocytosis (16) . Previous work from this laboratory has identified these
dense granules as the site of PRL phosphorylation(12) .
Subcellular fractionation produces a highly enriched fraction of
granules(11, 12) which can be used to monitor
phosphorylation of PRL by endogenous granule kinases. In intact cells
this phosphorylation is kept in check until exocytosis (12) making it very difficult to analyze phosphorylation in
vivo. The purity of the granule fraction is demonstrated in the
composite Fig. 2. In this particular preparation, PRL
represented 94% of the protein which could be stained by Coomassie Blue (lane 1). In a series of granule preparations, PRL constituted
between 94 and 97% of total protein. A number of other proteins are
present in small quantities some of which must be the
kinases(12) , a protease(17) , and a disulfide
isomerase (18) previously described as granule constituents.
PRL was identified by Western blot analysis of a duplicate to lane
1 (lane 2) and by approximate molecular mass as compared
to standards (lane 3).
Figure 2:
Granule preparation. Composite figure of
reducing SDS 10% gel analysis of proteins in the granule preparation
demonstrated by Coomassie Blue stain (lane 1) and Western blot
analysis using anti-rat PRL (lane 2). 3 µl (
Figure 3:
Phosphorylation of rat PRL by the
endogenous kinases of secretory granules. A, analysis of
radiolabeled proteins in 10% reducing SDS gels followed by
autoradiography (exposure for 3 days at -70 °C). An
autoradiogram is shown. Positions of molecular mass markers were
determined from a Coomassie Blue-stained gel. B, autoradiogram
of phosphoamino acid analysis (exposure for 9 days at -70
°C). P
Phosphoamino acid analysis of the excised, endogenously
phosphorylated PRL band showed phosphorylation on both serine and
threonine residues (Fig. 3B). No phosphotyrosine was
detected. The amount of phosphothreonine was larger than phosphoserine,
but this is likely due to the greater lability of phosphoserine. As
will be shown below, the major site of PRL phosphorylation was on a
serine. Tryptic phosphopeptide mapping of endogenously
phosphorylated PRL showed four labeled spots/areas. By a variety of
techniques, one spot was identified as free phosphate, two as distinct
peptides and one, at the origin, as poorly digested, C-terminal regions
of PRL. The vast majority of the incorporated label remained with the
poorly digested C-terminal region (Fig. 3C).
Figure 4:
Kallikrein cleavage following granule
phosphorylation. Autoradiogram of a reducing 20% SDS gel. Lane
1, no added kallikrein; lane 2, added kallikrein.
Relative units following densitometry of peptide band in lane 1, 1.28; lane 2, 1.71. Position of kallikrein peptide
determined by reference to silver-stained standard run in an adjacent
lane. 23- and 21-kDa products of kallikrein cleavage were assigned by
reference to the silver-stained gel and molecular mass standards. KKp, kallikrein undecapeptide
(Arg
To further analyze
phosphorylation at the C-terminal site, a 20-amino acid peptide
equivalent to amino acids 161-180 of PRL was synthesized. This
sequence, SKDLAFYNNIRCLRRDSHKV, was chosen so as to include the Arg six
places and Phe 11 places to the N terminus of the serine because
equivalent residues in the PKA inhibitor protein have a large positive
effect on affinity for PKA(20) . When included in the
endogenous granule phosphorylation reaction, this peptide became
phosphorylated (Fig. 5A). This is illustrated in lanes 1, 2, and 3 where 0, 18, or 36 µg of the
peptide were added, respectively. Lane 1 shows no labeled
20-aa peptide and lanes 2 and 3, increasing amounts
of the 20-aa peptide. The 20-aa peptide runs slower (rf value of 0.60)
than the KKp, produced by activity of the granular, endogenous
kallikrein. When the amount of radiolabeled intact PRL in lanes 1,
2, and 3 was quantified by densitometry (Fig. 5B), it was also clear that the added peptide
competed with intact PRL for phosphorylation by a granular kinase. If a
synthetic peptide containing the putative threonine phosphorylation
site (Leu
Figure 5:
Granular kinase phosphorylation of
synthetic PRL peptides. Autoradiogram of a reducing 20% SDS gel. Lane 1, no additions showing the endogenous production of the
kallikrein peptide (KKp); lane 2, plus 18 µg of
the 20-amino acid C-terminal peptide (20 aap); lane
3, plus 36 µg of the 20-amino acid C-terminal peptide; lane 4, plus 18 µg of the small synthetic peptide
containing threonine 63 (Tp). (Exposure for 2 days at
-70 °C.) The positions of the added peptides and kallikrein
peptide were confirmed by running these as standards in additional
lanes followed by silver-staining (silver-stained gel not shown). B, plot of the relative densitometry scans showing a decrease
in intact PRL labeling as a consequence of 20-amino acid peptide
addition.
