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(Received for publication, August 21, 1995) From the
Phosphorylated prolactin has been identified and isolated from
bovine pituitaries. The biological activity of this phosphoprotein is
severely reduced in comparison with nonphosphorylated prolactin. The
sites of phosphorylation are serines 26, 34, and 90, and the
stoichiometry is 1:1:10, respectively. In this report, the
phosphoserine residues have been individually replaced with glutamic
acid in recombinant methionyl bovine prolactins in order to mimic
phosphorylation at each site. Substitution of glutamic acid for serine
at positions 26, 34, and 90 reduced protein helical contents by 10, 6,
and 14%, respectively. UV absorbances for S26E and S34E bovine
prolactins were blue-shifted, similar to the biological isolates of
phosphorylated bovine prolactin, but the biological activities of the
S26E and S34E mutants (ED
Prolactin binds and activates receptors on the plasma membrane
of target cells(1) . The PRL ( The
three-dimensional structure of PRL has not been solved, but it is
likely to be a four-helix bundle motif (6) similar to the
up-up-down-down structure of hGH, a protein related by sequence able to
bind prolactin receptors and induce lactogenic actions. The sequence of
several variants of the PRL receptor have been reported; most variation
between receptor subtypes is observed in the intracellular
domain(7) . The three-dimensional structure of hGH bound to the
extracellular domain of the hPRL receptor in a 1:1 complex has been
reported(8) . These data have defined the site 1 surface area
and have shown that the four-helix bundle of hGH has undergone little
structural change when bound to either the hPRL or hGH receptor.
However, there is a major shift of the first ancillary helix of the
loop connecting helices 1 and 2. The surface areas of hGH that contact
the extracellular domains of either hPRL or hGH receptors substantially
overlap(9) . A limited number of residues within the contact
areas provide the specificity for the lactogenic and somatotropic
receptor binding by participating in specific interactions that provide
most of the free energy liberated during the binding
reaction(10) . Receptor binding site 2 for hPRL is proposed to
be within the channel between helices 1 and 3(11) . From
this information, one can hypothesize that similar surfaces of PRLs and
hGH constitute sites 1 and 2 that bind receptors to create the 1:2
hormone:receptor heterotrimeric complex. If this is the case, then the
most likely surfaces for receptor binding determinants for PRLs are on
helices 1, 3, and 4 and the long loop connecting helices 1 and 2.
Several studies with PRL have identified functional determinants that
are consistent with this idea(12, 13) . Phosphorylated PRL has been identified in rat, turkey, and
cow(6) . Phosphorylated PRLs appear to have altered structure,
because they have unique biological activities or are unable to bind or
activate PRL receptors to initiate biological
actions(14, 15) . The addition of exogenous
phosphorylated rat PRL to primary cultures of rat pituitary cells
reduces secretion of endogenous PRL(16) . Phosphorylated rat (17) or bovine (15) PRL is not active in the Nb2 rat
lymphoma bioassay(18) , whereas the nonphosphorylated form is
mitogenic. Enzymatic removal of the phosphate will restore the potency
of phosphorylated bPRL measured in this assay(15) .
Phosphorylated bPRL does not compete with Phosphorylated
bPRL has been isolated and characterized from bovine
pituitaries(20, 21) . Three sites of phosphorylation
were identified at serines 26, 34, and 90(20) . Phosphorylated
bPRL has physical properties that are different from the
nonphosphorylated form and are believed to be a consequence of
phosphorylation. Stoichiometric studies indicate that serine 90 is
phosphorylated approximately ten times more often than serines 26 or
34; thus, the unique physical and biological properties of
phosphorylated bPRL are most likely due to phosphorylation at this
residue. Serine 90 of bPRL is conserved in PRLs, GHs, and placental
lactogens, whereas serines 26 and 34 are only found in PRLs. Serine 90
is distal from the documented and proposed binding determinants of
lactogenic hormones(8, 13, 22) ; thus, if
phosphorylation at this site is responsible for the loss of biological
activity, then it must induce sufficient distal conformation change to
remove the binding determinants from their spatial relationship
required to successfully engage the PRL receptor. Elucidation of the
specific role of each phosphorylation site is not possible using the
biological isolates of bPRL as they contain mixtures of several
phosphorylated variants. Recently, several investigators have
substituted serine with acidic residues in recombinant proteins to
mimic the effects of phosphorylation. Substitution of glutamic or
aspartic acid for serine 113, the site of phosphorylation in isocitrate
dehydrogenase, blocks the binding of isocitrate in the active site of
this protein and reduces enzymatic activity(23, 24) .
