(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-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.
For many years prolactin (PRL) ()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) .
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 20-cm silica gel plate (EM
Separations, Gibbstown, NJ). This was followed by chromatography
(butanol:pyridine:acetic acid:H
O 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.
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
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.
For HPLC and sequencing
analysis of the radiolabeled kallikrein peptide, granule
phosphorylation reactions were increased proportionately to contain
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.
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 (3 µg)
of granules were loaded in lanes 1 and 2. Lane 3 shows Coomassie Blue-stained molecular mass markers. K,
kDa.
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, 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.
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-Lys
) labeled on serine 177. (Exposure
2 days at -70 °C.)
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-Glu
) was included (lane
4), no phosphorylation of it was seen. This is discussed in detail
below.
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. , 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%.
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 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.)
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 -elimination of the
cysteine.
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.