Figure 6:
Competition between the PepTag substrate
and the 20-amino acid C-terminal peptide of PRL. The amount of
phosphorylated PepTag substrate is expressed as percent total where 50%
is the amount phosphorylated in the absence of added C-terminal
peptide. P-PepTag, phosphorylated PepTag. Points represent the mean of
triplicate samples run on three different gels.
In vitro phosphorylation
of purified PRL (NIDDK I5) by the catalytic subunit of PKA is shown in Fig. 7A. The degree of PRL phosphorylation is dependent
on the amount of PKA catalytic subunit used. Under the conditions used,
a maximum of about 18% of the PRL is phosphorylated, as assessed by
incorporated moles of phosphate or densitometric analysis of the
phosphoprotein on a two-dimensional protein gel. Phosphoamino acid
analysis of PKA-phosphorylated PRL showed only the presence of
phosphoserine (Fig. 7B). Two-dimensional peptide maps
were the same as endogenously phosphorylated PRL (showing a
highly-labeled, poorly-digested spot at the origin) except that one of
the two distinct peptide spots (later identified as the threonine
peptide) was missing (not shown). HPLC analysis following extended
trypsin cleavage (40 h) showed the majority (90%) of the incorporated
radiolabel to be present in the flow-through of a reverse phase C18
column. (Free ATP was removed with AG-X1 resin (21) and its
removal was checked by thin layer chromatography.) This was anticipated
should phosphorylation mainly occur at serine 177. Deduced from the
sequences of predicted tryptic peptides (Fig. 1) (cut sites
determined by software of Genetics Computer Group, Madison, WI),
peptides in the region 172-179 would be expected to pass through
a C18 column. To test behavior on a C18 column, a peptide corresponding
to a less-than-ideal digestion of this region, constituting amino acids
173-180 (LRRDSHKV), was synthesized. The cysteine at position 172
(normally present in the tryptic peptide) was not included in order to
prevent potential problems with dimerization. The addition of a valine
at the C terminus restored approximate size and charge balance. Even
when not phosphorylated and even though larger than some potential
peptides in this region, this peptide passes through a C18 column (data
not shown). Thus, on the basis of incorporated counts most PKA
phosphorylation was at serine 177.
Figure 7:
In vitro phosphorylation of
purified rat PRL by PKA. A, autoradiogram of a reducing 10%
SDS gel and graph showing dose dependence of the phosphorylation.
(Exposure for 22 h at -70 °C.) Counts per minute in the PRL
band in the absence of PKA were 237. B, autoradiogram of
phosphoamino acid analysis. (Exposure for 24 h at -70 °C,
longer exposures showed no additional spots.) P-Ser,
phosphoserine; P-Thr, phosphothreonine; P-Tyr,
phosphotyrosine; ori, origin.
Figure 8:
Two-dimensional protein gel analysis of
PKA-phosphorylated purified PRL. A, silver-stained gel. 2, isoform 2 which is the unmodified translation product; 3, isoform 3 which is monophosphorylated PRL; 3`,
isoform 3` which is diphosphorylated PRL. B, autoradiogram
showing phosphate incorporation mainly into 3 with a much
lower incorporation into 3` in proportion to the amount of
silver-stained protein. PRL isoforms were identified by reference to pI
and M
Figure 9:
Mass analysis of phosphopeptide unique to
endogenously phosphorylated PRL. This peptide ran as a discrete spot
and was found only in phosphothreonine-containing endogenously
phosphorylated granular PRL. Matrix-assisted time-of-flight mass
analysis. Major peak has a mass of 1708 which equals mass of
phosphorylated AINDCPTSSLATPEDK allowing for
Further
analysis of this threonine phosphorylation site is complicated by the
presence of two threonines within a single peptide. On the basis of
consensus sequence analysis for known protein kinases, the most likely
phosphorylation site was judged to be threonine 63 and a peptide
representing amino acids 61-68 (Tp) was included in an endogenous
granule phosphorylation reaction, illustrated previously in Fig. 5(lane 4). No phosphorylation of this very small
peptide was observed. The threonine-containing peptide (Tp), as
determined from the silver-stained gel, ran slower than the kallikrein
peptide and faster than the 20-amino acid peptide and contained no
detectable radioactivity.