Trautwein et al.(25) have substituted aspartic acid
for serine 105 in the transcription activator NF-IL6/LAP and have
mimicked the effect of phosphorylation in activation of a
NF-IL6/LAP-regulated construct. In addition, alanine substitution of
serine 105 eliminated the enhancement of transcription observed with
the addition of exogenous kinase to an in vitro system. We
have substituted glutamic acid for serines 26, 34, and 90 to determine
if mimicry of phosphoserine residues will produce changes in the
properties and biological actions of bPRL that are similar to those
produced by phosphorylation. By this approach we hope to elucidate the
roles of individual phosphorylation sites.
Primers were designed to mutate serine 26, 34,
or 90 to glutamic acid and to add a unique restriction site (BglII, EcoRI, and AvaI for S26E, S34E, and
S90E bPRL, respectively). In vitro mutagenesis was performed
as described by Kunkel et al.(26) using T7 DNA
polymerase (catalog number 70017, U. S. Biochemical). The reaction
products were transformed into DH5
The
recombinant proteins were characterized for purity and size by
SDS-containing 12% acrylamide gel electrophoresis under reducing
conditions and by matrix-assisted laser desorption ionization-time of
flight mass analysis using an external standard (model G2025A,
Hewlett-Packard, Palo Alto, CA). An extinction coefficient was measured
by absorption at 280 nm in 10 mM ammonium bicarbonate and
related to the absolute amount of protein determined by quantitative
amino acid analysis(27) . Absorption, fluorescence, and far UV
circular dichroism spectra were measured at 22 °C for each
recombinant bPRL.
Stock solutions of recombinant bPRLs were prepared, their
optical densities were measured at 280 nm, and their protein
concentrations were determined by the bicinchoninic acid/copper sulfate
assay(28) . The bPRL preparations were diluted in medium and
added to 20,000 Nb2 cells in triplicate wells of 96-well culture plates
at final concentrations of 0-10 ng/ml. The cells were incubated
for 48 h. At the end of the growth period each well received 20 µl
of Alamar Blue (Alamar, Sacramento, CA) and was incubated for an
additional 4 h at 37 °C. The data provided by this vital dye method
were highly correlated to cell counts and demonstrated a reduced
variance within replicate determinations. At the completion of the
incubation the difference in absorbance of each well was measured at
570 and 600 nm. The difference was used to calculate an ED
The proteins were expressed and
purified with yields of final product varying between 3 and 12 mg/liter
of fermentation. Correlation coefficients varied between 0.86 and 0.91
when the moles of the individual amino acids of recombinant or National
Hormone and Pituitary Program bPRLs were correlated with the molar
ratios calculated from sequence(30) . The recombinant proteins
were greater than 95% homogeneous as observed on SDS-containing
polyacrylamide gel electrophoresis under reducing conditions (Fig. 1). The molecular weights as determined by mass
spectrometry were similar to the calculated values considering the
precision (0.1%) and accuracy (0.1%) of the measurement. No
contaminating proteins were observed in the mass spectra.
Figure 1:
SDS-gel
electrophoresis of recombinant bovine prolactins. 10 µg of each
bPRL were run on a 12% polyacrylamide gel under reducing conditions. NIH, NIH bPRL; WT, wild-type bPRL; S26E,
S26E bPRL; S34E, S34E bPRL; S90E, S90E
bPRL.
Figure 2:
Circular dichroism spectra for recombinant
bovine prolactins. Proteins were in 20 mM NaPO
The shape and
maxima of the fluorescence spectra (excitation 280 nm) (Fig. 3)
were similar for the wild-type, S26E, and S90E bPRLs, suggesting that
water exposure of the tryptophane residues were either not changed by a
mutation or were quenched by a mutation in the proximity of the
tryptophane (Trp
Figure 3:
Fluorescence spectra for recombinant
bovine prolactins. Proteins were in 20 mM NaPO
The UV
absorbance spectra of wild-type and S90E bPRLs are nearly identical
with maxima of 276 and 277 nm, respectively (Fig. 4). In
contrast, the mutation of serines 26 or 34 produce significant blue
shifts with small shifts of the maxima at 275 and 274 nm, respectively.
