(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 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.
Prolactin binds and activates receptors on the plasma membrane
of target cells(1) . The PRL ()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) .
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 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.
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 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.
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 by a four-parameter fit method (ALLFIT program of Munson and
Rodbard(29) ). The concentrations of the bPRLs were corrected
by their extinction coefficients.
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 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.
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 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.
Figure 3:
Fluorescence spectra for recombinant
bovine prolactins. Proteins were in 20 mM NaPO 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.
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 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.
Figure 5:
Biological activity of NIH and recombinant
bovine prolactins. Prolactins were placed in 10 mM
NHHCO
, 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.
The biological activity of S90E bPRL was
dramatically reduced. The calculated ED 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) .
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 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.
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 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.
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.