One
radiolabeled peptide was found representing 10% of incorporated
radioactivity. Sequencing established this peptide as IISQAYPEAK (amino
acids 131-140). When a synthetic peptide, EKIISQAY (amino acids
129-136 of PRL), containing the phosphorylatable residue, was
incubated with secretory granules, some of the peptide was found to
change its mobility on a C18 column, eluting at 24.83 rather than 25.5
min. In addition, by mass analysis, we observed an increase of 378 (946
to 1324 and not 80). These data are therefore more likely indicative of
disaccharide addition than phosphorylation. Thus this site may not be
phosphorylated in vivo. Also, inclusion of EKIISQAY in the
PepTag assay showed it to have no ability to compete for PKA
phosphorylation of the PepTag substrate (Fig. 6). From our previous work we had established that there were
apparently two sites of PRL phosphorylation based on the presence of
two, more acidic, phosphate-incorporating isomers of PRL on
two-dimensional gels(12) . The quantities of each produced, in
granule reactions, however, was very different. After a typical in
vitro granule reaction the apparent monophosphorylated variant
represented 30-40% of total PRL and the diphosphorylated variant
between 1 and 5%(12) . In experiments where radiolabeling was
not used, the production of the phosphorylated variants did not depend
on the addition of exogenous ATP and, although reduced, phosphorylation
was not eliminated by the removal of granule membranes(12) .
Granule membranes have been shown in another species to possess a
proton pump (23) . Since ATP does not have to be provided and
phosphorylation occurs in the absence of an ATP-generating pump, we
deduce that there is a store of ATP within the granules and assume that
this is one reason why we have not been able to radiolabel granule PRL
with endogenous kinases to very high specific activity. Nevertheless,
we have been sufficiently successful in unequivocally identifying the
major site of PRL phosphorylation. Analysis of the major site of PRL
phosphorylation did not yield to traditional approaches because of the
inefficient cleavage of the C terminus of PRL by trypsin. Twenty hours
of digestion left very large peptides which stayed at the origin when
subjected to two-dimensional peptide mapping. Even with complete
digestion, however, one would not be able to catch the phosphorylated
peptide by traditional means because it runs with free phosphate on a
two-dimensional peptide map and straight through a C18 column. It is
for this reason that we turned to kallikrein digestion of the molecule
and analysis on high percentage gels. This approach had the significant
advantage of producing fewer peptides and, from previous work from
Powers' (17) group using purified PRL (19) and
our group using PRL secretory granules, we knew the correct conditions
for efficient digestion. These conditions include reduction of
disulfide bonds and treatment with Triton X-100 to access the C
terminus of PRL, indicating the relative unavailability of this region
of the molecule. Based on the results of kallikrein digestion of
intragranular endogenously phosphorylated PRL, by far the largest
amount of radioactive phosphate is present in the C terminus of PRL in
the peptide RDSHKVDNYLK(175-185). Since the kallikrein digestion
experiments involved the least processing and no exposure to either
alkali or acid, this is strong evidence that serine 177 is the major
site of PRL phosphorylation. To confirm that this region of the
molecule was phosphorylated by the endogenous granule kinases, the
20-amino acid peptide representing amino acids 161-180 of PRL
(SKDLAFYNNIRCLRRDSHKV) was included during radiolabeling reactions. Not
only did this peptide become radiolabeled, but it prevented
phosphorylation of some intact PRL in the granules. This competition
demonstrates that the peptide was truly phosphorylated by PRL kinase
and not by a protein kinase on the cytoplasmic (outside membrane)
surface of the granules or by a contaminant of the granule fraction.
Serine 177 is a highly conserved residue among PRLs from different
species(24) . This region of the molecule has been determined
to be critical for biological activity(25) . Our previous
studies have demonstrated that monophosphorylated PRL is an antagonist
to non-phosphorylated PRL. Thus one might have predicted that this
would be an important site for phosphorylation. Based on consensus
sequence analysis for known protein kinases, serine 177 had been
predicted to be a possible site for PKA phosphorylation. For further
analysis of the biological activities of the monophosphorylated
variant, it was important that we identify a protein kinase which could
duplicate phosphorylation at serine 177. By analogy to the PKA
inhibitor protein, serine 177 of PRL may well have proven to be an
exceptional site for PKA phosphorylation because of the presence of the
Phe 11 places and Arg six places to its N-terminal side(20) .
Analysis in the commercial PepTag assay, however, showed it to be a
good substrate for PKA, but not an exceptional one. This is probably
due to the presence of the Asp in the X position of the
recognition sequence, RRXS. Ideally this should be a neutral
amino acid. Phosphoamino acid analysis of purified PRL
phosphorylated in vitro by PKA showed only the presence of
phosphoserine, consistent with only the duplication of the serine and
not the threonine phosphorylation site. Prolonged trypsin digestion of
PKA-phosphorylated PRL followed by HPLC on a C18 column to check for
additional sites of PKA phosphorylation showed 90% of the counts to be
present in flow-through peptides where they should be if
phosphorylation was at the same site as in vivo (serine 177)
and only 10% in one other peptide. This latter peptide was sequenced
and shown to contain amino acids 131-140 of PRL (IISQAYPEAK).