The aromatic peak of the S34E bPRL is approximately 50% broader than
the peaks of the other recombinant bPRLs.
Figure 4:
UV absorbance spectra for recombinant
bovine prolactins. Proteins were in 20 mM NaPO
Figure 5:
Biological activity of NIH and recombinant
bovine prolactins. Prolactins were placed in 10 mM
NH
The biological activity of S90E bPRL was
dramatically reduced. The calculated ED Replacement of serine 90 in bPRL with glutamic acid produced
a hormone that behaved similarly in the Nb2 biological assay to the
biological isolate of phosphorylated bPRL. These results confirm our
interpretation of previous sequence and stoichiometry studies (20) that demonstrated serine 90 to be the most frequently
phosphorylated and probably responsible for the reduced biological
activity of the phosphorylated hormone. The similar reductions of
ED The sites by which lactogenic
hormones interact and activate the lactogenic receptor are projected to
be on sections of PRL that are distal to serine
90(8, 12, 13) . Therefore, phosphorylation or
glutamic acid mimicry of bPRL at serine 90 must transduce structural
changes to distant receptor-binding determinants and induce a change in
their spatial relationships that preclude their productive interaction
with the PRL receptor. Several sets of structural elements may be
required for this mechanism to function. First, a kinase recognition
site must be present. Second, a set of local residues may be required
that would interact with the phosphoserine or glutamic acid in position
90 to induce a local change in structure. Finally, other structural
features must be present that respond to local alterations of structure
by affecting distal elements. The sequence surrounding serine 90 is
RSWNDP. If one assumes, by projecting from structures of related
proteins(8, 31) , that this sequence is a helix with a
proline-induced break, then an N+4 salt bridge between Arg In contrast, the S26E and S34E bPRLs had
biological activities that were indistinguishable from the wild-type
methionyl-bPRL. Both S26E and S34E bPRLs show evidence of tertiary
structural disruption by absorption and fluorescence spectroscopy.
These spectroscopic changes suggest that aromatic residue hydration is
increased, but the structural preturbations are neither sufficient nor
specific to affect the biological activity. In conclusion, glutamic
acid mutations of each of the serines that are phosphorylated in
vivo produce structural changes in bPRL that are observed by one
or more spectroscopic methods. Relative to wild-type bPRL, S26E and
S34E bPRLs appear to produce the blue shift in the absorption spectrum
seen with phosphorylated bPRL but do not affect biological activity,
whereas S90E bPRL produces a reduction in biological activity
equivalent to that observed with phosphorylated bPRL.
Volume 270,
Number 46,
Issue of November 17, 1995 pp. 27661-27665
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
values of 16.3 and 18.8
pM, respectively) were similar to that of wild-type prolactin
(ED
value of 18.6 pM) in the Nb2 rat lymphoma
assay. S90E bovine prolactin had the greatest reduction in helical
content but showed similar UV and fluorescent spectra to the wild-type
bovine prolactin. The biological activity of S90E bovine prolactin
(ED
value of 672 pM) was reduced to an activity
similar to that of phosphorylated bovine prolactin. The data indicate
that the phosphorylation of serine 90 is responsible for the reduction
in biological activity.
)receptor is a member
of the cytokine receptor superfamily(2) , receptors with
extracellular and intracellular domains connected by a single
transmembrane domain. Several receptors in this superfamily activate
cells by formation of receptor dimers brought about by the binding of
two receptors binding to unique surfaces of the ligand (sites 1 and
2)(3) . Receptor dimerization promotes the association and/or
activation of the Janus family of tyrosine kinases (4) and
phosphorylation of specific tyrosines of the receptor and substrate
proteins that regulate transcription in the nucleus or the activity of
other kinase systems. Substrate proteins appear to be presented to the
Janus kinases by binding to the phosphorylated loci of the
intracellular domain of the receptor(5) .I-labeled
nonphosphorylated bPRL for binding to the intermediate form of the PRL
receptor found in Nb2 cells(15) . The failure of phosphorylated
bPRL to bind to the PRL receptor suggests that phosphorylation removed
the binding determinants of site 1 from the spatial relationship
required for receptor binding(19) ; in other words,
phosphorylation has induced a conformation change.