Inclusion of a synthetic peptide (EKIISQAY), representing amino acids
129-136, in an endogenous granule phosphorylation produced a mass
change in the peptide from 946 to 1324, more indicative of disaccharide
addition than phosphorylation. This peptide also showed no ability to
compete for PKA phosphorylation in the PepTag assay. We propose
therefore that this very minor site of PKA phosphorylation is an
artifact of the use of very high enzyme concentrations, forcing the
phosphorylation of a site normally reserved for O-linked
glycosylation. Consistent with the major phosphorylation site in
vivo being at serine 177 and its duplication by PKA are the
results of two-dimensional gel analysis following PKA in vitro phosphorylation of already partially phosphorylated purified PRL.
This purified PRL provided by the NIDDK, is extracted from pituitaries
and contains non-phosphorylated, monophosphorylated, and
diphosphorylated PRL. These run as charge isomers designated 2, 3, and
3`, respectively, in Fig. 8. If PKA phosphorylated a site
different from those used in vivo, one would predict the
production of three phosphorylated spots running as 3, 3`, and 3". This
was not seen. Only radiolabeled 3 and 3` were observed. The highest
specific activity (deduced from the size of the autoradiographic spot versus the silver-stained spot) was seen in spot 3,
representing the conversion of non-phosphorylated PRL to
monophosphorylated PRL. By contrast, as judged by the specific activity
of the diphosphorylated variant, much of the monophosphorylated
material present before the reaction was unavailable for conversion to
the diphosphorylated variant by PKA, thereby suggesting that the PKA
site was already phosphorylated. What radioactivity is present in the
diphosphorylated spot could be due to correct phosphorylation of
deamidated PRL since deamidation creates a negative charge and
preparations of PRL always contain some deamidated hormone.
Alternatively, this could be the proposed artifactitious
overphosphorylation discussed above. It appears therefore that
phosphorylation of serine 177 can be duplicated by PKA although some
care needs to be exercised so as not to overphosphorylate the molecule.
To this extent, one PRL kinase is PKA-like. It remains to be
determined, however, whether it resembles PKA in any other way. PKA is
a cytosolic enzyme with no known mechanism to enable it to enter the
secretory pathway. Cyclic AMP-dependent phosphorylation of bovine
adenohypophyseal proteins has been reported previously (26) as
has the association of PKA with anterior pituitary
granules(27) . In both cases, however, the phosphorylated
granule proteins were not identified and it is not clear whether the
PKA was on the cytosolic face of the granule membrane, responsible for
phosphorylating proteins involved in membrane fusion during exocytosis,
or inside the granule responsible for phosphorylating hormone and/or
other granule constituents. As previously mentioned, PRL can also be
diphosphorylated. From the phosphoamino acid analysis we conclude that
the second phosphorylation site was on a threonine. In fact, because of
the relative stability of the ester bond on phosphothreonine (22, 28) and the possible inclusion of amounts of the
21-kDa protein in the excised band of these heavily loaded 10% gels,
this appeared at first to be the major site. Traditional analysis by
two-dimensional tryptic peptide mapping was successful in isolating
this site as a discrete peptide. Mass analysis was consistent with a
phosphopeptide containing two threonines, but the phosphorylation of
only one. A synthetic peptide containing only threonine 63 was made in
an attempt to discriminate between the two threonines. Addition of this
peptide to granule phosphorylation reactions did not result in
phosphorylation of the peptide. This could be because phosphorylation
is at threonine 58, or the peptide was too small for efficient
recognition by the protein kinase. Further analysis of this minor site
will require an alternate approach. It is important to note that
phosphorylation of this site did not occur with PKA since
PKA-phosphorylated PRL only contained phosphoserine. Thus there are
apparently two PRL kinases subject to individual control. This is
consistent with our earlier studies demonstrating changes in specific
phosphorylated species in response to physiologic
stimuli(29, 30) . It should therefore be possible to
reproduce both phosphorylations in vitro either separately or
together as desired. In summary we have established the primary site
of PRL phosphorylation as serine 177. Phosphorylation at this site has
a major effect on biological activity causing the phosphorylated PRL to
become an antagonist to the non-phosphorylated
hormone(8, 9) . Phosphorylation at this site can be
duplicated by PKA and an intragranular PKA-like enzyme may be integral
to the regulation of PRL structure and function in vivo.
Volume 271,
Number 5,
Issue of February 2, 1996 pp. 2462-2469
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-Lys
. Thus the major site of
prolactin phosphorylation was determined to be serine 177. Using a
synthetic peptide equivalent to this region of the molecule
(Ser
-Val
), serine 177 was demonstrated to
be a substrate for protein kinase A as well as for one of the
endogenous granule kinases. Inclusion of the synthetic peptide in an
endogenous granule phosphorylation reaction resulted in competition for
the kinase and reduced phosphorylation of prolactin. Protein kinase A
phosphorylation of purified prolactin resulted in the production of
only phosphoserine and primarily the most abundant (monophosphorylated)
variant. We conclude that serine 177 is the major in vivo phosphorylation site of rat prolactin and that phosphorylation of
this site can be reproduced by protein kinase A in vitro. The
minor threonine phosphorylation site was demonstrated by
two-dimensional tryptic peptide mapping and mass analysis to be either
threonine 58 or 63, both of which are contained within a single
peptide.
)was considered an
unmodified polypeptide hormone. It is now clear, however, that
post-translational processing of PRL causes it to be phosphorylated (e.g.(1) and (2) ), glycosylated (e.g.(3) ), and variously proteolytically cleaved (e.g.(4, 5, 6) ). The phosphorylation of PRL
within pituitary cells has been demonstrated to occur in vivo in the rat(1) , chicken(7) , and cow(2) .
Phosphate analysis of purified preparations of PRLs from different
species showed them to be variously phosphorylated with molar ratios of
hormone to phosphate of 1.0:0.2 for ovine and rat and 1.0:0.7 for
turkey(7) .
Secretory Granule Preparation and
Characterization
PRL secretory granules were isolated from
female rat anterior pituitaries as described by Zanini and Giannattasio (11) and modified by Greenan et al.(12) . This
procedure results in a highly enriched fraction of large, dense PRL
granules(11, 12) . Three to five µl (4-6
µg) of granules (suspension in 0.32 M sucrose) were
subjected to SDS electrophoresis under reducing conditions on a 10% gel
(30 mA, 3.5 h). Separated proteins were stained with Coomassie Blue and
the amount of protein present in the PRL band determined by laser
densitometry. The PRL band was identified by (a) blotting a
duplicate lane onto nitrocellulose, probing with rabbit anti-PRL, and
development of a peroxidase-conjugated second antibody according to
previously published methods(13) , and (b) by
reference to co-run and stained molecular mass markers.Endogenous Phosphorylation of PRL in Secretory
Granules
Frozen secretory granules were sonicated on ice for 10
s at position 4 using a Sonifier Cell Disrupter (Heat
Systems-Ultrasonics, Plainview, NJ). Typically 40 µg of secretory
granules were incubated with 250 µCi of
[-
P]ATP (4500 Ci/mmol, ICN, Irvine, CA) in
60 mM Tris-HCl, pH 7.4, in a total of 80 µl at 37 °C
for 16 h. 6 mM
-Glycerophosphate was included during this
6-h incubation to reduce phosphatase activity. For analysis on gels,
the reaction was terminated by boiling for 3 min with an equal volume
of 2 
sample buffer (to give 6.2 mM Tris, 2% SDS, 5%
-mercaptoethanol, 10% glycerol, pH 6.8). Electrophoresis in 10%
gels was followed by Coomassie Blue staining and either exposure to
autoradiographic film after drying or in the wet state (with 10%
methanol to prevent cracking) if further processing was to take place.
Film used was Kodak X-Omat (Kodak, Rochester, NY) and exposure was at
-70 °C.Phosphoamino Acid Analysis and Phosphopeptide Mapping of
Endogenously Labeled Intragranular PRL
After phosphorylation by
endogenous granule kinases and separation on a 10% gel, the
phosphorylated PRL band was excised and sliced into 1-mm cubed pieces.
These were rinsed 6 times (10 min each) in double distilled deionized
water to remove SDS and once in 50 mM NH
OH, pH
8.0. L-1-Tosylamido-2-phenylethyl chloromethyl ketone-treated
sequencing grade trypsin (Promega, Madison, WI) was used to digest the
PRL (1:10, w/w) in 200-250 µl of 50 mM NHOH, pH 8.0, at 37 °C for 20-40 h. Five
µl of glacial acetic acid was then added to terminate the reaction.
The gel suspension was then separated into component parts by passage
through a 10-kDa filtration membrane (Ultrafree-MC, Millipore, Bedford,
MA). Peptides below 10 kDa (undigested PRL is 24 kDa) were lyophilized
and dissolved in either 10 µl of electrophoresis buffer
(pyridine:acetic acid:H
O at 10:100:1890, pH 3.5) for
peptide mapping or 0.5 ml of 6 N sequencing grade HCl (Pierce)
for phosphoamino acid analysis. 20-cm silica gel plate (EM
Separations, Gibbstown, NJ). This was followed by chromatography
(butanol:pyridine:acetic acid:HO at 60:40:12:48) until the
solvent was 2 cm from the top of the plate, according to the procedure
of Boyle et al.(14) . The position of free phosphate
was determined by duplicate runs of 3 10
cpm
[P]H
PO (ICN). Plates
were allowed to dry and were then exposed to film at -70 °C.