Plasmids and Bacterial Strains
Plasmid
pT7-7 was kindly provided by S. Tabor (Harvard Medical School,
Boston, MA). An f origin of replication was
ligated into the plasmid at the ClaI site to obtain both
positive and negative strand vectors. (
)The positive strand
pT7-7 phagemid was used in all studies. E. coli strains
DH5
, RZ1032 dutung
, and
BL21(DE3) were used for cloning, production of uridine-substituted
single-stranded DNA for in vitro mutagenesis, and protein
expression, respectively.
Preparation of Recombinant Bovine
Prolactin
Total RNA was isolated from bovine anterior
pituitary using RNAsol B (Tel-Test, Inc., Friendswood, TX). cDNA was
prepared with reverse transcription with random hexamers. The 5` primer
for polymerase chain reaction was designed to eliminate the leader
sequence, add a methionyl residue at the start of the mature sequence,
and provide an NdeI site at the methionyl residue. The first 7
residues were converted to bacterially preferred codons by design of
this primer. The 3` primer introduced a HindIII restriction
site following the termination codon. The polymerase chain reaction
product was digested with NdeI and HindIII and
ligated into the pT7-7 phagemid previously cut by the same
restriction enzymes. The product was transformed into DH5 cells,
and colonies were selected after growth on LB agar plates containing
ampicillin. Selected colonies were initially analyzed by agarose gel
electrophoresis. Positive colonies contained a 3623-base pair DNA.Site-directed Mutagenesis
The wild-type
bPRL phagemid was transformed into RZ1032 cells, and clones were
selected by ampicillin resistance. A colony was grown in LB media, and
a portion was transferred to 2 
TY media (16 g of tryptone, 10 g
of yeast extract, 5 g of NaCl in 1 liter of water) containing
ampicillin and grown to a density of 0.3 at A. The culture was infected with R408 helper phage at a
multiplicity of infection of 20. Cells containing both vectors were
selected by resistance to kanamycin. After an overnight growth, the
single-stranded uridine-substituted phagemid was purified from the
media(26) .
cells and grown on LB agar
plates containing ampicillin. Several colonies for each mutant were
grown in LB media and subsequently analyzed by restriction digests.
Colonies that were cut by the enzyme for the restriction site added
during mutagenesis were selected, and the mutation was confirmed by
sequence analysis.Production and Purification of Recombinant Bovine
Prolactins
Phagemids containing the wild-type or the S26E,
S34E, and S90E mutants of DNA coding for methionyl-bPRL were
transformed into BL21 cells, and positive clones were selected. One
liter LB media cultures were grown to an optical density of A = 0.3. Protein expression was
induced by the addition of
isopropyl-
-D-thiogalatopyranoside to 0.4 mM, and
incubation of the culture was continued for 4 h. The cells were
collected, resuspended in 100 mM Tris, pH 7.5, containing 1
mM phenylmethylsulfonyl fluoride and 25 mM dithiothreitol. Cells were broken by two passages through a French
pressure cell. The inclusion bodies were collected by centrifugation,
resuspended in 4.5 M deionized urea, 100 mM Tris, pH
11.3, and dissolved for 2 h at room temperature. The solubilized
protein was incubated at 4 °C for 2 days and then dialyzed into 20
mM Tris, pH 7.4. The bPRLs were purified on a disposable DE52
anion exchange matrix (Whatman, Clifton, NJ) developed with a
0-750 mM NaCl gradient in the same buffer. The proteins
were dialyzed into 5 mM ammonium bicarbonate and lyophilized.
Each of the proteins was prepared under identical conditions.Characterization of Recombinant DNA and
Proteins
The pT7-7 phagemid DNAs containing the
wild-type or the S26E, S34E or S90E mutations were each sequenced from
primers annealing to phagemid sequences surrounding the polycloning
region. The reactions used fluorochrome-labeled termination bases with
T7 DNA polymerase (Sequinase, U. S. Biochemical), and the separation
and data acquisition were performed by a fluorescence DNA sequencer
(Applied Biosystems model 373A, Foster City, CA). Two reactions,
reading opposite strands, sequence the 604-base pair insert.Nb2 Rat Lymphoma Bioassay
The biological
activity of each bPRL was measured in the Nb2 rat lymphoma biological
assay(18) . Nb2 cells were grown in Fischer's medium
containing penicillin (50 units/ml), streptomycin (50 µg/ml), 0.1
mM 2-mercaptoethanol and supplemented with 10% horse serum and
10% fetal bovine serum. The cells were grown in reduced serum (10%
horse and 1% fetal bovine serum) for the 24 h immediately preceding the
assay. by a four-parameter fit method (ALLFIT program of Munson and
Rodbard(29) ). The concentrations of the bPRLs were corrected
by their extinction coefficients.