20-cm silica gel plate. Electrophoresis was for 4 h at 400 V with
cooling water at 6 °C. Plates were air-dried, sprayed with
ninhydrin (0.1% in butanol) to identify the location of the standards,
and then exposed to film at -70 °C.Extraction of Phosphopeptides from Silica Gel Plates
after Two-dimensional Mapping
For further analysis of the
phosphopeptides, an area of silica equivalent to the radiolabeled area
on the autoradiogram was carefully scraped off and then sequentially
extracted with pyridine (0.5 M, 1 ml, 10 min), HO
(1 ml, 10 min), and acetic acid (0.5 M, 1 ml, 10 min). This
material was filtered through a 10-kDa membrane, the filtrate was
lyophilized, dissolved in water, and re-lyophilized. The resultant
preparation was then analyzed by matrix-assisted time-of-flight mass
spectrometry in the Biotechnology Instrumentation Facility at the
University of California, Riverside.Phosphorylation of Synthetic PRL Peptides by Granule
Kinases and Kallikrein Digestion of Phosphorylated PRL following
Endogenous Granule Phosphorylation
To granule phosphorylation
reactions containing 18 µg of sonicated PRL granules in 0.32 M sucrose (18 µl) were added 0, 18, or 36 µg of
synthetic PRL peptide dissolved in buffer (0.2 M Tris-HCl, pH
7.5, 0.02 M MgCl
, 0.1 M NaF) (18 µl)
and 18 µl of [-
P]ATP (10 mCi/ml, 4500
Ci/mmol) (ICN). Synthetic peptides were obtained from Coast Scientific,
San Diego, CA, or Multiple Peptide Systems, San Diego, CA. After 16 h
at 37 °C,
-glycerophosphate was added to reach a final
concentration of 5 mM. To some of the tubes without added
peptide was added 300 ng of kallikrein (kindly provided by Dr. Julie
Chao, Medical University of South Carolina, Charleston, SC) and Triton
X-100 and -mercaptoethanol to give final concentrations of 0.15%
and 6 mM, respectively. After a further 4-h incubation at 37
°C, samples were boiled with an equal volume of 2 sample
buffer and applied to 20% Tris glycine gels. Gels were prepared and run
as described in the Hoeffer (San Francisco, CA) catalog according to
Giulian and Graham. Gels were fixed in 50% methanol, 10% acetic acid
for 1 h and then in 10% gluteraldehyde overnight. Following extensive
washing, the gels were placed in 10% methanol prior to freezing and
exposure to autoradiographic film at -70 °C. After
autoradiography, the gels were silver-stained according to the method
of Oakley et al.(15) .50 µg of sonicated PRL granules. After the kallikrein
incubation, free ATP was removed with AG-X1 resin (21) (removal
was checked by thin layer chromatography) and the granule peptides were
collected in the flow through of a 10-kDa cut-off centricon (peptides
washed through with 0.01 N acetic acid), lyophilized, and
subjected to HPLC on a reverse phase 0.5-mm Reliasil C18 column.
Elution was with a 2-85% acetonitrile gradient containing 0.05%
trifluoroacetic acid. Three discrete peaks were eluted as monitored by
OD at 214 nm. The peak containing most of the radiolabel was sequenced.
PKA Substrate Activity of a Synthetic C-terminal Peptide
Equivalent to aa 161-180 of PRL
To determine whether
serine 177 phosphorylation could be duplicated by PKA, a
non-radioactive PKA kit, obtained from Promega was utilized. After
establishing the concentration of PKA catalytic subunit required to
phosphorylate 50% of the provided fluorescent pink PepTag
substrate, potentially competing peptides were titrated. These included
a peptide equivalent to amino acids 161-180 in PRL with the
sequence SKDLAFYNNIRCLRRDSHKV (Fig. 1) as well as others of
similar size without a phosphorylatable residue or those with a
phosphorylatable residue, but without a consensus sequence for PKA.
Phosphorylated PepTag was quantified as described in the kit. In our
hands we found it necessary to use twice the recommended concentration
of PepTag to achieve reproducibly quantifiable results. The PepTag
substrate had the sequence LRRASLG. It should be noted that the
161-180 PRL peptide is toward the C terminus, but is not the last
20 amino acids of the molecule.
In Vitro Phosphorylation of Purified PRL by the Catalytic
Subunit of PKA
To determine the dose dependence of
phosphorylation, reaction mixtures containing 2 µg of PRL (NIDDK
NHPP rat PRL I 5) were incubated with 0.1-1.0 µg of PKA
catalytic subunit (kindly provided by Dr. William H. Fletcher, V. A.
Medical Center, Loma Linda, CA) in phosphorylation buffer (50 mM Tris, pH 7.4, 10 mM MgCl, 0.4 mM EGTA, 0.2 mM unlabeled ATP) with 6 µCi of
[-
P]ATP at 37 °C for 30 min. Reactions
were terminated by the addition of EDTA and unlabeled ATP to final
concentrations of 10 mM. One-dimensional gel analysis,
two-dimensional phosphopeptide analysis, and phosphoamino acid analysis
were performed as described earlier. For two-dimensional gel analysis,
reactions contained 4 µg of PRL and 1 µg of catalytic subunit.
For microsequencing, 60 µg of PRL were used. Microsequencing was
performed in the Biotechnology Facility at the University of
California, Riverside. Two-dimensional gel electrophoresis of PRL was
as described previously(13) .
3 µg)
of granules were loaded in lanes 1 and 2. Lane 3 shows Coomassie Blue-stained molecular mass markers. K,
kDa.
Intragranular Phosphorylation of PRL
Phosphorylation of
PRL by the endogenous kinases of secretory granules is illustrated in Fig. 3A. Under these conditions about 30% of the PRL is
phosphorylated, as assessed by densitometric analysis of the
phosphoprotein on two-dimensional protein gels. Besides PRL, which is a
major phosphoprotein, a phosphoprotein at approximately 21 kDa, which
is most likely a previously characterized cleaved fragment of PRL
produced by the granular kallikrein(17) , is seen (see later
for further analysis). This 21-kDa protein is produced in granules
which have been incubated at 37 °C and hence it is not present in
unincubated granules such as those illustrated in Fig. 2. The
other phosphoproteins may represent the protein kinases and/or other
very highly phosphorylated minor proteins of the granules.
, free phosphate; P-Ser,
phosphoserine; P-Thr, phosphothreonine; ori, origin. C, autoradiogram of two-dimensional phosphopeptide map
(exposure for 6 days at -70 °C). O, origin; 1 and 2, phosphopeptides; P, free
phosphate.
Serine 177 Phosphorylation Site
To overcome the
major problem of incomplete trypsin digestion, which occurred with or
without alkylation of the cysteines, and because consensus sequence
analysis suggested that one phosphorylation site was close to the C
terminus, we took advantage of the well defined C-terminal cleavage of
PRL by glandular kallikrein and analyzed the peptide products and
remnant PRL on high percentage Tris glycine gels. Glandular kallikrein
cleaves at 3 sites at the C terminus of PRL (Fig. 1) resulting
in the production of an undecapeptide, RDSHKVDNYLK (amino acids
175-185), a tripeptide FLR (amino acids 186-188), a
nonapeptide, CQIVHKNNC, and the remaining approximately 21-kDa remnant
portion of the PRL molecule(19) . An intermediate band of 23
kDa represents loss of only the nonapeptide. The only potential site
for serine or threonine phosphorylation within this region is present
in the undecapeptide at serine 177. Following intragranular
phosphorylation by endogenous kinases, digestion with exogenous
kallikrein was initiated. Kallikrein is a natural component of PRL
secretory granules (17) and so some cleavage was expected in
the samples to which exogenous kallikrein was not added as soon as the
requirement for -mercaptoethanol and Triton X-100 was fulfilled in
the experimental protocol (necessary for efficient kallikrein cleavage
of PRL)(19) . Electrophoresis and autoradiographic analysis of
this control (Fig. 4, lane 1) therefore indicated a
band with an rf value equivalent to the undecapeptide, which for the
sake of simplicity we hence refer to as the kallikrein peptide (KK
peptide). The tripeptide is not retained on the gel and the
nonapeptide, which has a different rf value, has no site for
phosphorylation. It is clear from Fig. 4, however, that the
addition of exogenous kallikrein increased the production of the
radiolabeled KK peptide, which travels with an rf value of 0.62. The
average increase from four experiments was 35.6 ± 3%.
Concomitant with an increase in label in KKp was a loss of label in the
PRL band. As judged by analysis of lane 2, by far the greatest
amount of radioactivity was present in the peptide and not in the
remainder of the molecule (21-kDa radiolabeled band). This identifies
the major site of PRL phosphorylation as serine 177 and the minor site
as N-terminal to amino acid 175. HPLC separation of the individual
peptides liberated by kallikrein confirmed the presence of only one
completely cleaved labeled peptide. Sequencing of this peptide showed
it to be RDSHKVDNYLK (Table 1).
-Lys
) labeled on serine 177. (Exposure
2 days at -70 °C.)