Characterization of the Recombinant DNA and
Proteins
The wild-type bPRL phagemid DNA insert coded for a
methyionyl residue followed by the complete DNA sequence of mature bPRL (30) . Each of the mutants was completely sequenced, and each
contained the sequence appropriate for a silent restriction site and
serine to glutamic acid mutation.
Spectroscopy of the Recombinant
Proteins
The concentrations of the wild-type, S26E, S34E,
and S90E bPRLs used to measure circular dichroism spectra were
determined by applying the molar extinction coefficients to the
absorbance values. S26E, S34E, and S90E bPRLs had molar elipticities at
222 nm of 90, 94, and 86%, respectively, when compared with that of the
wild-type bPRL (Fig. 2); these data suggest small reductions in
helical contents resulting from the glutamic acid mutations. The 208 nm
elipticity of the S90E bPRL is increased relative to the wild-type
bPRL, suggesting a reduction in helical content.
buffer, pH 7.5, with 150 mM NaCl. Concentrations
determined by the extinction coefficients and the 280 nm absorbance
were between 4.9 and 8.8 uM. Spectra were obtained at room
temperature with a 0.0009684-cm pathlength after calibration with
camphosulfonic acid in a Jasco Model J-500A
spectropolarimeter.
and S90E). In contrast, S34E bPRL
displayed an 8.5 nm red shift (at half-peak height), indicative of a
greater water exposure of the tryptophane residues.
buffer, pH 7.5, with 150 mM NaCl. The emission spectra
were measured with a 280 nm excitation in a Perkin-Elmer model LS-50B
fluorimeter at ambient temperature. The raw data (inset) were
normalized as a fraction of the maximum
intensity.
buffer, pH 7.5, with 150 mM NaCl. The absorbance spectra
were measured in a Uvicon Model 930 spectrophotometer at ambient
temperature. The raw data (inset) were normalized to 280
nm.
Biological Activity of the Recombinant
Proteins
The ED values of the bPRL
preparations were calculated from the Nb2 biological assay data (Fig. 5) without fixing any of the four variables of the
curve-fitting method (minimum, maximum, slope, and ED
).
ED
s of wild-type, S26E, and S34E bPRLs were 18.6, 16.3,
and 18.8 pM, respectively. The coefficient of variation of
these measurements averaged 11.8%. The biological activities of the
recombinant bPRLs were slightly less than the biological isolate of
National Hormone and Pituitary Program bPRL (ED
=
7.0 pM).
HCO
, and the concentration was calculated
using the extinction coefficient. The Nb2 bioassay was performed as
described under ``Materials and Methods'' using a vital dye
to measure relative cell numbers.
of S90E bPRL was
1578 pM with none of the variables fixed, but the accuracy of
this value is difficult to assess because the doses used in the assay
failed to induce a full biological response. When the maximum and
minimum variables were set as parameters, the ED
for S90E
bPRL was 672 pM with an 11% coefficient of variation. The
activity of S90E bPRL is similar to the activity of our biological
isolate of phosphorylated bPRL (727 pM) in the Nb2
bioassay(15) .
values of the S90E and phosphorylated bPRLs suggested
that glutamic acid was fully capable of functionally replacing
phosphoserine. The presence of a negative charge is the most striking
common structural feature of these two preparations. We suggest that
this charge induces changes in the bPRL structure that disallows the
protein to productively engage the receptor. The mechanism by which
this occurs remains to be elucidated.
and Asp
stabilizes the helical structure just
N-terminal to Pro
. The introduction of a negative charge
at Ser
between Arg
and Asp
might
disrupt the secondary structure in this region. This may account for
the 14% reduction in helical content observed between the wild-type and
S90E bPRLs. Further studies will be required to conclude if the
introduction of charge in an N+4 salt bridge is the local
mechanism of transduction.
))
Bovine prolactin was provided by the National Hormone
and Pituitary Program. We acknowledge and appreciate the contributions
of Michael Zehfus and Mark Maciejewski of the College of Pharmacy for
aid in obtaining and interpreting the circular dichroism data.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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