-Glu
) was included (lane
4), no phosphorylation of it was seen. This is discussed in detail
below.
In Vitro PKA Phosphorylation of Purified PRL and the
20-aa C-terminal Peptide
Despite the likelihood, based on
consensus sequence analysis, that serine 177 was a site for PKA
phosphorylation we wished to determine this unequivocally. As
demonstrated in Fig. 6, the 20-aa peptide representing amino
acids 161-180 of PRL competes with the provided PepTag substrate
in the commercial PKA assay kit. With limiting enzyme, 24 µg of the
PRL peptide inhibited phosphorylation of 4 µg of PepTag substrate
by 50%. On a molar basis this is translated to 2 mol of PRL
peptide competing equally with 1 mol of PepTag. This demonstrates that
although the Arg six places and Phe 11 places N-terminal to serine 177
in the PRL peptide may increase affinity for PKA, the Asp in the X position of the recognition sequence, RRXS, in PRL must
be less than ideal. Otherwise, one would have predicted that the PRL
peptide would have been a much better substrate than the PepTag.
Nevertheless, it is clear that this region of PRL is an excellent
substrate for PKA. Other non-phosphorylatable peptides and
phosphorylatable peptides without a PKA consensus sequence had no
effect on PepTag phosphorylation.
, result of
competition with EKIISQAY (amino acids 129-136) or CQIVHKNNC
(amino acids 189-197) or IISRAKEIEEQNKRLLEGIE (amino acids
110-129); 
, competition with 20-amino acid C-terminal
peptide. S.E. never greater than 8%.
Two-dimensional Gel Analysis of PKA in Vitro
Phosphorylated PRL
Two-dimensional gel analysis of
PKA-phosphorylated purified PRL (Fig. 8, A and B) showed radiolabeled phosphate incorporation predominantly
into the monophosphorylated variant. Since purified PRL already
contains unlabeled mono- and diphosphorylated PRL as well as
non-phosphorylated PRL and phosphorylation increases the charge on the
PRL, this indicates that the phosphorylation site is available in
non-phosphorylated PRL, but not in mono- or diphosphorylated PRL (see
``Discussion'' for a more detailed explanation). It is
therefore the first and major phosphorylation.
markers. The autoradiogram is of the dried,
silver-stained gel shown in A. Correct isoform assignment in
the autoradiogram was assured by triangulating radiolabeled markers at
the edge of the gel. (Exposure was for 22 h at -70
°C.)
Preliminary Analysis of the Minor, Threonine
Phosphorylation Site
Evidence thus far presented has confirmed
that the major site for phosphorylation of PRL in vivo is
serine 177. In addition, we know that in vivo phosphorylated
PRL also contains phosphothreonine. Two-dimensional phosphotryptic
peptide mapping of endogenously labeled PRL showed a phosphopeptide not
present in the PKA phosphorylated material. Since the amount of this
peptide was limiting, only mass analysis was carried out. The results
are shown in Fig. 9. The mass of 1708 obtained can only be
ascribed to the tryptic peptide Ala-Lys
,
having the sequence AINDCPTSSLATPEDK. No other peptide, even allowing
for incomplete tryptic digestion and any known artifacts of tryptic
digestion or subsequent processing, have a comparable mass. The
H
peptide has a mass of 1662.
elimination of
cysteine, which occurs very readily in alkaline
conditions(22) , reduces this to 1628. This particular
preparation of PRL was not alkylated and trypsin digestion occurs in an
alkaline environment. Phosphorylation of one residue in the sequence
raises the mass to 1708. Since (a) there are no threonine
residues in the poorly digested C-terminal region of PRL, (b)
this phosphopeptide is only present in the PRL phosphorylated by
endogenous kinases, and (c) only endogenously phosphorylated
PRL contains phosphothreonine, it is likely that the phosphorylation
site is threonine 58 or 63 rather than serine 59 or 60.
-elimination of the
cysteine.
Specificity of PKA Phosphorylation of Purified
PRL
Besides the peptides around serine 177, all other serine and
threonine-containing PRL tryptic peptides should be retained on a C18
column and hence HPLC analysis of tryptic digests of the in vitro PKA-phosphorylated material was a good test for additional and,
from our point of view, undesirable phosphorylation sites.
)
We are grateful for the advice and help provided by
Drs. Polygena Tuazon, Jolinda Traugh, Gary Hathaway, and Donal Walsh.
Special thanks go to Dr. Mary Lorenson for critical review of the
manuscript. The expert secretarial assistance of Nancy Price is
gratefully acknowledged as are the contributions of Vinita Rangnekar
who helped to optimize the 20% gel separations. The purified PRL was
provided by the National Hormone and Pituitary Program of the National
Institutes of Diabetes and Digestive and Kidney Diseases.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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